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Polarized epithelium and localization of ion channels


I'm trying to learn more about polarized epithelial cells of the gut. I am familiar with classic brush border transporters localized to the apical memebrane to facilitate nutrient absorption. I am wondering though, where are ion channels located? I would guess basolaterally since they would be exposed to the extracellular space. I would appreciate a primary reference showing the location of voltage-gated channels in particular as I could not find them myself.


Well, that's a first for me. I wouldn't have guessed gut cells would have voltage-gated channels. This article describes voltage-gated sodium channels on both the luminal and basolateral membranes:

Barshack, I., Levite, M., Lang, A., Fudim, E., Picard, O., Ben Horin, S., & Chowers, Y. (2008). Functional voltage-gated sodium channels are expressed in human intestinal epithelial cells. Digestion, 77(2), 108-117. http://www.ncbi.nlm.nih.gov/pubmed/18391489


Ion channel

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Ion channel, protein expressed by virtually all living cells that creates a pathway for charged ions from dissolved salts, including sodium, potassium, calcium, and chloride ions, to pass through the otherwise impermeant lipid cell membrane. Operation of cells in the nervous system, contraction of the heart and of skeletal muscle, and secretion in the pancreas are examples of physiological processes that require ion channels. In addition, ion channels in the membranes of intracellular organelles are important for regulating cytoplasmic calcium concentration and acidification of specific subcellular compartments (e.g., lysosomes).


Polarized targeting of ion channels in neurons

Since the time of Cajal it has been understood that axons and dendrites perform distinct electrophysiological functions that require unique sets of proteins [Cajal SR Histology of the nervous system, Oxford University Press, New York, (1995)]. To establish and maintain functional polarity, neurons localize many proteins specifically to either the axonal or the somatodendritic compartment. In particular, ion channels, which are the major regulators of electrical activity in neurons, are often distributed in a polarized fashion. Recently, the ability to introduce tagged proteins into neurons in culture has allowed the molecular mechanisms underlying axon- and dendrite-specific targeting of ion channels to be explored. These investigations have identified peptide signals from voltage-gated Na+ and K+ channels that direct trafficking to either axonal or dendritic compartments. In this article we will discuss the molecular mechanisms underlying polarized targeting of voltage-gated ion channels from the Kv4, Kv1, and Nav1 families.

Journal

Pflügers Archiv European Journal of Physiologyl of Physiology &ndash Springer Journals


Mechanisms of polarized sorting

A polarized surface distribution of proteins and lipids can be achieved by targeted delivery or selective retention (Yeaman et al., 1999 Matter, 2000). Targeted delivery relies on the segregation of cargo molecules destined for different plasma membrane domains before they reach the cell surface. Selective retention works by trapping them once they arrive - for instance, by anchoring to a domain-specific cytoskeletal scaffold. Proteins delivered to the wrong surface domain are removed by endocytosis and are then degraded or undergo another round of delivery. Targeted delivery and selective retention can be combined such that selective retention enhances the accuracy of intracellular sorting (Mays et al., 1995).

The basolateral as well as the apical sorting pathway has been suspected to involve bulk flow, but it is now clear that neither is a general default pathway for the surface delivery of proteins. Instead, apical and basolateral sorting are both governed by sorting determinants embedded in cargo proteins.

Sorting determinants

Sorting determinants in membrane proteins can reside in the cytoplasmic domain, membrane anchor or extracellular domain (Table 1). Cytoplasmic domain determinants include the basolateral-targeting, tyrosine-based and dileucine motifs (Matter and Mellman, 1994), PDZ-domain-binding motifs and a growing list of unrelated sequences (Altschuler et al., 2003 Muth and Caplan, 2003). Determinants in membrane anchors include the transmembrane domains of some apically targeted viral proteins (Kundu et al., 1996 Lin et al., 1998) and glycosylphosphatidylinositol (GPI) anchors. The latter usually confer localization to the apical membrane (Brown et al., 1989 Lisanti et al., 1989), but alone are not always sufficient (Brown and London, 1998 Benting et al., 1999a). Both N- and O-glycosylation of the extracellular domain have been implicated in apical targeting (Scheiffele et al., 1995 Yeaman et al., 1997 Gut et al., 1998 Spodsberg et al., 2001). Finally, oligomerization of membrane proteins may be an important sorting determinant, particularly for apical transport. Evidence in epithelial cells is lacking so far, but sorting of the voltage-gated potassium channel Kv1 into the axonal pathway of neurons, which is related to the apical pathway of epithelial cells (Horton and Ehlers, 2003), requires oligomerization (Gu et al., 2003).

Determinants for the polarized sorting of membrane proteins

Site . Determinant . Polarity . Examples * .
Cytoplasmic domain Tyrosine-based motif Basolateral LDL receptor, transferrin receptor, vesicular stomatitis virus glycoprotein
Dileucine motif Basolateral IgG Fc receptor, E-cadherin
PDZ-binding motif Apical/basolateral CFTR (a), BGT-1 GABA transporter (bl), ERbB-2 receptor tyrosine kinase (bl)
Others Apical/basolateral H,K-ATPase α-subunit (a), megalin (a), GAT-2 GABA transporter (bl)
Membrane anchor Transmembrane domain Apical Influenza virus hemagglutinin and neuraminidase
GPI-anchor Apical Placental alkaline phosphatase, decay-accelerating factor, Thy-1
Extracellular domain N-glycosylation Apical Occludin (truncated version), FcLR (Fc receptor-LDL receptor chimera), GLYT2 glycine transporter
O-glycosylation Apical Neurotrophin receptor, sucrase isomaltase
Oligomerization domain? Apical? Kv 1 potassium channel (neurons)
Site . Determinant . Polarity . Examples * .
Cytoplasmic domain Tyrosine-based motif Basolateral LDL receptor, transferrin receptor, vesicular stomatitis virus glycoprotein
Dileucine motif Basolateral IgG Fc receptor, E-cadherin
PDZ-binding motif Apical/basolateral CFTR (a), BGT-1 GABA transporter (bl), ERbB-2 receptor tyrosine kinase (bl)
Others Apical/basolateral H,K-ATPase α-subunit (a), megalin (a), GAT-2 GABA transporter (bl)
Membrane anchor Transmembrane domain Apical Influenza virus hemagglutinin and neuraminidase
GPI-anchor Apical Placental alkaline phosphatase, decay-accelerating factor, Thy-1
Extracellular domain N-glycosylation Apical Occludin (truncated version), FcLR (Fc receptor-LDL receptor chimera), GLYT2 glycine transporter
O-glycosylation Apical Neurotrophin receptor, sucrase isomaltase
Oligomerization domain? Apical? Kv 1 potassium channel (neurons)

Overall, the known apical sorting determinants are more diverse and not as strict as prototypical basolateral determinants. Basolateral determinants seem to dominate over apical ones, because addition of a tyrosine-based or dileucine motif usually redirects apical proteins to the basolateral membrane. Such a hierarchy of sorting determinants could help to ensure stringent sorting of basolateral proteins away from the apical membrane. However, there are also examples of recessive basolateral determinants (Monlauzeur et al., 1995 Jacob et al., 1999 Ihrke et al., 2001), indicating that the hierarchy of sorting determinants is more complicated or that their relative strength depends on the protein in which they are present.

Sorting by receptor-mediated cargo capture

Certain cytoplasmic domain determinants are recognized by specific sorting receptors. Best characterized are the adaptor protein (AP) complexes, which sort proteins along many intracellular trafficking routes (Robinson, 2004). Several tyrosine-based motifs interact with the epithelia-specific μt1B subunit of the AP-1 complex, and this interaction is crucial for the basolateral sorting of the low-density lipoprotein (LDL) receptor and the transferrin receptor (Folsch et al., 1999 Sugimoto et al., 2002). Other adaptors implicated in the recognition of basolateral determinants are the AP-3 and AP-4 complexes (Nishimura et al., 2002 Simmen et al., 2002). How the binding of adaptor complexes to cargo proteins is coupled to the machinery responsible for the formation and movement of transport carriers is not understood. The ability of the AP-1 complex to bind to clathrin makes an involvement of clathrin-coated vesicles in basolateral transport plausible, but direct evidence has been difficult to obtain. Adaptor complexes can also bind to motor proteins, which then move cargo containers towards the cell periphery (Nakagawa et al., 2000 Setou et al., 2000). Alternatively, motor proteins themselves may serve as receptors for cytoplasmic domain determinants, as is the case for the dynein-mediated apical transport of rhodopsin (Tai et al., 2001). In any event, receptor-mediated cargo capture by specific protein-protein interactions provides a stringent mechanism for the inclusion of proteins into transport carriers.

Whether cytoplasmic PDZ-domain-binding motifs are recognized by sorting receptors or mediate selective retention at the target membrane is not clear. In the case of the cystic fibrosis transmembrane conductance regulator (CFTR), a PDZ-domain-binding motif seems to be responsible for its selective apical retention after initial transport to both membrane domains (Swiatecka-Urban et al., 2002). The PDZ protein NHERF links CFTR to the actin network beneath the apical membrane (Short et al., 1998), and NHERF probably plays this scaffolding role for various apical proteins (Shenolikar and Weinman, 2001 Altschuler et al., 2003). Similarly, the epithelial gamma aminobutyric acid (GABA) transporter BGT-1 and the receptor tyrosine kinase ErbB-2 have PDZ-domain-binding motifs that mediate basolateral retention by interacting with the PDZ protein LIN-7 (Perego et al., 1999 Shelly et al., 2003). However, there are reports arguing that the PDZ-domain-binding motif is dispensable for the apical targeting of CFTR (Benharouga et al., 2003 Ostedgaard et al., 2003), and basolateral targeting of BGT-1 and ErbB-2 still occurs when the motifs are deleted. Localization of these proteins therefore appears to involve a combination of targeting and retention mechanisms.

Lipid rafts as apical sorting platforms

Sorting receptors that engage in direct protein-protein interactions with apical sorting determinants in membrane anchors or extracellular domains have not been identified. Instead, the sorting of many apical proteins may be governed by lipid-lipid and lipid-protein interactions. The enrichment of sphingolipids in the apical membrane, together with their propensity to associate with cholesterol to form lipid rafts, has led to the concept that rafts preferentially traffic to the apical membrane after intracellular assembly. Since certain proteins associate with rafts during apical transport, rafts could act as apical sorting platforms (Simons and Ikonen, 1997).

Support for this hypothesis has come from two types of experiment. First, apical sorting is particularly sensitive to depletion of cholesterol and sphingolipds (Mays et al., 1995 Keller and Simons, 1998 Hansen et al., 2000 Lipardi et al., 2000). However, this is only indirect evidence, because depletion of these raft lipids could also affect apical trafficking in a more general way. Second, certain apically targeted proteins such as the GPI-anchored placental alkaline phosphatase (PLAP) and influenza virus hemagglutinin, but not typical basolateral protein, enter rafts before they reach the cell surface (Skibbens et al., 1989 Brown and Rose, 1992). Apical sorting of these proteins thus correlates with a dramatic change in their lipid environment during surface transport, but whether this change is required for accurate targeting is unclear. In addition, in these experiments, rafts were defined as detergent-resistant membranes (DRMs), i.e. membranes that resist solubilization with mild detergents such as Triton X-100. Although useful, detergent resistance is a rather crude criterion to determine raft association (London and Brown, 2000 Schuck et al., 2003). Indeed, several endogenous apical proteins in the well-characterized Madin-Darby canine kidney (MDCK) cell line are fully detergent soluble, and some DRM-associated proteins still reach the apical membrane when rendered detergent soluble by mutation or cholesterol depletion (Lin et al., 1998 Lipardi et al., 2000). Detergent extraction probably disrupts weak interactions of proteins with raft domains, but these interactions might nonetheless be important for sorting (Shvartsman et al., 2003). Thus, a lack of suitable methods has hampered the quest for conclusive evidence for or against sorting by raft association.

Sorting by cargo recruitment into clustered rafts

Recent work has revealed the ability of individual rafts to cluster selectively into large domains, and this allows us to propose a more comprehensive model for the role of rafts in polarized sorting (Fig. 1). To introduce this model, we first examine the clustering of rafts at the cell surface. We then discuss how raft clustering might be utilized for sorting, how known apical sorting determinants fit into this picture and what kind of protein machinery could mediate raft clustering. Last, we argue that different raft clusters can be produced.

Raft clustering and domain-induced budding. Before clustering, proteins associate with rafts (grey) to various extents. A GPI-anchored protein (gold) resides exclusively in rafts, a doubly acylated protein (red) is mainly in rafts, a transmembrane protein (green) is mainly outside rafts, and another transmembrane protein (pink) is excluded from rafts (A). Clustering is induced, for example, by the binding of a multimeric protein of the annexin type (blue) to the cytoplasmic face of rafts. The strongly raft-associated GPI-anchored and doubly acylated proteins partition into clustered rafts. The weakly raft-associated transmembrane protein is driven into clustered rafts by crosslinking with a divalent interaction partner, e.g. a lectin (black). Note that the recruitment of the weakly raft-associated transmembrane protein is not complete, nor are all rafts clustered. For simplicity, only one type of raft cluster is shown, i.e. differential clustering of rafts into separate domains with different constituents is not depicted (B). Growth of the clustered raft domain beyond a critical size induces budding (C). Finally, a transport container consisting of raft components pinches off from the parent membrane by fission at the domain boundaries (D).

