Ion-gated ion channels

Today I've heard for the first time of calcium-gated ion channels but find it hard to get an idea how they work, where they are located, and which role they play.

I assume calcium-gated ion channels are just a special form of ion-gated ion channels, which would be a fourth group of ion channels next to voltage-, lipid-, and ligand-gated ion channels (with ligands being some larger molecules). But on ion-gated ion channels even less is to be found on the internet.

One specific paper that mentions ion-gated ion channels is this (Experimental Neurology, 2001)

One paper that mentions calcium-gated ion channels is this (New England Journal of Medicine, 2015)

Where do I find a general introduction into ion-gated ion channels - if they exist?

The main families of calcium-gated channels I am aware of are the calcium-gated potassium channels and the internal ryanodine receptors involved in calcium-induced calcium release - you could start from those linked Wikipedia articles.

I wouldn't really make these a fourth category, they should either be thought of as voltage-gated channels that are modulated by intracellular calcium, or as ligand-gated channels - the ligand just happens to be a calcium ion.

Some channels in this category are not influenced by calcium directly but via calmodulin, which is a common mediator of calcium-driven intracellular influences.

Ion channels

Ion channels are pore-forming protein complexes that facilitate the flow of ions across the hydrophobic core of cell membranes. They are present in the plasma membrane and membranes of intracellular organelles of all cells, performing essential physiological functions including establishing and shaping the electrical signals which underlie muscle contraction/relaxation and neuronal signal transmission, neurotransmitter release, cognition, hormone secretion, sensory transduction and maintaining electrolyte balance and blood pressure. They are usually classified by gating i.e. the stimulus that 'opens' the channel, be it chemical or mechanical stimuli.

Most Na + , K + , Ca 2+ and some Cl - channels are gated by voltage, whereas others (such as some K + and Cl - channels, TRP channels, ryanodine receptors and IP3 receptors) are relatively voltage-insensitive and are gated by second messengers and other intracellular and/or extracellular mediators.

Many ion channels (e.g. K + , Na + , Ca 2+ , HCN and TRP channels) share several structural similarities which suggests that they have evolved from a common ancestor. This group of ion channels can be classified together as the ‘voltage-gated-like (VGL) ion channel chanome’ (Figure 1).

Figure 1. Representation of the amino acid sequence relations of the minimal pore regions of the voltage-gated ion channel superfamily. This global view of the 143 members of the structurally related ion channel genes highlights seven groups of ion channel families and their membrane topologies. Four-domain channels (CaV and NaV) are shown as blue branches, potassium selective channels are shown as red branches, cyclic nucleotide–gated channels are shown as magenta branches, and transient receptor potential (TRP) and related channels are shown as green branches. Background colors separate the ion channel proteins into related groups: light blue, CaV and NaV light green, TRP channels light red, potassium channels, except KV10–12, which have a cyclic nucleotide–binding domain and are more closely related to CNG and HCN channels light orange, KV10–12 channels and cyclic nucleotide–modulated CNG and HCN channels (from Yu and Catterall, 2004)

Other ion channels, such as Cl channels, aquaporins and connexins, have evolved separately and possess completely different structural properties to the VGL channels.

Defects in ion channel function cause a wide range of disorders termed 'channelopathies' which include conditions resulting from mutations in ion channels (e.g. cystic fibrosis, long QT syndrome, short QT syndrome- inherited ion channel diseases are reviewed by Lieve and Wilde (2015)) and acquired diseases caused by autoimmune attack on ion channels (e.g. myasthenia gravis and possibly multiple sclerosis- autoimmunity and channelopathy are reviewed by RamaKrishnan and Sankaranarayanan (2016)).

Ion channel modulators are an extremely successful drug class, second only to drugs targeting G protein-coupled receptors, with amlodipine, zolpidem, alprazolam, the sulfonylureas, repaglinide and nateglinide amassing huge returns for their developers. Technical advances in high-throughput screening methodology and high resolution crystal structures of ion channels should enable development of the ion channel drugs of the future. Potential new ion channel drug targets are discussed in Bagal et al. (2013).