Raft clustering and domain-induced budding. Before clustering, proteins associate with rafts (grey) to various extents. A GPI-anchored protein (gold) resides exclusively in rafts, a doubly acylated protein (red) is mainly in rafts, a transmembrane protein (green) is mainly outside rafts, and another transmembrane protein (pink) is excluded from rafts (A). Clustering is induced, for example, by the binding of a multimeric protein of the annexin type (blue) to the cytoplasmic face of rafts. The strongly raft-associated GPI-anchored and doubly acylated proteins partition into clustered rafts. The weakly raft-associated transmembrane protein is driven into clustered rafts by crosslinking with a divalent interaction partner, e.g. a lectin (black). Note that the recruitment of the weakly raft-associated transmembrane protein is not complete, nor are all rafts clustered. For simplicity, only one type of raft cluster is shown, i.e. differential clustering of rafts into separate domains with different constituents is not depicted (B). Growth of the clustered raft domain beyond a critical size induces budding (C). Finally, a transport container consisting of raft components pinches off from the parent membrane by fission at the domain boundaries (D).

The size and stability of rafts in the unperturbed state is controversial, but there is a growing consensus that individual rafts can be induced to form large, stable clusters (Kusumi et al., 2004 Simons and Vaz, 2004). Raft clustering can be initiated by the oligomerization of raft components. At the cell surface, clustering can be brought about artificially by antibody crosslinking, but it also occurs naturally, for example when interactions between the T-cell receptor and peptides bound to major histocompatibility complexes (MHC) on antigen-presenting cells trigger the clustering of rafts during formation of the immunological synapse (Harder and Engelhardt, 2004). As a consequence of raft clustering, raft association of proteins and lipids that have an affinity for liquid-ordered domains is stabilized, whereas non-raft components are excluded. For example, antibody crosslinking of the GPI-anchored PLAP or the apical transmembrane protein gp114 at the plasma membrane of MDCK cells strengthens their raft association, as judged by increased detergent resistance (Harder et al., 1998 Verkade et al., 2000). This happens because oligomerization by crosslinking multiplies the tendency of these proteins to associate with raft domains. In thermodynamic terms, the partitioning coefficient of oligomers between the raft and the non-raft phase is given by the product of the partitioning coefficients of the monomers they are composed of. This should lead to an exponential increase in raft affinity as oligomer size increases (Simons and Vaz, 2004).

Importantly, antibody crosslinking of PLAP leads to co-clustering of the doubly acylated raft protein Fyn, a peripheral membrane protein that resides on the opposite side of the membrane. Thus, the crosslinking of one raft protein moves whole lipid microdomains together in both membrane leaflets (Harder et al., 1998 Prior et al., 2003). The same is true for raft lipids, as shown by crosslinking of the glycosphingolipid GM1 with the pentameric cholera toxin, which leads to co-clustering with various raft proteins (Janes et al., 1999). This presumably occurs because lipids and proteins within raft microdomains associate tightly enough to allow these domains to behave as stable entities (Pralle et al., 2000). In contrast to raft components, non-raft proteins, such as the transferrin receptor, are excluded from clustered rafts, even when crosslinked (Harder et al., 1998 Janes et al., 1999). These examples show that the clustering of individual rafts reinforces the segregation of the raft and the non-raft phase, and sharpens the distribution of membrane components between the two phases. Thus, raft clustering is a mechanism for the selective recruitment of proteins and lipids that have an affinity for liquid-ordered domains, as well as the efficient exclusion of non-raft proteins.

We envisage that the clustering of lipid rafts is used as a cellular sorting mechanism to recruit certain cargo proteins yet exclude others. Stabilization of rafts should attract proteins and lipids that have a strong affinity for ordered domains. Indeed, clustered rafts might exhibit an even greater degree of lipid ordering than individual rafts, thus providing a preferable environment for these molecules. Recruitment into clustered rafts could be assisted by additional interactions in the case of weakly raft-associated proteins. Crosslinking by a multivalent interaction partner outside rafts could augment their raft affinity. Also, they could bind to other proteins that drag them into clustered rafts. There, they could become entrapped by further modifications that strengthen their raft association, such as palmitoylation, oligomerization or a conformational change (Bagnat et al., 2001 Cherukuri et al., 2004). Note that certain proteins and lipids that have raft affinity might still partially localize to non-raft membranes or non-clustered rafts. Nevertheless, clustering should create membrane patches consisting of a select group of molecules whose trafficking fates are connected.

Certain apical sorting determinants are able to facilitate raft association, especially when rafts are clustered. First, GPI-anchored proteins generally associate with lipid rafts on the basis of the favourable packing of the GPI anchor into liquid-ordered domains. However, GPI anchors are structurally diverse, and so this might not be true for all GPI-linked proteins (Benting et al., 1999b Mayor and Riezman, 2004). Interestingly, raft association of GPI-anchored proteins might need to be stabilized by oligomerization for them to be sorted apically (Paladino et al., in press). Second, transmembrane domains known to act as apical sorting determinants also mediate raft association (Scheiffele et al., 1997 Barman and Nayak, 2000). The principles underlying the association of transmembrane proteins with rafts are poorly understood. One element is binding to raft lipids and, possibly, conformational changes induced by specific protein-lipid interactions (Simons and Vaz, 2004). Another factor might be the length of the transmembrane domain. It has been proposed that the targeting of transmembrane proteins to different cell membranes is based on the length of the membrane-spanning region (Bretscher and Munro, 1993). Long transmembrane domains could have a preference for raft membranes, whereas proteins that have short transmembrane domains might be excluded. Indeed, owing to the ordering of lipid side chains, raft membranes are probably thicker than non-raft membranes. In addition, they have different elastic properties, e.g. they are more difficult to bend or compress. Recently, a theoretical study showed that these elastic properties, rather than membrane thickness, provide the primary driving force for the exclusion of proteins that have short transmembrane domains from cholesterol-enriched membranes (Lundbaek et al., 2003). If clustering were to increase the lipid order of rafts and make them even less elastic, raft clustering would potentiate the effect of transmembrane domain length on the raft association of transmembrane proteins. Third, oligomerization should amplify the affinity of proteins for rafts by the same mechanism as antibody crosslinking does. This could explain why oligomerization might be an important determinant for raft-mediated apical sorting.

How might raft clustering be induced? From what is known about the clustering process, any oligomerization of raft components could be sufficient. Several proteins might promote raft clustering, although the available evidence is circumstantial. One type of `clustering agent' might be represented by VIP17/MAL and the closely related MAL2. Both are tightly raft-associated integral membrane proteins and play a role in apical targeting (Puertollano et al., 1999 Cheong et al., 1999 de Marco et al., 2002). Moreover, VIP17/MAL can form oligomers, which might function to cluster rafts at the sites at which sorting takes place. Similarly, caveolins, flotillins and stomatin are raft-associated membrane proteins that form oligomers (Monier et al., 1995 Snyers et al., 1999 Neumann-Giesen et al., 2004). Caveolins clearly have the ability to cluster rafts, acting as scaffolds for caveolae (van Deurs et al., 2003). Sotgia et al. have proposed that caveolin-1 is required for transport of GPI-anchored proteins to the plasma membrane (Sotgia et al., 2002), but evidence from our laboratory argues against a major role of caveolin-1 in polarized sorting in MDCK cells (A. Manninen, J. Füllekrug and K.S., unpublished).

Raft clustering might be promoted in a slightly different way by annexin 13b and annexin 2, which are cytosolic proteins that facilitate apical transport in MDCK cells (Lafont et al., 1998 Jacob et al., 2004). Both are unusual annexins because they preferentially associate with cholesterol-rich membranes (Lafont et al., 1998 Rescher and Gerke, 2004). Again, they could act as membrane organizers through their ability to oligomerize, since some annexins, including annexin 2, form large, two-dimensional ordered arrays (Oling et al., 2001). Little is known about the cytoplasmic side of rafts, but it might contain enough cholesterol to serve as a preferred docking site for annexin 13b and annexin 2. Although rafts are primarily thought of as assemblies in the exoplasmic membrane leaflet, there clearly is leaflet coupling such that raft domains on the exoplasmic side are matched by raft domains on the cytoplasmic side (Harder et al., 1998 Korlach et al., 1999 Prior et al., 2003). Thus, clustering of raft lipids on the cytoplasmic side by annexin oligomers should cluster raft components in the exoplasmic leaflet.

Lectins of the galectin family might be involved in driving glycolipids and glycoproteins into clustered rafts. Galectins are usually multivalent owing to self-association and can organize glycoproteins into large regular arrays (Brewer et al., 2002). Galectin 4 tightly associates with rafts on the outside of the brush border membrane of intestinal cells and stabilizes raft domains (Braccia et al., 2003). Recent studies implicate galectin 4 in apical sorting in enterocytes (D. Delacour, V. Gouyer, A. Manninen, K.S. and G. Huet, unpublished). Galectins are secreted from cells by a non-classical mechanism that bypasses the Golgi complex. How they could enter the secretory pathway is therefore unclear. However, they might be re-internalized by endocytosis and thus gain access to intracellular sorting sites.

Finally, it is important to realize that rafts can be clustered differentially. For example, migrating T lymphocytes are able to set up separate raft domains that have distinct compositions at the leading edge and the uropod (Gomez-Mouton et al., 2001). Likewise, although the mating projection in yeast is a specialized membrane domain that might arise by raft clustering, raft proteins are also found elsewhere in the plasma membrane (Bagnat and Simons, 2002). The immunological synapse formed by activated T cells is viewed as a raft cluster but nevertheless excludes raft proteins that have no function in immune signalling (Harder and Kuhn, 2000 Bunnell et al., 2002). In all these processes, the actin cytoskeleton plays a key role. However, even when raft components are clustered artificially, selective co-clustering of raft components is still observed (Wilson et al., 2004). Hence, differential raft clustering appears to be a general principle. In the context of intracellular sorting, the differential clustering of lipid rafts might be used to segregate raft components that have different destinations.

Generation of transport carriers by raft clustering

Raft clustering could also provide a mechanism for the generation of transport carriers. If phases with different properties coexist in the same membrane, as in the case of liquid-ordered microdomains in a membrane mainly in the liquid-disordered phase, there is line tension at the phase boundaries. Line tension is the two-dimensional equivalent of surface tension and arises from the immiscibility of membrane components that prefer different phases. The energetic cost of line tension can be reduced by decreasing the contact between phases, i.e. by decreasing the boundary length of domains that constitute the minority phase. This is achieved most effectively when domains that are part of the minority phase bud out of the majority phase and eventually detach from the parent membrane. Line-tension-driven budding is opposed by the energy required to bend the membrane of the incipient bud. Importantly, the balance between line tension and bending energy depends on the size of the membrane domain in the minority phase. The line tension increases with increasing domain size, whereas the bending energy is independent of the domain size. Small domains give rise to buds that have higher degrees of curvature than large buds, but the energy to deform the membrane into a sphere is the same. As a result, a growing domain within a membrane will reach a critical size beyond which budding becomes energetically favourable (Lipowsky, 1993 Lipowsky, 2002). This mechanism, termed domain-induced budding, was originally postulated on theoretical grounds but has recently received experimental support from studies of model membranes (Baumgart et al., 2003). As predicted, domains in the minority phase were observed to bud out of the surrounding membrane, with fission occurring at the phase boundaries.

In cell membranes, individual rafts usually form a dispersed minority phase within a continuous non-raft phase (Simons and Toomre, 2000 Prior et al., 2003). Therefore, clustering of small rafts into large domains should induce budding, followed by fission at the domain boundaries and the generation of vesicles strongly enriched in raft components (Fig. 1C,D). However, the factors governing the budding size of clustered raft domains are likely to be more complex in cell membranes. The line tension between raft and non-raft membranes could be modulated by surfactancy effects of lipids and proteins at the domain interfaces (Simons and Vaz, 2004). The spontaneous curvature of raft domains that results from lipid asymmetry between the two membrane leaflets could play a part in controlling the budding process (Huttner and Zimmerberg, 2001). Moreover, budding must be regulated in cells by proteins and probably also be supported by traction forces generated by molecular motors, and scission of the bud neck could be assisted by proteins such as dynamin (Kreitzer et al., 2000).


Apical sorting mechanisms

Apical sorting signals

Apical sorting signals are required to direct the transport of newly synthesized proteins to the apical cell surface. Remarkably, sorting signals have been localized to all the portions of apical proteins: extracellular, transmembrane and cytoplasmic domains 8,60 .

A well-studied apical sorting signal is the glycosylphosphatidylinositol (GPI) anchor. GPI-anchored proteins (GPI-APs) are preferentially localized to the apical membrane of epithelial cells 61 . Supporting evidence for the role of GPI anchors in apical localization comes from the fact that not only endogenous GPI-APs but also chimeric GPI-APs 62,63,64 in polarized MDCK cells localize apically. However, the relative strength of this sorting signal and what determines in detail whether a GPI-AP will be routed to the apical membrane remain not completely understood. For example, the GPI-anchored prion protein was shown to localize basolaterally in MDCK cells 65 , and GPI-APs are preferentially targeted to the basolateral surface in Fischer rat thyroid epithelial cells 66 . Importantly, clustering of GPI-AP is necessary for efficient apical targeting 67,68 . Furthermore, the GPI-attachment sequences 69 and the remodeling of the fatty-acid chains 70 seem to play important roles in membrane targeting 8 .

N- and O-linked protein glycosylation are other apical sorting signals 8,71,72 . Glycan structures are extraordinarily diverse, thus having considerable information potential, nevertheless the molecular mechanism for apical sorting of glycosylated proteins has not been determined yet 73 , although their functional interactions with lectins during sorting at the TGN were postulated 72 . The sequential addition of one to five N-glycans to the basolaterally located Na + /K + -ATPase β1-subunit caused a gradual redirection of this subunit to the apical domain in HGT-1 cells 74 . Similarly, the O-glycosylated stalk domain in neurotrophin receptor p75 (p75NTR) is necessary for its apical targeting. An internal deletion of 50 amino acids that removes this stalk domain from p75NTR causes this protein to be sorted to the basolateral plasma membrane 71 . Oligomerization and apical sorting of glycosylated GPI-APs may not involve N- and O-glycans directly, but may depend on a lipid raft-associated glycosylated interactor 75 .

Also, proteoglycan-sorting determinants have been identified 76 . Proteoglycans with chondroitin sulfate are preferentially sorted to the apical membrane, while those carrying heparan sulfate are routed basolaterally.

Transmembrane apical sorting signals have been identified in influenza virus hemagglutinin (HA) and neuraminidase, but so far little work has been done to uncover the underlying sorting principles 77,78 .

Other apical sorting signals have been found, e.g., in rhodopsin 79 , megalin 80 , M2 muscarinic acetylcholine receptor 81 , the copper transporting P-type ATPase (ATP7B) 82 and the Na-K-Cl cotransporter (NKCC2) 83 , and they ranged from short motifs of a few amino acids to up to 30 amino acids long stretches.

The diversity of apical sorting determinants implies that several different mechanisms are employed to route the apical proteins to their destination. One such mechanism in MDCK cells involves lipid rafts as apical sorting platform in the Golgi complex 84 .

Lipid rafts in apical sorting

A role of lipid rafts in polarized epithelial sorting was suggested long ago. This was the origin of the lipid raft concept: apical proteins were postulated to be sorted through their affinity for microdomains of glycosphingolipids and cholesterol, assembled in the Golgi complex to form apical transport carriers 4,84 . The concept was generalized into a dynamic sub-compartmentalization principle, making use of sphingolipids and sterols to form small fluid membrane entities (lipid rafts) with specific proteins included. Lipid rafts are now defined as dynamic, nanometer-sized, sterol-sphingolipid-enriched, tightly packed lipid–protein assemblies that fluctuate on a sub-second time scale 85,86,87,88,89 . These assemblies can be induced to cluster to form more stable, specific ordered lipid raft platforms, which exert functions in membrane trafficking, cell polarization, signaling and other membrane processes 88,89 .

The best studied apical cargo that employs lipid rafts to be delivered to the apical membrane is the influenza virus HA. HA becomes detergent-resistant after entering the Golgi complex 90,91,92 . Obviously, detergent-resistant membranes (DRMs) cannot be directly equated with lipid rafts, as has often been the case 93,94 , though DRM analysis is a useful method to determine a protein's raft association potential when changes in DRM composition are induced by biochemically/physiologically meaningful events 94,95 . However, HA lipid raft association was also demonstrated by several other studies involving different methods. First, depletion of raft lipids, such as cholesterol and sphingolipids, resulted in the missorting of HA on its way to the apical domain of MDCK cells 96,97,98 . Second, antibody-mediated cross-linking of HA, GPI-proteins or non-raft proteins led to cholesterol-dependent co-patching of HA with GPI-proteins, while excluding non-raft proteins 99 . Third, photonic force microscopy demonstrated that HA was moving as a cholesterol-dependent assembly with a size of 50 nm in the plasma membrane 100 . In these experiments, beads containing antibodies that bound the HA protein were immobilized by an optical trap. Although binding to more than one HA protein was prevented, the force field applied to the cell and the immobilization of the protein by the trap could have altered the lifetime of the nanoscale HA-protein assemblies and caused them to grow larger than in the resting state. Nevertheless, this was clear demonstration that the HA protein was associated with lipids. Fourth, studies employing quantitative electron microscopy and fluorescence spectroscopy also showed that HA was present in microdomains of different sizes, which could be modulated by cholesterol and sphingolipid depletion 101,102 . Fifth, fluorescence photoactivation localization microscopy demonstrated that HA was present in nanoscale domains of different sizes 103 . And finally sixth, FRET microscopy showed that HA clustered with GPI-proteins on the cell surface in a cholesterol-dependent manner 104 . Altogether, these various experiments demonstrated that the HA protein is present in dynamic cholesterol-dependent assemblies, which is in the agreement with the lipid raft concept.

What is still missing from showing that rafts are directly involved in transport from the TGN to the apical membrane is the demonstration that the apical transport carriers are enriched in raft lipids as predicted by the concept. In yeast, Klemm et al. 105 used a lipid raft-associated plasma membrane protein as bait to isolate TGN-derived vesicles and subsequently characterized their lipid composition by mass spectrometry. Their results showed that yeast sphingolipids and ergosterol (the equivalent to cholesterol in animal cells) are sorted at the TGN and transported in specific secretory vesicles to the cell surface. This was the first time that a transport carrier involved in a lipid raft-dependent pathway has been isolated and characterized. The finding that raft lipids are enriched in these carriers brought convincing support to the raft concept as originally postulated. Further experiments with additional yeast plasma membrane proteins as baits showed that sorting of raft lipids is a generic feature of vesicles carrying transmembrane and GPI-protein cargoes to the plasma membrane 106 .

For a long time, a disturbing issue in the field has been the lack of genetic evidence for the lipid raft-sorting model in the generation and maintenance of the apical membrane. Why has all the work on different model organisms failed to identify lipid raft elements in the genetic screens of mutations affecting epithelial polarity? However, this gap has been closed recently. Through a combination of genetic screens, lipid analysis and imaging methods, it was established that glycosphingolipids indeed play a role in mediating apical sorting in the gut of Caenorhabditis elegans 107 .

Generation of apical transport carriers

After sorting in the plane of the membrane, cargo must be selectively incorporated into specific transport carriers. Membrane curvature has to be generated to form cargo-containing membrane buds or tubules, followed by subsequent scission to release the transport carrier from the donor membrane.

Since the advent of the lipid raft concept, raft clustering was postulated as a major driving force in the generation of transport carriers. In cellular membranes, nanoscale rafts are usually dispersed in a continuous non-raft phase 108,109 . In model membranes, coexistence of liquid-ordered and liquid-disordered phases results in line tension at the phase boundary, which arises from the immiscibility of membrane components that prefer different phases 110 . Clustering of small rafts into larger domains further increases the line tension, which in three dimensional system can be relieved by domain budding from the donor membrane, followed by fission at the phase boundaries, resulting in the generation of vesicles enriched in raft components 60 (Figure 1). The growing curvature of a membrane close to the demixing point (phase separation) further induces lipid sorting based solely on their underlying connectivity, which is greatly amplified by their clustering 111 . Since curvature of a membrane can also drive protein sorting, a growing bud can generate a feedback system whereby curvature-preferring proteins would be recruited to a growing lipid raft platform, further increasing the propensity to generate curvature 112,113,114,115 . Once a curved membrane is generated, phase separation in membrane tubes can trigger membrane fission arising from the difference in elastic constants between the domains 115,116 .

A scheme for apical transport carrier formation by domain-induced budding. (A) Nanoscale dynamic rafts surrounded by non-raft membrane. (B) Growing rafts are selectively induced by galectin–glycolipid–glycoprotein interactions into a budding domain, while non-raft components are excluded. Raft clustering results in increased line tension. (C) Insertion of hydrophobic or amphipathic protein domains (red) promotes membrane bending. FAPP2 could play this role for membrane deformation. (D) Fission at the domain boundary (possibly aided by fission proteins) results in the release of an apical transport carrier. For simplicity GPI-APs, cholesterol and other (e.g., palmitoylated proteins) proteins are not shown and cytoskeletal elements are omitted as well.

Supporting this domain-budding hypothesis, it was shown that the interaction of the B-subunit of Shiga toxin with the plasma membrane glycosphingolipid Gb3 is sufficient for clustering, which increases the bilayer order in these regions 117 . This, together with an asymmetric membrane stress imposed by the toxin, results in negative curvature of the membrane and induced tubule formation. Similarly, membrane invaginations are induced by Simian virus 40 binding to GM1 gangliosides 118 . Therefore, multivalent binding of specific lipids and clustering can result in membrane tube formation and a similar mechanism might be at work at the TGN.

Galectins, annexins and VIP17/MAL proteins, involved in apical trafficking and with a potential to cluster or array, are possible mediators of lipid clustering upon the exit from the TGN. For the raft-mediated pathway in MDCK cells, galectin-9 is the strongest candidate for a clustering function 64 due to its binding to the Forssman glycolipid 50 .

The process of carrier generation probably does not rely solely on the lipid clustering. Bending proteins are likely to be essential for successful transport carrier formation (Figure 1). There are two principal mechanisms of protein-induced membrane curvature. BAR domain–containing proteins are 'banana-shaped' and thus confer curvature by direct membrane scaffolding 119,120 . They bind to membranes by their positively charged concave face and therefore are able to sense, stabilize, and generate membrane curvature. Recently, Willenborg et al. reported that the sorting nexin 18 (SNX-18), a BAR domain-containing protein, together with the Rab11 GTPase-binding protein FIP5, which enhances its tubulation potential, is involved in the formation of podocalyxin-containing apical carriers 121 . The other mechanism relies on the insertion of a small amphipathic or hydrophobic wedge to induce membrane asymmetry resulting in curvature 122 . Recently, the FAPP2 protein, involved in the transport of apical cargo in polarized MDCK cells, was shown to possess phosphatidylinositol 4-P-dependent membrane tubulation activity, which could be attributed to a hydrophobic wedge in its PH domain 123,124 .

Proteins secreted apically have been shown to depend on N-glycans to be sorted correctly 125 . Galectin-3 seems not to be involved in this pathway 126 . Whether other galectins such as galectin-9 in MDCK cells plays a role in transporting secretory proteins to the apical side of the epithelium remains to be analyzed. Also in the basolateral direction, binding proteins or sorting receptors would be needed. However, little is so far known about how this is accomplished. Probably, each basolaterally secreted protein will need its own receptor because so far no general sorting signals have been identified. However, for basolateral transmembrane proteins specific cytoplasmic sorting signals have been identified.


Results

The discovery of STIM1 and Orai1 and their prominent role in SOCs raised the question of their role and localization in Ca 2+ signaling in specialized polarized cells like secretory cells. The Ca 2+ signal in secretory cells is unique in that it is highly polarized, always initiating at the apical pole and propagating to the basal pole 1, 2 . That the molecular basis for the polarized Ca 2+ signal is highly polarized and restricted localization of all Ca 2+ signaling proteins examined has been firmly established 1 . This would predict polarized expression and recruitment of Orai1 and STIM1, respectively, in polarized cells. Moreover, in response to cell stimulation and store depletion, the expressed ER-localized STIM1 and plasma membrane-localized Orai1 are recruited to form punctae at which STIM1 and Orai1 show perfect colocalization 19, 21, 26, 40, 41 . It is not known whether there is a similar perfect colocalization of the native STIM1 and Orai1. This is of particular importance because STIM1 also gates the TRPC channels 22-25 that have prominent role in receptor-stimulated Ca 2+ influx in secretory cells 30, 31 . The present studies which were set to address these questions resulted in several unexpected findings with significant implications for the mechanism of Ca 2+ influx in secretory cells.