Ligand-gated ion channels

Ligand-gated ion channels (LGICs) mediate passive ion flux driven by the electrochemical gradient for the permeant ions. LGICs are gated by the binding of a specific ligand to an orthosteric site(s) that triggers a conformational change that results in the conducting state, or by binding of endogenous, or exogenous, modulators to allosteric sites. LGICs are responsible for fast synaptic transmission in the nervous system and at the somatic neuromuscular junction.

This group contains the excitatory, cation-selective, nicotinic acetylcholine (nAch), 5-HT3, ionotropic glutamate (NMDA, AMPA and kainate receptors) and P2X receptors and the inhibitory, anion-selective, GABAA and glycine receptors as well as acid-sensing (proton-gated) ion channels (ASICs), epithelial sodium channels (ENaC), IP3 receptor and the zinc-activated channel (ZAC). LGICs are generally heteromultimers, with subunits encoded by multiple genes. Multimeric combinatorial diversity leads to the wide variety of receptors reported, with differing pharmacological and biophysical properties and varying patterns of expression within the nervous system and other tissues. The pharmaceutical industry is striving to use this heterogeneity to develop new therapeutic agents with improved discrimination between receptor isoforms and reduced off-target effects.

The epithelial sodium channels (ENaC) mediate sodium reabsorption principally in the aldosterone-sensitive distal part of the nephron and the collecting duct of the kidney, and also in the lung epithelia. In the kidney, these channels are involved blood pressure regulation and are associated with many cardiovascular diseases. The ‘potassium-sparing’ diuretics amiloride and triamterene are ENaC channel blockers.

Acid-sensing (proton-gated) ion channels (ASICs) are involved in fear conditioning, memory formation, and pain sensation

Aberrant LGIC function is associated with many diseases. For example, overactivation of NMDA glutamate receptors may play a part in causing neurotoxic damage in the development of neurodegenerative disorders.

Many prescription drugs exert their effects by modulating the activity of LGICs. Some of the more common drug groups are described below:

Volatile anesthetic drugs used for general anesthesia primarily block the activity of GABA-, glutamate- and glycine-gated LGICs, but different agents have different effects on each receptor, and may block other LGICs. For example, sevoflurane is thought to act as a positive allosteric modulator of the GABAA receptor, but also acts as an NMDA receptor antagonist, potentiates glycine receptor currents, as well as nACh and 5-HT3 receptor currents. Desflurane also acts as a positive allosteric modulator of the GABAA and glycine receptors and as a negative allosteric modulator of nACh receptors. Sevoflurane and desflurane have largely replaced isoflurane except in economically undeveloped areas, where their high cost precludes use. Nitrous oxide, used in surgery and dentistry for its anaesthetic and analgesic effects, directly modulates a broad range of ligand-gated ion channels, and this likely plays a major role in many of its effects. It moderately blocks NMDA and β2-subunit-containing nACh channels, weakly inhibits AMPA, kainate, GABAC, and 5-HT3 receptors, and slightly potentiates GABAA and glycine receptors (see Emmanouil and Quock (2007): Advances in understanding the actions of nitrous oxide).

Gamma-aminobutyric acid analogs (the 'gabapentinoids') such as gabapentin and its conjugated prodrug gabapentin enacarbil (both used to treat epilepsy, neuralgic/neuropathic pain) and pregabalin (used to treat generalised anxiety disorder (GAD), neuropathic pain, post herpetic neuralgia) were thought to act as GABAA receptor agonists and cause inhibitory action like GABA. However, more recent analysis reveals high-affinity gabapentin binding sites on neuronal membranes, subsequently demonstrated to represent the α2δ protein, an accessory component of L-type calcium channels, encoded by gene CACNA2D1 or CACNA2D2- see Rogawski and Bazil (2007): New molecular targets for antiepileptic drugs: alpha(2)delta, SV2A, and K(v)7/KCNQ/M potassium channels. But whatever the mechanism, the effect of GABA analogs is to inhibit release of monoamine neurotransmitters including norepinephrine, substance P, and glutamate.

Acamprosate (used alongside behavioural therapy to manage alcohol abstinence in alcohol-dependent patients) is another GABA-like drug which appears to stimulate GABAergic inhibitory neurotransmission and antagonise the effects of excitatory amino-acids, particularly glutamate (via antagonism of NMDA receptors).