Role of endogenous STIM1 and Orai1 in secretory cell Ca 2+ signaling

First, we have sought to establish a role for STIM1 and Orai1 in Ca 2+ signaling by secretory cells. As the use of siRNA probes cannot be employed in native acini that lose polarity within 12 h in culture, the recent development of probes that inhibit the activity of STIM1 and Orai1 offers a reasonable alternative. The STIM1(445–475) fragment inhibits the native CRAC current when infused into Jurkat cells 40 . [Ca 2+ ]i in pancreatic acinar cells was followed by recording the Ca 2+ -activated Cl − current 11 to allow infusing the cells with peptides through the patch pipette. Figure 1A,B shows that infusion of STIM1(445–475) into pancreatic acinar cells reduces the frequency by about 35% of Ca 2+ oscillations triggered by weak receptor stimulation. Inhibition of the fully activated Ca 2+ influx (plateau phase) by STIM1(445–475) did not reach statistical significance, likely because the peptide is a weak inhibitor of Ca 2+ influx 40 . Nevertheless, even partial inhibition of Ca 2+ influx was sufficient to reduce the frequency of Ca 2+ oscillations. A five-residues Orai1(153–157) fragment was reported to strongly inhibit the current mediated by expressed STIM1-Orai1 42 . Figure 1C,E shows that infusion of this fragment into pancreatic acinar cells inhibited Ca 2+ influx by about 50% and reduced the frequency of the Ca 2+ oscillations by about 70%. Hence, there is a good correlation between inhibition of Ca 2+ influx and reduced Ca 2+ oscillation frequency, highlighting the crucial role of Ca 2+ influx in sustaining the oscillation and determining their frequency.

Inhibition of Ca 2+ signaling by STIM1 and Orai1 inhibitory peptides in pancreatic acini and by siRNA knockdown in parotid ducts. [Ca 2+ ]i is monitored as changes in the Ca 2+ -activated Cl − current, which faithfully reports changes in pancreatic acinar cells [Ca 2+ ]i. Acinar cells were infused with pipette solution (A, control) containing 50 µ m of the STIM1 inhibitory peptide STIM1(445–475) (B), or 50 µ m of the Orai1 inhibitory peptide Orai1(153–157) (C), for 7 min before stimulation with 0.75 µ m carbachol to induce Ca 2+ oscillations and then with 100 µ m carbachol to maximally activate Ca 2+ influx. The average oscillation frequency is shown in (D) and the plateau in (E). The dashed lines are the zero current. The plateau (marked by a line with double arrows) was normalized relative to the initial increase in current because of Ca 2+ release from the ER. The columns show the mean ± SEM of seven experiments. The efficiency of the Orai1 (F) and STIM1 (G) siRNA probes was determined by RT-PCR in mouse keratinocytes. Panels (H and J) show the response of sealed parotid ducts treated with scrambled (dark traces and columns) or with Orai1 siRNA and stimulated with 100 µ m ATP in Ca 2+ -free media and then exposed to media containing 2 m m Ca 2+ to evaluate Ca 2+ influx. The ducts were then perfused with Ca 2+ -containing media for 10 min to load the stores before treatment with 25 µ m CPA in Ca 2+ -free media and finally exposed to media containing Ca 2+ to evaluate SOC activity. (I and K) Parotid ducts treated with scrambled or STIM1 siRNA were treated with 25 µ m CPA in Ca 2+ -free media and then exposed to Ca 2+ -containing media to measure SOC activity. Panels J and K show the mean ± SEM of four ducts. * denotes P < 0.01 or better.

To further demonstrate the role of STIM1 and Orai1 in Ca 2+ signaling in secretory cells we used an independent assay to test the effect of their knockdown on Ca 2+ influx in parotid gland sealed ducts in primary culture, in which siRNA effectively knockout the desired genes 43-45 . Ducts were prepared from the parotid gland to show the role of STIM1 and Orai1 in another secretory cell type. Figure 1F,G shows the efficiency of the knockdown of Orai1 and STIM1 by three siRNA probes. The second probe for each gene was used for the experiments shown, but we also tested the effect of the siRNA1 for Orai1 and siRNA3 for STIM1 to exclude off-target effects and obtained similar results. Figure 1H,J shows that knockdown of Orai1 have reduced Ca 2+ influx activated by receptor stimulation or by store depletion by about 65% and Figure 1I,K shows that knockdown of STIM1 has reduced SOC by about 85%.

Polarized localization of native Orai1

The specificity of the antibody used to localize Orai1 has been validated in a recent report showing that the immunohistochemical signal obtained in wild-type tissues is eliminated in tissues obtained from patient and mice with deletion of Orai1 34 . Figure 2A shows that this anti-Orai1 antibody detected Orai1 in pancreatic and SG extracts. Figure 2B shows that Orai1 localization is restricted to the lateral membrane of pancreatic acinar cells (arrows), similar to the localization of a number of key Ca 2+ signaling proteins 1 . Staining is also observed at the lateral membrane most adjacent to the apical pole (dotted arrows) and there is no detectable staining of Orai1 in the basal plasma membrane region, suggesting that the level of Orai1, if it is expressed at this site, is very low.

Localization of Orai1 in pancreatic acini. The anti-Orai1 antibody detects Orai1 in pancreatic and submandibular gland extracts (A). To deplete the ER Ca 2+ store acini in Ca 2+ -free media containing 1 m m EGTA were stimulated with 0.5 m m carbachol and treated with 25 µ m CPA for 10 min. The acini were then fixed and co-stained for Orai1 and IP3R3 (B and C) or ZO1 (D). In (B and C), membranes next to the apical pole are marked with solid arrows and the lateral membranes are marked with dashed arrows. In (E), images acquired from resting and stimulated cells that were treated with vehicle or with 50 µ m 2APB were used to determine the overlap of Orai1 with IP3R3 and with ZO1. The results are the mean ± SEM of the number of cells indicated in the columns. There is no significant difference between all conditions.

The luminal plasma membrane in the apex of acinar cells is small but folds along the lateral membrane at the apical pole, where the two membranes are separated by tight junctions 3, 46 . Staining of the tight junctions in pancreatic acini resulted in an image very similar to that obtained with Orai1 3 . Indeed, co-staining with the tight junction protein ZO1 shows nearly perfect colocalization of Orai1 and ZO1 (Figure 2D) with 94 ± 5% overlap (n = 98 cells).

Another Ca 2+ signaling protein expressed in this region of the cell is the ER-located IP3R. Accordingly, localization of Orai1 significantly overlaps with IP3R3 (Figure 2D, 93 ± 6%, n = 76 cells). Although a significant portion of Orai1 transfected in pancreatic acinar cells is targeted to the apical pole, localization of the expressed Orai1 was more homogeneous at the plasma membrane 33 . We cannot fully explain the difference between the present and previous findings. However, it might be because of over-expression of Orai1 overwhelming the cellular protein sorting and trafficking machinery.

Cell stimulation and depletion of ER Ca 2+ result in clustering of expressed Orai1 19, 21, 26, 27, 40, 41 . However, cell stimulation together with inhibition of the SERCA pumps have had no dramatic effect on the unique polarized localization of native Orai1 in acinar cells (Figure 2C). Additionally, treating the cells with 2APB had no effect on the localization of Orai1 (Figure 2D). The images in Figure 2 were acquired at a plane showing the maximal expression of Orai1 and IP3R3 and ZO1. However, similar tight overlap between Orai1 and IP3R3 and ZO1 was observed in all cellular planes. A possible implication of this finding is that in the polarized acinar cells, Orai1 is already targeted to its site of function and further targeting or relocation is not needed. This may ensure the highly polarized Ca 2+ signal in these cells.

Polarized recruitment of native STIM1

Given the polarized localization of Orai1 in Figure 2 and the tight coclustering of heterologously expressed STIM1 and Orai1 in response to store depletion 19, 21, 26, 40, 41 , we expected STIM1 to show a similar localization as Orai1 in stimulated cells. We examined the localization of the native STIM1 using two different anti-STIM1 antibodies with established specificity. One antibody raised against the conserved STIM1(657–685) detected the native STIM1 and, importantly, the signal was eliminated in tissues from patients who lack STIM1 34 . Figure S1 shows that this antibody reported diffused STIM1 staining in resting acinar cells with more intense staining at the basal pole. Cells stimulation did not result in massive redistribution of this staining to the basal pole, but rather induced recruitment of STIM1 to the apical (turquoise arrowheads) and lateral regions close to the plasma membranes (yellow arrowheads) (Figure S1B). The SOC inhibitor 2APB that disrupts clustering of STIM1 with native Orai1 37-39 had no obvious effect on STIM1 localization in resting cells, but markedly reduced accumulation of STIM1 at the apical and lateral plasma membrane regions (Figure S1C). Another notable finding is that the recruited STIM1 and IP3R3 show only partial colocalization along the lateral membrane (turquoise squares in Figure S1B). This is different from the findings with Orai1 (see also below), where there was >90% overlap with IP3R3.

Similar, but superior, staining was obtained with a second anti-STIM1 antibody. Staining with the Protein-Tech antibody was eliminated in tissue from STIM1−/− mice 35 . We further validated the use of this antibody in Figure S2 by showing that it recognizes the native and expressed STIM1 in HeLa cells (panel A), showing reduced signal in STIM1 siRNA-treated HeLa cells (panels B and D), and giving the expected STIM1 staining in resting and stimulated cells (panels B, C).

Figure 3 shows the localization of STIM1 relative to IP3R3 obtained with this antibody. Resting cells show primarily diffuse STIM1 localization (Figure 3A). Treating resting cells with 2APB did not have any effect on this localization (Figure 3B,E). Cell stimulation and store depletion recruited STIM1 primarily to the apical pole (solid arrows) and lateral cellular regions (dashed arrows), with small amount of STIM1 punctae at the basal membrane region (turquoise arrowheads) (Figure 3C). STIM1 at the apical pole shows excellent colocalization with IP3R3 (Figure 3C). Unexpectedly, intense accumulation of STIM1 at the lateral membrane region was at a region free of IP3Rs (turquoise squares). Hence, STIM1 showed only 43 ± 6% (n = 117) overlap with IP3R3 (Figure 3E). An important control is shown in Figure 3D,E, in which treatment with 2APB disrupted STIM1 accumulation, reducing the overlap with IP3R3 to 13 ± 3% (n = 64), which is not different from the overlap in resting cells.

Localization of STIM1 in pancreatic acini. Resting acini (A and B) were maintained in solution A, whereas stimulated acini (C and D) are acini in Ca 2+ -free media containing 1 m m EGTA that were stimulated with 0.5 m m carbachol and treated with 25 µ m CPA for 10 min. Control and stimulated acini were also treated with 50 µ m 2APB (B and D) that was added just before cell stimulation. The acini were then co-stained for STIM1 and IP3R3. In (C), membranes next to the apical pole are marked with solid arrows and lateral membranes are marked with dashed arrows. Punctae at the basal and lateral membranes are marked with turquoise arrows. The turquoise squares in all images depict area showing high expression of STIM1 and no IP3R3. In (E), images collected from resting and stimulated cells that were treated with vehicle or with 50 µ m 2APB were used to determine the overlap of STIM1 with IP3R3. Panel (F) compares the overlaps of Orai1 and STIM1 with IP3R3. The results are the mean ± SEM of the indicated number of cells. * denote p < 0.01 from resting cells.

To further confirm the unexpected differential overlap of Orai1 and STIM1 with IP3R3 (summarized in Figure 3F) we compared colocalization of STIM1 and Orai1 with other marker proteins. In Figure 4A,B the basal membrane was stained with laminin to show more clearly the very low level or no localization of STIM1 and Orai1 at this site. Figure 4C shows the partial localization of STIM1 at the tight junction and Figure 4D shows quantification of the dramatic difference in the colocalization of Orai1 and STIM1 with ZO1. We also measured the area of the basolateral membrane that overlaps with the Orai1 and STIM1. The basolateral membrane was visualized with E-cadherin. The images in Figure 4E,F were acquired in planes optimal to observe the basolateral membrane with E-cadherin. Figure 4E,G shows that Orai1 was detected in 28 ± 3.3% (n = 84 cells) of the basolateral membrane domain, whereas Figure 4F,G shows that STIM1 was detected in 67 ± 4.5% (n = 63 cells) of the basolateral membrane domain.

Plasma membrane area occupied by Orai1 and STIM1 in pancreatic acini. Store-depleted and receptor-stimulated acini were stained with the basal membrane marker laminin (A and B), the tight junction marker ZO1 (C) and the basolateral membrane E-cadherin (E and F) (all red).The acini were co-stained with Orai1 (A and E) or STIM1 (B, C and F). Panel (D) Compares the overlap of STIM1 and Orai1 with ZO1 and in (G) images similar to those in (E) and (F) were used to determine the % basolateral membrane expressing Orai1 or with adjacent STIM1. The results are the mean ± SEM of the number of cells indicated in the columns.

Finally, we tagged the anti-STIM1 antibodies with Alexa Fluor® 647 and used it to determine localization of STIM1 and Orai1 in the same stimulated cells. Figure 5 shows that only about 50% of STIM1 colocalizes with Orai1, which confirms the results obtained with IP3R3 and E-cadherin. Thus, the remaining STIM1 likely forms complexes with other Ca 2+ influx channels.

Colocalization of STIM1 and Orai1 in the same cells. Store-depleted acini were labeled with Orai1 (A) and then with the Alexa Fluor ® 647-tagged STIM1 (B) as detailed in Methods. The merged image is shown in (C). The double labeling procedure resulted in a significant background staining in the form of dots, mainly from the Orai1 antibodies. These were excluded from calculation of % overlap of STIM1 and Orai1. Overlap was restricted to the plasma membrane region and is shown in (D).

Polarized localization of native TRPC1

The distinct lateral regions that express STIM1 but not Orai1 raise the question of whether STIM1-interacting TRPC channels are localized at this region. Of particular interest is TRPC1 that is localized at the lateral membrane of SG acinar cells 30 . Unfortunately, detection of TRPC1 in pancreatic acini with the antibodies requires fixation with paraformaldehyde that precludes detection of IP3Rs. Reclaiming detection of the IP3Rs after paraformaldehyde fixation required incubation with cold MeOH, but this resulted in a non-specific intense staining of the secretory granules that overwhelmed the staining for TRPC1. Nevertheless, fixation with paraformaldehyde suggested that TRPC1 is localized at the apical and lateral regions of the basolateral membrane of pancreatic acinar cells (Figure S3A). Pancreatic acinar cells from Trpc1−/− mice showed reduced Ca 2+ influx (Figure S3B) and Ca 2+ oscillation frequency (Figure S3C), indicating that TRPC1 mediates a significant portion of Ca 2+ influx in these cells.

It was possible to obtain reasonable images of TRPC1 and IP3Rs in parotid acini treated with paraformaldehyde and MeOH. Figure 6A shows staining of the lateral membranes with the anti-TRPC1 antibody and Figure 6B shows that the staining is markedly reduced in Trpc1−/− parotid acini. Co-staining of Trpc1 and IP3R3 revealed lateral membrane regions with no overlap between them (turquoise squares), as was found with STIM1 (Figures 3 and S1). Although the fixation procedure reduced the resolution of the IP3Rs images and precluded reliable calculation of overlap, the findings in Figure 6 suggest that part of STIM1 is recruited to a plasma membrane domain expressing TRPC1, STIM1 and no Orai1. However, all the proteins, Orai1, TRPC1, IP3R3 as well as STIM1 colocalize in the lateral membrane close to the apical pole following cell stimulation.

Localization of TRPC1 in parotid gland acini. Acini from wild-type (A) and Trpc1−/− mice (B) were co-stained for TRPC1 (green) and IP3R3 (red). The turquoise squares in all images depict area showing high expression of TRPC1 and no IP3R3.

Co-IP of STIM1/Orai1/TRPC1/IP3 R3

Colocalization by confocal microscopy is not sufficient to determine if the proteins exist in the same Ca 2+ signaling complex. This can be tested more directly by co-IP assays. First, we determined if proteins expressed in model systems can be co-IPed. A recent study reported that cell stimulation enhances co-IP of expressed TRPC3–Orai1–STIM1–IP3R1 47 . Figure S4 extends these findings by showing that the co-IP is enhanced by cell stimulation and it is reduced by treating the cells with 2APB. co-IP of expressed proteins can be markedly affected by their over-expression. Therefore, when possible, it is important to demonstrate co-IP of the native proteins. Therefore, next we examined the reciprocal co-IP of all native Ca 2+ signaling proteins in pancreatic acinar cells. The co-IP of TRPC3 was examined because it shows close colocalization with IP3R3 7 , interacts with TRPC1 25 and contributes to Ca 2+ influx in acinar cells 31 . Figure 7A shows that IP of STIM1 co-IPs Orai1. Figure 7B,C shows the reciprocal co-IP of IP3R3 and STIM1 and Figure 7D,E shows the reciprocal co-IP of IP3R3 and Orai1. TRPC3 co-IPs with IP3R3 (Figure 7F,G) and with Orai1 (Figure 7G).

Co-IP of STIM1, Orai1, IP3R3, TRPC3 and TRPC1 is enhanced by cell stimulation and disrupted by 2APB. For each set of experiments, extracts were prepared from the pancreas of two mice. Control lanes are without primary antibodies and inputs are 5% of the initial material used for the IP. Extracts were used to IP STIM1 (A and B) and probe for co-IP Orai1 (A) and IP3R3 (B) IP IP3R3 (C, E and G) and probe for co-IP of STIM1 (C), Orai1 (E) and TRPC3 (G) IP Orai1 (D and G) and probe for co-IP of IP3R3 (D) and TRPC3 (G). In (H, I) part of the cells were not stimulated (rest) and part were stimulated by incubation in Ca 2+ -free media and treatment with 0.5 m m carbachol and 25 µ m CPA for 10 min at 37°C. Portions of resting and of stimulated cells were treated with 50 µ m 2APB. Extracts were prepared from all four conditions and used to IP STIM1 (two upper blots in H) and probe for co-IP of Orai1 and IP3R3 and for IP IP3R3 (two middle blots in H) and probe for co-IP of STIM1 and Orai1. The bottom blots are the inputs. Extracts were also used to determine the reciprocal co-IP of STIM1 and TRPC1 (I). Note that cell stimulation enhances the co-IP, whereas treatment with 2APB reduces the co-IP.

Significantly, Figure 7H shows that cell stimulation enhances the co-IP of IP3Rs with STIM1 and Orai1 and the co-IP is inhibited by treatment with 2APB. Figure 7I shows the mutual co-IP of STIM1 and TRPC1, enhancement of the co-IP by cell stimulation and its disruption by treating the acini with 2APB. Hence, the native Orai1, STIM1, IP3Rs, TRPC3 and TRPC1 are in Ca 2+ signaling complexes and STIM1 may stabilize the complexes, because the complexes are disrupted by 2APB. Because of the partial Orai1/STIM1 overlap and the overlap of TRPC3 and TRPC1 with STIM1 in regions that do not express STIM1, it is likely that STIM1 stabilizes complexes of several compositions: Orai1-STIM1 TRPC1/TRPC3-STIM1 and Orai1-STIM1-TRPC1/TRPC3. It will be of particular interest to test whether the various complexes mediate spatially different Ca 2+ influx pathways that may regulate specific cellular functions.

Polarized initiation of Ca 2+ influx

To begin to evaluate the possibility that STIM1 may mediate several Ca 2+ influx pathways that operate in different cellular microdomains, we analyzed initiation of Ca 2+ influx in cells with fully depleted stores and thus maximally activated Ca 2+ influx. In addition, the stores were depleted passively so as to minimize propagation of the Ca 2+ signal because of cell stimulation. Figure 8 shows that the consequence of the localization of TRPC channels, Orai1 and the recruited STIM1 at the apical pole next to the tight junctions is the generation of a defined Ca 2+ influx site (Figure 8, left images). As these cells maintain polarity even when dissociated to single cells, we have also measured Ca 2+ influx in single acinar cells to ensure uniform Ca 2+ access to all parts of the plasma membrane. Figure 8 (right) images show that also in single pancreatic acinar cells Ca 2+ influx initiates at the apical pole. The rate of cytoplasmic Ca 2+ increase because of Ca 2+ influx is very slow, indicating that the Ca 2+ signal does not propagate. Diffusion of Ca 2+ in the cytoplasm is quite slow 48 , and thus cytoplasmic Ca 2+ increase at later times likely reflects Ca 2+ influx through sites different from the initial site where Orai1 is present. These sites likely mediate Ca 2+ entry through TRPC channels that are present at the lateral membrane.

Site of Ca 2+ entry in pancreatic acinar cells. A three-cell acinus (left panels) and a single acinar cell (right panels) loaded with Fura2 were incubated in Ca 2+ -free media and were treated with 0.5 m m carbachol and 25 µ m CPA to activate Ca 2+ influx. When Ca 2+ returned to basal level the cells were exposed to media containing 5 m m Ca 2+ and images were collected every 0.3 seconds to detect the initial sites of Ca 2+ influx. The traces show the time–course of the changes in Ca 2+ recorded in the regions of interest indicated in the first fluoresce image and the bright field image.


Materials and Methods

Molecular Biology

Our HA-, myc- and biotin ligase acceptor peptide (BLAP)-tagged KCa3.1 (also referred to as IK1 or SK4) constructs, as well as the bicistronic plasmid (pBudCE4.1) expressing both BLAP-KCa3.1 and BirA-KDEL have been previously described [22], [23], [24]. BLAP-KCa3.1 was also subcloned in to the pAdlox (SwaI modified) adenoviral shuttle plasmid using EcoRI and SalI restriction sites. In order to biotinylate KCa3.1 within the endoplasmic reticulum before trafficking to the plasma membrane, we subcloned BLAP-KCa3.1 as well as BirA-KDEL (generously provided by Dr. Alice Ting, Massachusetts Institute of Technology, Cambridge, MA) into the bicistronic adenoviral shuttle plasmid DUALCCM-CMW-MCS2 (Vector Biolabs Philadelphia, PA). In this construct, both cDNAs are located behind unique CMV promoters. BLAP-KCa3.1 and BirA-KDEL were sequentially subcloned using EcoRI/XhoI and NheI/SalI restriction sites, respectively. BLAP-KCa3.1 and BirA-KDEL/BLAP-KCa3.1 replication deficient adenoviruses were generated by the University of Pittsburgh Vector Core facility and Vector Biolabs, respectively. The transferrin receptor (TfnR) adenovirus was generously provided by Dr. Ora Weisz (University of Pittsburgh, Pittsburgh, PA). Generation of the GFP- and Flag-tagged variants of Rabs 1, 2, 6 and 8 have been previously described [25], [26], [27]. Rab 10 was purchased from Addgene (Cambridge, MA). Wild type (WT) and dominant negative (DN) GFP-tagged receptor-mediated endocytosis-1 (RME-1) constructs were generously provided by Dr. Barth Grant (Rutgers University, New Brunswick, NJ).

Cell Culture

Madin-Darby canine kidney (MDCK) and pig epithelial (LLC-PK1) cells were cultured in α-MEM medium, human epithelial colorectal adenocarcinoma (Caco-2) and human embryonic kidney (HEK293) cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) and Fischer rat thyroid (FRT) epithelial cells were grown in F12 (Sigma-Aldrich, St. Louis, MO). All media were supplemented with 10% fetal calf serum, and 1% penicillin/streptomycin. Both the wild type LLC-PK1 cells and the LLC-PK1 cell line stably expressing the FLAG-tagged 𻰛 subunit of the AP1 adaptor complex was generously provided by Dr. Michael Caplan (Yale University, New Haven, CT) [28]. The MDCK, Caco-2, HEK293, LLC-PK1 and FRT cell lines were obtained from the ATCC (Manassas, VA). All cells were grown in a humidified 5% CO2/95% O2 incubator at 37ଌ. A stable FRT cell line was generated by transfecting in the pBudCE4.1 bicistronic plasmid expressing both BLAP-KCa3.1 and BirA-KDEL using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions and selecting a stable cell line using zeocin (850 μg/ml) (protocol approved by the University of Otago Institutional Biological Safety Committee, approval code GMD003298-33).

HEK293 cells were transfected using Lipofectamine 2000 following the manufacturer's instructions. MDCK, Caco-2, LLC-PK1 and FRT cells were seeded onto Transwell® permeable supports (Corning Inc., Corning, NY) and grown to confluence forming a polarized epithelium (3𠄴 days post seeding). Polarized MDCK, Caco-2, LLC-PK1 or FRT cells were transduced with the BLAP-KCa3.1 or BirA-KDEL/BLAP-KCa3.1 adenoviruses, as indicated for each experiment. Briefly, well-polarized cells were washed three times with Ca 2+ -free PBS followed by addition of adenovirus to the apical side of the filter. After 1 hr of incubation at 37ଌ in a 5% CO2/95% O2 atmosphere, cells were washed once with PBS and allowed to recover until the next day in normal growth media. In some experiments, 6 hrs after adenoviral addition in MDCK cells the cells were further transfected with WT or DN RME-1 or appropriate Rab construct using Lipofectamine 2000, following the manufacturer's instructions.

Biotinylation of KCa3.1 using recombinant biotin ligase

BLAP-tagged KCa3.1 was either biotinylated using recombinant biotin ligase (BirA), as described [22] or by co-expression with BirA-KDEL [23]. Plasma membrane BLAP-KCa3.1 was then labeled with streptavidin-Alexa555 (0.01 mg/ml, Invitrogen) for 15 min at 4ଌ. The cells were extensively washed to remove unbound streptavidin and incubated for various periods of time at 37ଌ as indicated or immediately fixed and permeabilized with 2% paraformaldehyde plus 0.1% Triton X-100 [22]. The apical plasma membrane was labeled with WGA-Alexa488 (wheat germ agglutinin, 5 μg/ml), (Invitrogen). Nuclei were labeled with DAPI (Sigma-Aldrich). Cells were imaged by laser-scanning confocal microscopy (Olympus FluoView 1000 system) using a 63× oil immersion lens as described [19]. Z-stacks were taken to cover the entire thickness of the cell in a step size of 0.5 μm.

Antibodies

GFP antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Monoclonal α-streptavidin antibody was obtained from Abcam (Cambridge, MA). Monoclonal Flag, α-tubulin and β-actin were purchased from Sigma-Aldrich. Antibodies against Myc and HA were obtained from Covance (Princeton, NJ) and Rab8 antibody was purchased from BD Transduction laboratories (San Jose, CA).

Immunoprecipitations and immunoblots

Our immunoprecipitation (IP) and immunoblot (IB) protocols have been described [15], [16]. Briefly, cells were lysed and equivalent amounts of total protein were pre-cleared with protein G-agarose beads (Invitrogen) followed by incubation with the indicated antibody. Normal IgG was used as negative control. Immune complexes were precipitated with protein G-agarose beads, washed extensively, and resuspended in Laemmli sample buffer. Proteins were resolved by SDS-PAGE and transferred to nitrocellulose for IB analysis. To eliminate interference by the heavy and light chains of the immunoprecipitating antibody in the IP, mouse or rabbit IgG Trueblot ULTRA (eBioscience, San Diego, CA) were used as a secondary antibody for the detection of immunoprecipitated proteins in the IB.

Determination of degradation rate for plasma membrane of KCa3.1

The degradation rate for endocytosed membrane KCa3.1 was determined as described [22]. Briefly, KCa3.1 in polarized MDCK, Caco-2 or FRT cells was specifically biotinylated using BirA and labeled with non-conjugated streptavidin after which the cells were incubated for various periods of time at 37ଌ, as indicated. In some experiments, the lysosomal protease inhibitors leupeptin (100 μM) and pepstatin (1 μg/ml Leu/Pep) (Sigma-Aldrich), the proteasome inhibitor lactacystin (10 μM, Lacta) or a general deubiquitylase (DUB) inhibitor PR-619 (50 μM) (LifeSensors Inc., Malvern, PA) were added to both apical and BL membranes prior to the 37ଌ incubation step. The cells were then lysed and equivalent amounts of total protein were separated by SDS-PAGE, followed by IB for streptavidin. Bands were quantified by densitometry using ImageJ software (NIH http://rsb.info.nih.gov/ij/). The obtained band intensities for the various time points were normalized relative to the intensity at time 0 (T =𠂠) and are reported. The blots were also probed for α-tubulin and β-actin as a protein-loading control.

Pulldown of ubiquitylated KCa3.1 using TUBEs

To determine the ubiquitylation state of KCa3.1 at the plasma membrane, and following endocytosis, we used GST-tagged tandem-repeat ubiquitin-binding entities (TUBEs) (LifeSensors Inc.), as described [20]. Caco-2 cells were enzymatically biotinylated and streptavidin labeled at the plasma membrane after which the cells were lysed in the presence of GST-TUBEs (200 μg/ml) or returned to 37ଌ for various periods of time to allow endocytosis to occur, and then lysed in the presence of TUBEs. The TUBEs were subsequently pulled down on glutathione agarose beads followed by SDS-PAGE. The resulting IB was probed with α-streptavidin Ab. In this way, only the streptavidin-tagged KCa3.1, which was ubiquitylated and hence bound to TUBEs, was detected [20].

Recycling endosome ablation assay

MDCK cells were transduced with TfnR and BirA-KDEL/BLAP-KCa3.1 adenoviruses. After 24 hrs, recycling endosome ablation was carried out based on the methods of Ang et al. [29]. MDCK cells were incubated with 0.010 mg/ml Tfn-HRP and Tfn-Alexa488 in serum starved media for 45 min at 37ଌ, washed twice in serum-free αMEM media, and incubated for 20 min at 37ଌ. Cells were then washed twice on ice-cold PBS. Surface-bound Tfn-HRP and Tfn-alexa488 were removed by two 5 min washes in 0.15 M NaCl plus 20 mM citric acid, pH 5.0. After washes with ice-cold PBS, the cells were incubated in PBS containing 0.1 mg/ml diaminobenzidine (DAB Sigma-Aldrich) in the absence (control) or presence (ablation) of H2O2 (0.025%) for 1 hr on ice in the dark. The Tfn-HRP reacts with DAB and H2O2 forming an insoluble precipitate that prevents the fusion of post-Golgi vesicles with the recycling endosome. The ablation reaction was stopped by washing cells twice in PBS with 1% BSA. Following recycling endosome ablation, neutravidin biotin binding protein (600 μg/ml) (Thermo Scientific, Rockford, IL) was added to the control and ablated cells for 2 hrs at 4ଌ such that all plasma membrane localized BLAP-KCa3.1 channels would be bound and 𠇋locked” from binding to subsequently added streptavidin. The cells were then incubated in media with cyclohexamide (400 μg/ml, Sigma-Aldrich) for 90 min at 37ଌ to allow ER/Golgi-resident KCa3.1 channels to potentially traffic to the plasma membrane. To determine whether ER/Golgi-resident channels had trafficked to the plasma membrane following recycling endosome ablation we labeled with streptavidin-Alexa555 (0.01 mg/ml, Invitrogen) for 15 min at 4ଌ.

KCa3.1 trafficking from Golgi to the plasma membrane

MDCK cells were transduced with BirA-KDEL/BLAP-KCa3.1 adenovirus and subsequently transfected with GFP-tagged WT or DN RME-1. Plasma membrane localized and biotinylated BLAP-KCa3.1 was 𠇋locked” with neutravidin (600 μg/ml), as above. In order to allow accumulation of channels in the Golgi, cells were incubated for 2 hrs at 19ଌ in the continued presence of neutravidin. Cells were then washed twice on ice-cold PBS, warm media was added and the cells were incubated for 30 min or 2 hrs at 37ଌ to allow channels to traffic to the plasma membrane. Finally, the newly resident plasma membrane channels were labeled with streptavidin-Alexa555, for IF localization studies or with non-conjugated streptavidin followed by IB with α-streptavidin antibody to quantify the rate of plasma membrane KCa3.1 appearance. Similarly, HEK293 cells were transfected with BirA-KDEL/BLAP-KCa3.1 and each Rab construct followed by neutravidin 𠇋lock”, incubation at 19ଌ to allow KCa3.1 Golgi accumulation and subsequent incubation at 37ଌ for the indicated periods of time to allow trafficking of KCa3.1 from the ER/Golgi to the plasma membrane. Plasma membrane localized KCa3.1 was then labeled with non-conjugated streptavidin, followed by IB with α-streptavidin antibody to quantify plasma membrane appearance.

Statistical analysis

All data are presented as means ± SEM, where n indicates the number of experiments. Statistical analysis was performed using a Student's t-test. To compare the normalized values of the IB band intensities, statistical analysis was performed using the non-parametric Kruskal-Wallis test. The traffic from ER/Golgi to plasma membrane was analyzed using a two-way Anova test followed by Bonferroni post-test comparing WT and DN at each time point. A value of pπ.05 is considered statistically significant and is reported.


Results

The discovery of STIM1 and Orai1 and their prominent role in SOCs raised the question of their role and localization in Ca 2+ signaling in specialized polarized cells like secretory cells. The Ca 2+ signal in secretory cells is unique in that it is highly polarized, always initiating at the apical pole and propagating to the basal pole 1, 2 . That the molecular basis for the polarized Ca 2+ signal is highly polarized and restricted localization of all Ca 2+ signaling proteins examined has been firmly established 1 . This would predict polarized expression and recruitment of Orai1 and STIM1, respectively, in polarized cells. Moreover, in response to cell stimulation and store depletion, the expressed ER-localized STIM1 and plasma membrane-localized Orai1 are recruited to form punctae at which STIM1 and Orai1 show perfect colocalization 19, 21, 26, 40, 41 . It is not known whether there is a similar perfect colocalization of the native STIM1 and Orai1. This is of particular importance because STIM1 also gates the TRPC channels 22-25 that have prominent role in receptor-stimulated Ca 2+ influx in secretory cells 30, 31 . The present studies which were set to address these questions resulted in several unexpected findings with significant implications for the mechanism of Ca 2+ influx in secretory cells.

Role of endogenous STIM1 and Orai1 in secretory cell Ca 2+ signaling

First, we have sought to establish a role for STIM1 and Orai1 in Ca 2+ signaling by secretory cells. As the use of siRNA probes cannot be employed in native acini that lose polarity within 12 h in culture, the recent development of probes that inhibit the activity of STIM1 and Orai1 offers a reasonable alternative. The STIM1(445–475) fragment inhibits the native CRAC current when infused into Jurkat cells 40 . [Ca 2+ ]i in pancreatic acinar cells was followed by recording the Ca 2+ -activated Cl − current 11 to allow infusing the cells with peptides through the patch pipette. Figure 1A,B shows that infusion of STIM1(445–475) into pancreatic acinar cells reduces the frequency by about 35% of Ca 2+ oscillations triggered by weak receptor stimulation. Inhibition of the fully activated Ca 2+ influx (plateau phase) by STIM1(445–475) did not reach statistical significance, likely because the peptide is a weak inhibitor of Ca 2+ influx 40 . Nevertheless, even partial inhibition of Ca 2+ influx was sufficient to reduce the frequency of Ca 2+ oscillations. A five-residues Orai1(153–157) fragment was reported to strongly inhibit the current mediated by expressed STIM1-Orai1 42 . Figure 1C,E shows that infusion of this fragment into pancreatic acinar cells inhibited Ca 2+ influx by about 50% and reduced the frequency of the Ca 2+ oscillations by about 70%. Hence, there is a good correlation between inhibition of Ca 2+ influx and reduced Ca 2+ oscillation frequency, highlighting the crucial role of Ca 2+ influx in sustaining the oscillation and determining their frequency.

Inhibition of Ca 2+ signaling by STIM1 and Orai1 inhibitory peptides in pancreatic acini and by siRNA knockdown in parotid ducts. [Ca 2+ ]i is monitored as changes in the Ca 2+ -activated Cl − current, which faithfully reports changes in pancreatic acinar cells [Ca 2+ ]i. Acinar cells were infused with pipette solution (A, control) containing 50 µ m of the STIM1 inhibitory peptide STIM1(445–475) (B), or 50 µ m of the Orai1 inhibitory peptide Orai1(153–157) (C), for 7 min before stimulation with 0.75 µ m carbachol to induce Ca 2+ oscillations and then with 100 µ m carbachol to maximally activate Ca 2+ influx. The average oscillation frequency is shown in (D) and the plateau in (E). The dashed lines are the zero current. The plateau (marked by a line with double arrows) was normalized relative to the initial increase in current because of Ca 2+ release from the ER. The columns show the mean ± SEM of seven experiments. The efficiency of the Orai1 (F) and STIM1 (G) siRNA probes was determined by RT-PCR in mouse keratinocytes. Panels (H and J) show the response of sealed parotid ducts treated with scrambled (dark traces and columns) or with Orai1 siRNA and stimulated with 100 µ m ATP in Ca 2+ -free media and then exposed to media containing 2 m m Ca 2+ to evaluate Ca 2+ influx. The ducts were then perfused with Ca 2+ -containing media for 10 min to load the stores before treatment with 25 µ m CPA in Ca 2+ -free media and finally exposed to media containing Ca 2+ to evaluate SOC activity. (I and K) Parotid ducts treated with scrambled or STIM1 siRNA were treated with 25 µ m CPA in Ca 2+ -free media and then exposed to Ca 2+ -containing media to measure SOC activity. Panels J and K show the mean ± SEM of four ducts. * denotes P < 0.01 or better.

To further demonstrate the role of STIM1 and Orai1 in Ca 2+ signaling in secretory cells we used an independent assay to test the effect of their knockdown on Ca 2+ influx in parotid gland sealed ducts in primary culture, in which siRNA effectively knockout the desired genes 43-45 . Ducts were prepared from the parotid gland to show the role of STIM1 and Orai1 in another secretory cell type. Figure 1F,G shows the efficiency of the knockdown of Orai1 and STIM1 by three siRNA probes. The second probe for each gene was used for the experiments shown, but we also tested the effect of the siRNA1 for Orai1 and siRNA3 for STIM1 to exclude off-target effects and obtained similar results. Figure 1H,J shows that knockdown of Orai1 have reduced Ca 2+ influx activated by receptor stimulation or by store depletion by about 65% and Figure 1I,K shows that knockdown of STIM1 has reduced SOC by about 85%.

Polarized localization of native Orai1

The specificity of the antibody used to localize Orai1 has been validated in a recent report showing that the immunohistochemical signal obtained in wild-type tissues is eliminated in tissues obtained from patient and mice with deletion of Orai1 34 . Figure 2A shows that this anti-Orai1 antibody detected Orai1 in pancreatic and SG extracts. Figure 2B shows that Orai1 localization is restricted to the lateral membrane of pancreatic acinar cells (arrows), similar to the localization of a number of key Ca 2+ signaling proteins 1 . Staining is also observed at the lateral membrane most adjacent to the apical pole (dotted arrows) and there is no detectable staining of Orai1 in the basal plasma membrane region, suggesting that the level of Orai1, if it is expressed at this site, is very low.

Localization of Orai1 in pancreatic acini. The anti-Orai1 antibody detects Orai1 in pancreatic and submandibular gland extracts (A). To deplete the ER Ca 2+ store acini in Ca 2+ -free media containing 1 m m EGTA were stimulated with 0.5 m m carbachol and treated with 25 µ m CPA for 10 min. The acini were then fixed and co-stained for Orai1 and IP3R3 (B and C) or ZO1 (D). In (B and C), membranes next to the apical pole are marked with solid arrows and the lateral membranes are marked with dashed arrows. In (E), images acquired from resting and stimulated cells that were treated with vehicle or with 50 µ m 2APB were used to determine the overlap of Orai1 with IP3R3 and with ZO1. The results are the mean ± SEM of the number of cells indicated in the columns. There is no significant difference between all conditions.

The luminal plasma membrane in the apex of acinar cells is small but folds along the lateral membrane at the apical pole, where the two membranes are separated by tight junctions 3, 46 . Staining of the tight junctions in pancreatic acini resulted in an image very similar to that obtained with Orai1 3 . Indeed, co-staining with the tight junction protein ZO1 shows nearly perfect colocalization of Orai1 and ZO1 (Figure 2D) with 94 ± 5% overlap (n = 98 cells).

Another Ca 2+ signaling protein expressed in this region of the cell is the ER-located IP3R. Accordingly, localization of Orai1 significantly overlaps with IP3R3 (Figure 2D, 93 ± 6%, n = 76 cells). Although a significant portion of Orai1 transfected in pancreatic acinar cells is targeted to the apical pole, localization of the expressed Orai1 was more homogeneous at the plasma membrane 33 . We cannot fully explain the difference between the present and previous findings. However, it might be because of over-expression of Orai1 overwhelming the cellular protein sorting and trafficking machinery.

Cell stimulation and depletion of ER Ca 2+ result in clustering of expressed Orai1 19, 21, 26, 27, 40, 41 . However, cell stimulation together with inhibition of the SERCA pumps have had no dramatic effect on the unique polarized localization of native Orai1 in acinar cells (Figure 2C). Additionally, treating the cells with 2APB had no effect on the localization of Orai1 (Figure 2D). The images in Figure 2 were acquired at a plane showing the maximal expression of Orai1 and IP3R3 and ZO1. However, similar tight overlap between Orai1 and IP3R3 and ZO1 was observed in all cellular planes. A possible implication of this finding is that in the polarized acinar cells, Orai1 is already targeted to its site of function and further targeting or relocation is not needed. This may ensure the highly polarized Ca 2+ signal in these cells.

Polarized recruitment of native STIM1

Given the polarized localization of Orai1 in Figure 2 and the tight coclustering of heterologously expressed STIM1 and Orai1 in response to store depletion 19, 21, 26, 40, 41 , we expected STIM1 to show a similar localization as Orai1 in stimulated cells. We examined the localization of the native STIM1 using two different anti-STIM1 antibodies with established specificity. One antibody raised against the conserved STIM1(657–685) detected the native STIM1 and, importantly, the signal was eliminated in tissues from patients who lack STIM1 34 . Figure S1 shows that this antibody reported diffused STIM1 staining in resting acinar cells with more intense staining at the basal pole. Cells stimulation did not result in massive redistribution of this staining to the basal pole, but rather induced recruitment of STIM1 to the apical (turquoise arrowheads) and lateral regions close to the plasma membranes (yellow arrowheads) (Figure S1B). The SOC inhibitor 2APB that disrupts clustering of STIM1 with native Orai1 37-39 had no obvious effect on STIM1 localization in resting cells, but markedly reduced accumulation of STIM1 at the apical and lateral plasma membrane regions (Figure S1C). Another notable finding is that the recruited STIM1 and IP3R3 show only partial colocalization along the lateral membrane (turquoise squares in Figure S1B). This is different from the findings with Orai1 (see also below), where there was >90% overlap with IP3R3.

Similar, but superior, staining was obtained with a second anti-STIM1 antibody. Staining with the Protein-Tech antibody was eliminated in tissue from STIM1−/− mice 35 . We further validated the use of this antibody in Figure S2 by showing that it recognizes the native and expressed STIM1 in HeLa cells (panel A), showing reduced signal in STIM1 siRNA-treated HeLa cells (panels B and D), and giving the expected STIM1 staining in resting and stimulated cells (panels B, C).

Figure 3 shows the localization of STIM1 relative to IP3R3 obtained with this antibody. Resting cells show primarily diffuse STIM1 localization (Figure 3A). Treating resting cells with 2APB did not have any effect on this localization (Figure 3B,E). Cell stimulation and store depletion recruited STIM1 primarily to the apical pole (solid arrows) and lateral cellular regions (dashed arrows), with small amount of STIM1 punctae at the basal membrane region (turquoise arrowheads) (Figure 3C). STIM1 at the apical pole shows excellent colocalization with IP3R3 (Figure 3C). Unexpectedly, intense accumulation of STIM1 at the lateral membrane region was at a region free of IP3Rs (turquoise squares). Hence, STIM1 showed only 43 ± 6% (n = 117) overlap with IP3R3 (Figure 3E). An important control is shown in Figure 3D,E, in which treatment with 2APB disrupted STIM1 accumulation, reducing the overlap with IP3R3 to 13 ± 3% (n = 64), which is not different from the overlap in resting cells.

Localization of STIM1 in pancreatic acini. Resting acini (A and B) were maintained in solution A, whereas stimulated acini (C and D) are acini in Ca 2+ -free media containing 1 m m EGTA that were stimulated with 0.5 m m carbachol and treated with 25 µ m CPA for 10 min. Control and stimulated acini were also treated with 50 µ m 2APB (B and D) that was added just before cell stimulation. The acini were then co-stained for STIM1 and IP3R3. In (C), membranes next to the apical pole are marked with solid arrows and lateral membranes are marked with dashed arrows. Punctae at the basal and lateral membranes are marked with turquoise arrows. The turquoise squares in all images depict area showing high expression of STIM1 and no IP3R3. In (E), images collected from resting and stimulated cells that were treated with vehicle or with 50 µ m 2APB were used to determine the overlap of STIM1 with IP3R3. Panel (F) compares the overlaps of Orai1 and STIM1 with IP3R3. The results are the mean ± SEM of the indicated number of cells. * denote p < 0.01 from resting cells.

To further confirm the unexpected differential overlap of Orai1 and STIM1 with IP3R3 (summarized in Figure 3F) we compared colocalization of STIM1 and Orai1 with other marker proteins. In Figure 4A,B the basal membrane was stained with laminin to show more clearly the very low level or no localization of STIM1 and Orai1 at this site. Figure 4C shows the partial localization of STIM1 at the tight junction and Figure 4D shows quantification of the dramatic difference in the colocalization of Orai1 and STIM1 with ZO1. We also measured the area of the basolateral membrane that overlaps with the Orai1 and STIM1. The basolateral membrane was visualized with E-cadherin. The images in Figure 4E,F were acquired in planes optimal to observe the basolateral membrane with E-cadherin. Figure 4E,G shows that Orai1 was detected in 28 ± 3.3% (n = 84 cells) of the basolateral membrane domain, whereas Figure 4F,G shows that STIM1 was detected in 67 ± 4.5% (n = 63 cells) of the basolateral membrane domain.

Plasma membrane area occupied by Orai1 and STIM1 in pancreatic acini. Store-depleted and receptor-stimulated acini were stained with the basal membrane marker laminin (A and B), the tight junction marker ZO1 (C) and the basolateral membrane E-cadherin (E and F) (all red).The acini were co-stained with Orai1 (A and E) or STIM1 (B, C and F). Panel (D) Compares the overlap of STIM1 and Orai1 with ZO1 and in (G) images similar to those in (E) and (F) were used to determine the % basolateral membrane expressing Orai1 or with adjacent STIM1. The results are the mean ± SEM of the number of cells indicated in the columns.

Finally, we tagged the anti-STIM1 antibodies with Alexa Fluor® 647 and used it to determine localization of STIM1 and Orai1 in the same stimulated cells. Figure 5 shows that only about 50% of STIM1 colocalizes with Orai1, which confirms the results obtained with IP3R3 and E-cadherin. Thus, the remaining STIM1 likely forms complexes with other Ca 2+ influx channels.

Colocalization of STIM1 and Orai1 in the same cells. Store-depleted acini were labeled with Orai1 (A) and then with the Alexa Fluor ® 647-tagged STIM1 (B) as detailed in Methods. The merged image is shown in (C). The double labeling procedure resulted in a significant background staining in the form of dots, mainly from the Orai1 antibodies. These were excluded from calculation of % overlap of STIM1 and Orai1. Overlap was restricted to the plasma membrane region and is shown in (D).

Polarized localization of native TRPC1

The distinct lateral regions that express STIM1 but not Orai1 raise the question of whether STIM1-interacting TRPC channels are localized at this region. Of particular interest is TRPC1 that is localized at the lateral membrane of SG acinar cells 30 . Unfortunately, detection of TRPC1 in pancreatic acini with the antibodies requires fixation with paraformaldehyde that precludes detection of IP3Rs. Reclaiming detection of the IP3Rs after paraformaldehyde fixation required incubation with cold MeOH, but this resulted in a non-specific intense staining of the secretory granules that overwhelmed the staining for TRPC1. Nevertheless, fixation with paraformaldehyde suggested that TRPC1 is localized at the apical and lateral regions of the basolateral membrane of pancreatic acinar cells (Figure S3A). Pancreatic acinar cells from Trpc1−/− mice showed reduced Ca 2+ influx (Figure S3B) and Ca 2+ oscillation frequency (Figure S3C), indicating that TRPC1 mediates a significant portion of Ca 2+ influx in these cells.

It was possible to obtain reasonable images of TRPC1 and IP3Rs in parotid acini treated with paraformaldehyde and MeOH. Figure 6A shows staining of the lateral membranes with the anti-TRPC1 antibody and Figure 6B shows that the staining is markedly reduced in Trpc1−/− parotid acini. Co-staining of Trpc1 and IP3R3 revealed lateral membrane regions with no overlap between them (turquoise squares), as was found with STIM1 (Figures 3 and S1). Although the fixation procedure reduced the resolution of the IP3Rs images and precluded reliable calculation of overlap, the findings in Figure 6 suggest that part of STIM1 is recruited to a plasma membrane domain expressing TRPC1, STIM1 and no Orai1. However, all the proteins, Orai1, TRPC1, IP3R3 as well as STIM1 colocalize in the lateral membrane close to the apical pole following cell stimulation.

Localization of TRPC1 in parotid gland acini. Acini from wild-type (A) and Trpc1−/− mice (B) were co-stained for TRPC1 (green) and IP3R3 (red). The turquoise squares in all images depict area showing high expression of TRPC1 and no IP3R3.

Co-IP of STIM1/Orai1/TRPC1/IP3 R3

Colocalization by confocal microscopy is not sufficient to determine if the proteins exist in the same Ca 2+ signaling complex. This can be tested more directly by co-IP assays. First, we determined if proteins expressed in model systems can be co-IPed. A recent study reported that cell stimulation enhances co-IP of expressed TRPC3–Orai1–STIM1–IP3R1 47 . Figure S4 extends these findings by showing that the co-IP is enhanced by cell stimulation and it is reduced by treating the cells with 2APB. co-IP of expressed proteins can be markedly affected by their over-expression. Therefore, when possible, it is important to demonstrate co-IP of the native proteins. Therefore, next we examined the reciprocal co-IP of all native Ca 2+ signaling proteins in pancreatic acinar cells. The co-IP of TRPC3 was examined because it shows close colocalization with IP3R3 7 , interacts with TRPC1 25 and contributes to Ca 2+ influx in acinar cells 31 . Figure 7A shows that IP of STIM1 co-IPs Orai1. Figure 7B,C shows the reciprocal co-IP of IP3R3 and STIM1 and Figure 7D,E shows the reciprocal co-IP of IP3R3 and Orai1. TRPC3 co-IPs with IP3R3 (Figure 7F,G) and with Orai1 (Figure 7G).

Co-IP of STIM1, Orai1, IP3R3, TRPC3 and TRPC1 is enhanced by cell stimulation and disrupted by 2APB. For each set of experiments, extracts were prepared from the pancreas of two mice. Control lanes are without primary antibodies and inputs are 5% of the initial material used for the IP. Extracts were used to IP STIM1 (A and B) and probe for co-IP Orai1 (A) and IP3R3 (B) IP IP3R3 (C, E and G) and probe for co-IP of STIM1 (C), Orai1 (E) and TRPC3 (G) IP Orai1 (D and G) and probe for co-IP of IP3R3 (D) and TRPC3 (G). In (H, I) part of the cells were not stimulated (rest) and part were stimulated by incubation in Ca 2+ -free media and treatment with 0.5 m m carbachol and 25 µ m CPA for 10 min at 37°C. Portions of resting and of stimulated cells were treated with 50 µ m 2APB. Extracts were prepared from all four conditions and used to IP STIM1 (two upper blots in H) and probe for co-IP of Orai1 and IP3R3 and for IP IP3R3 (two middle blots in H) and probe for co-IP of STIM1 and Orai1. The bottom blots are the inputs. Extracts were also used to determine the reciprocal co-IP of STIM1 and TRPC1 (I). Note that cell stimulation enhances the co-IP, whereas treatment with 2APB reduces the co-IP.

Significantly, Figure 7H shows that cell stimulation enhances the co-IP of IP3Rs with STIM1 and Orai1 and the co-IP is inhibited by treatment with 2APB. Figure 7I shows the mutual co-IP of STIM1 and TRPC1, enhancement of the co-IP by cell stimulation and its disruption by treating the acini with 2APB. Hence, the native Orai1, STIM1, IP3Rs, TRPC3 and TRPC1 are in Ca 2+ signaling complexes and STIM1 may stabilize the complexes, because the complexes are disrupted by 2APB. Because of the partial Orai1/STIM1 overlap and the overlap of TRPC3 and TRPC1 with STIM1 in regions that do not express STIM1, it is likely that STIM1 stabilizes complexes of several compositions: Orai1-STIM1 TRPC1/TRPC3-STIM1 and Orai1-STIM1-TRPC1/TRPC3. It will be of particular interest to test whether the various complexes mediate spatially different Ca 2+ influx pathways that may regulate specific cellular functions.

Polarized initiation of Ca 2+ influx

To begin to evaluate the possibility that STIM1 may mediate several Ca 2+ influx pathways that operate in different cellular microdomains, we analyzed initiation of Ca 2+ influx in cells with fully depleted stores and thus maximally activated Ca 2+ influx. In addition, the stores were depleted passively so as to minimize propagation of the Ca 2+ signal because of cell stimulation. Figure 8 shows that the consequence of the localization of TRPC channels, Orai1 and the recruited STIM1 at the apical pole next to the tight junctions is the generation of a defined Ca 2+ influx site (Figure 8, left images). As these cells maintain polarity even when dissociated to single cells, we have also measured Ca 2+ influx in single acinar cells to ensure uniform Ca 2+ access to all parts of the plasma membrane. Figure 8 (right) images show that also in single pancreatic acinar cells Ca 2+ influx initiates at the apical pole. The rate of cytoplasmic Ca 2+ increase because of Ca 2+ influx is very slow, indicating that the Ca 2+ signal does not propagate. Diffusion of Ca 2+ in the cytoplasm is quite slow 48 , and thus cytoplasmic Ca 2+ increase at later times likely reflects Ca 2+ influx through sites different from the initial site where Orai1 is present. These sites likely mediate Ca 2+ entry through TRPC channels that are present at the lateral membrane.

Site of Ca 2+ entry in pancreatic acinar cells. A three-cell acinus (left panels) and a single acinar cell (right panels) loaded with Fura2 were incubated in Ca 2+ -free media and were treated with 0.5 m m carbachol and 25 µ m CPA to activate Ca 2+ influx. When Ca 2+ returned to basal level the cells were exposed to media containing 5 m m Ca 2+ and images were collected every 0.3 seconds to detect the initial sites of Ca 2+ influx. The traces show the time–course of the changes in Ca 2+ recorded in the regions of interest indicated in the first fluoresce image and the bright field image.


Basolateral Potassium Channels and Epithelial Ion Transport

The two-membrane hypothesis, proposed by Koefoed-Johnsen and Ussing (1) in 1958, formalized the concepts that the inner and outer membranes of the frog-skin epithelium are functionally different and that both membranes participate in active sodium absorption. The model described the frog skin as a homogenous intracellular compartment bounded by an apical and basolateral membrane with different ion transport properties. The apical membrane is sodium conductive (sodium channels), and the basolateral membrane contains Na + ,K + –adenosine triphosphatase (ATPase) pumps and is potassium conductive (potassium channels) (Figure 1 ). Approximately 20 years later (2), a similar model was developed to explain Cl − secretion by the dogfish rectal gland (Figure 2 ). Important modifications included (1) addition of a basolateral membrane Cl − entry step, and (2) replacement of the apical membrane Na + channel by a cyclic adenosine monophosphate (cAMP)–activated Cl − channel. Again, the apical and basolateral cell membranes are functionally different, and both membranes participate in active Cl − secretion. Several years earlier, Olver and colleagues (3) showed that canine airway epithelium absorbed Na + and secreted Cl − . A model that incorporates each of the transport elements found in the frog skin and shark rectal gland can explain both sodium absorption and chloride secretion by the airway epithelium (4).

Fig. 1. Schematic representation of the two-membrane hypothesis (1) for transepithelial sodium absorption by frog skin. The apical membrane contains sodium channels, and the basolateral membrane contains Na + ,K + -ATPase pumps and potassium channels.

Fig. 2. Transport model for epithelial chloride secretion. The basic model proposed by Silva and colleagues (2) is modified to include a basolateral Na + ,K + ,2Cl − cotransporter, rather than a NaCl cotransporter, as originally described.

A major impetus for the study of ion transport in nonrenal tissues is the desire to understand and treat secretory diarrheal diseases and cystic fibrosis (CF), disorders that result from abnormal salt and fluid secretion (5, 6). The discovery that CF is caused by the loss of an apical membrane, cAMP-activated Cl − conductance (7, 8), coupled with the knowledge that cholera toxin activates adenylate cyclase and stimulates intestinal secretion, focused attention on apical membrane Cl − channels in gastrointestinal and airway epithelia. The first epithelial ion channel to be identified and cloned was the CF transmembrane conductance regulator (CFTR) (9). Accordingly, we now know a great deal about the expression, regulation, and function of apical CFTR Cl − channels and their role in Cl − secretion (10). Several other Cl − channels have been cloned during the past decade, but the importance of the so-called ClC protein family members in epithelial Cl − secretion is uncertain (11). There are, however, other non-CFTR apical membrane Cl − channels that do participate in epithelial Cl − secretion, but the molecular identity and many of the details of regulation of these transport proteins are yet to be established (12, 13, 14). The sodium channel that is present in the apical membrane and that mediates amiloride-sensitive sodium absorption by frog skin, colon, airway, and numerous other epithelia was recently cloned and is known as ENaC (15). The molecular identity of the basolateral isoform of the Na + ,K + ,2Cl − cotransporter, responsible for accumulation of intracellular Cl − for secretion, is also known (16). The Na + ,K + -ATPase, located at the basolateral membrane of nearly all epithelial cells, is crucial for generating and maintaining appropriate intracellular activities of sodium and potassium. The molecular identity of Na + ,K + -ATPase, as well as detailed knowledge of the regulation and mechanism of transport by the pump, are well established (17). The transport models that describe sodium absorption and Cl − secretion (Figures 1 and 2 , respectively) both include basolateral potassium channels however, the molecular identity of the proteins (channels) responsible for basolateral potassium conductance in epithelial cells remains largely unknown.

The basolateral K + conductance pathway is critical in both secretory and absorptive epithelia, and inhibition of basolateral K + conductance with nonselective K + channel blockers (e.g., barium) will reduce transepithelial transport. Basolateral K + channels recycle the potassium that enters the cell in exchange for sodium via the Na + ,K + -ATPase (Figures 1 and 2 ) and also the potassium that enters the cell across the basolateral membrane coupled to Na + and Cl − (Figure 2 ) in Cl − -secreting epithelia. Not only do K + channels recycle potassium, but they contribute to the membrane potential of the cell, an important determinant of the electrochemical driving force for passive ion movement across the plasma membrane. In the two transport models depicted above, Na + influx and Cl − efflux are both driven in part by the electrical potential difference that exists across the apical membrane. Although it is not necessarily obvious, the apical and basolateral membranes in epithelia are electrically coupled by an intraepithelial current loop that arises due to the existence of a finite ionic conductance across the paracellular pathway (18). In other words, depolarization or hyperpolarization of the basolateral cell membrane will influence the opposite membrane and vice versa. It has long been argued that sustained Cl − secretion requires activation of basolateral K + conductance to facilitate K + recycling and to hyperpolarize the apical cell membrane away from the chloride equilibrium potential (E Cl− ). Intracellular microelectrode measurements of canine and human airway epithelia demonstrated that the initial depolarization of the apical membrane potential upon stimulation with beta agonists was followed by partial repolarization (8, 19, 20). The accompanying changes in membrane resistance and apical/basolateral resistance ratio suggested that the secondary repolarization resulted from activation of basolateral K + conductance. The properties of basolateral K + channels expressed in airway epithelial cells have received little attention, and the genes that encode these proteins are not known.

Since the first K + channel was cloned a little more than a decade ago from Drosophila (21), molecular techniques have provided us with a plethora of potassium ion channel genes (22). Genomic sequence data will undoubtedly reveal additional K + channel genes. Four broad classes of mammalian K + channels have been identified: (1) voltage-gated K + channels, (2) calcium-activated K + channels, (3) “leak” K + channels, and (4) inward rectifier K + channels (22). Several of the cloned potassium channels from these families have properties similar to native epithelial K + channels. For example, the Kir1.1 (ROMK1) inward rectifying K + channel shares many of the features of the native small conductance secretory K + channel, located in the apical membrane of cortical collecting-duct principal cells and the thick ascending limb of Henle cells (23). An ATP-sensitive K + channel found in the basolateral membrane of mammalian proximal tubule cells is thought to respond to changes in intracellular ATP and thereby coordinate apical solute entry, Na + pump activity, and basolateral K + conductance (24, 25). The molecular identity of this channel is not yet established however, several members of the Kir family of channels acquire ATP sensitivity when coexpressed with the sulfonylurea receptor (22). A calcium-activated K + channel found on the basolateral membrane of the T84 colonic cell line is activated by 1-ethyl-2-benzimidazolinone (1-EIBO) and inhibited by clotrimazole (26). A channel with identical properties is encoded by the SK4 (also known as hIK1) gene, and mRNA for SK4 is expressed in T84 cells (27, 28). Thus, it appears that SK4 potassium channels comprise at least a portion of the basolateral K + conductance in colonic epithelial cells. Greger and coworkers described a K + channel in the basolateral membrane of colonic crypts that resembles the SK4 gene product (29). In addition, they found a cAMP-regulated small-conductance K + channel that was too small to be resolved by single channel recording (probably < 3 pS) (30). The conductance was inhibited by chromanol 293B and is likely due to voltage-gated, KvLQT1 channels (22).

In this issue, Mall and associates (31) show that cAMP-dependent stimulation of human airway epithelial Cl − secretion causes parallel activation of basolateral K + conductance. The secretory response, as well as the conductance increase, are blocked by chromanol 293B, a compound known to inhibit KvLQT1 K + channels (32). Furthermore, they demonstrate by RT-PCR that mRNA for KvLQT1 is expressed in airway epithelium. Although KvLQT1 mRNA was detected in CF nasal polyp cells, chromanol 293B did not inhibit transport in the CF epithelium. These results suggest that the depolarization of the apical and basolateral cell membranes upon activation of CFTR is necessary to stimulate basolateral, chromanol 293B–sensitive K + channels. The authors conclude that KvLQT1 potassium channels are important for maintaining cAMP-dependent secretion in the human airway and that pharmacologic activators of these channels may be useful for the treatment of CF patients. However, activation of basolateral potassium conductance in native CF airway epithelium may be counterproductive. Since CF airway epithelia exhibit excessive sodium absorption, activation of basolateral K + channels would tend to increase sodium absorption and may further deplete airway-surface liquid volume (33, 34). Therefore, it may be necessary to fully inhibit sodium absorption prior to activation of basolateral K + conductance in the CF airway. In contrast, CF epithelia, in which loss of cAMP-stimulated anion secretion is the dominant phenotype (e.g., pancreas, biliary epithelium, intestine), may prove to be better targets for activation of basolateral K + conductance as a mechanism promoting anion secretion. Residual activity of mutant CFTR and/or stimulation of Cl − secretion via non-CFTR apical membrane Cl − channels might be enhanced in this way.

It has recently been demonstrated that isoflavonoids and flavonoids activate normal and mutant CFTR, and a phase I clinical trial of genistein is currently underway (35, 36). One potential problem associated with the use of isoflavonoids or flavonoids to stimulate CFTR-dependent anion secretion is that genistein inhibits basolateral K + channels in a colonic epithelial cell line, HT29 (37). We have recently found that genistein inhibits cAMP- and calcium-stimulated anion secretion in immortalized, murine, pancreatic-duct epithelial cells (38). The inhibition of anion secretion appears to result from an effect of genistein on several different potassium channels present in the basolateral membrane of these cells. These observations, along with the findings of Mall and colleagues (31), highlight the necessity of considering ion transport pathways in the context of a polarized epithelial cell rather than in isolation.


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