NMDA receptor antagonists (or channel blockers) such as ketamine (an anesthetic), dextromethorphan (a widely used OTC cough suppressant), phencyclidine (PCP, withdrawn from use as an anesthetic pharmaceutical), and nitrous oxide act principally via inhibition of glutamate NMDA receptors. Dextromethorphan is metabolised to the NMDA antagonist dextrorphan by CYP2D6. Many drugs of this class are used recreationally because of their psychoactive and dissociative effects.

nAch receptor modulators such as nicotine itself (as nicotine replacement therapy) and the nAch receptor antagonist varenicline are used to aid smoking cessation.

Part 2: Calcium-Activated Chloride Channel (CaCC) in the Enigmatic TMEM16 Family

00:00:0723 Hi.
00:00:0823 I am Lily Jan.
00:00:1023 In this second of the two-part series on our studies of ion channels, I will tell you about
00:00:1924 calcium-activated chloride channels.
00:00:2225 This is part of a long-term collaboration I have had with Yuh-Nung Jan.
00:00:2820 Calcium-activated chloride channels have only been molecularly identified in this millennium,
00:00:3620 about a decade ago, even though these channels have been studied ever since the 1980s
00:00:4318 and they have been associated with a number of different functions that are important.
00:00:5317 In this talk, I will first go over the ways we went about identifying the channel molecule,
00:01:0016 and then tell you what we have learned about the function of these channels.
00:01:0706 For a channel of interest, where we know about the function but not the molecules that form these channels,
00:01:1715 one general approach is to identify a rich source for this channel and
00:01:2509 inject pools of RNA into Xenopus oocytes so that the channel activity can be detected
00:01:3402 with recording from the oocytes.
00:01:3705 And we can then subdivide these pools of cDNAs until we end up with a single clone for the channel.
00:01:4613 For this approach to work, however, the Xenopus oocytes used as the expression system
00:01:5503 cannot be expressing the channel of interest.
00:01:5727 So, if we inject water into the Xenopus oocytes, we should see no channel activity.
00:02:0701 This approach of expression cloning was initially pioneered by Julius and Nakanishi.
00:02:1521 And in their early studies using this approach, they cloned a G protein-coupled receptor
00:02:2413 that activates a signaling pathway including the activation of phospholipase C and
00:02:3223 the release of calcium from internal stores.
00:02:3607 And they relied on the calcium-activated chloride channels that are endogenous to the
00:02:4217 Xenopus oocytes to report the activation of this whole signaling pathway.
00:02:5108 And we know that these calcium-activated chloride channels in the Xenopus oocytes
00:02:5703 serve an important function, to prevent polyspermia.
00:03:0124 And these channels actually have been studied in the oocyte ever since the 1980s.
00:03:0806 And for this reason, we know Xenopus oocytes cannot be used as the expression system
00:03:1505 for expression cloning of CaCC.
00:03:1812 And instead, Bjorn Schroeder went to the axolotl oocytes that are physiologically polyspermic.
00:03:2902 And after finding very little endogenous CaCC expression in the axolotl ooctyes, Bjorn used
00:03:3716 these oocytes as the expression system, and Xenopus oocytes as the source of RNA for CaCC
00:03:4708 to clone a calcium-activated chloride channel.
00:03:5125 And that led to the identification of Xenopus TMEM16A as a CaCC.
00:03:5816 And he then tested the mammalian homologues and found that of. in the family with ten members,
00:04:0509 TMEM16A and 16B formed calcium-activated chloride channels.
00:04:1122 And around the same time, Oh's group in Korea and Galietta's group in Italy independently
00:04:2117 came to the conclusion that TMEM16A forms calcium-activated chloride channels,
00:04:2816 but using very different approaches.
00:04:3306 From recent studies, we see that TMEM16A is very broadly expressed in the periphery,
00:04:4125 including epithelial cells and smooth muscle cells.
00:04:4604 And TMEM16B is expressed in multiple brain regions, and also in sensory neurons for
00:04:5420 odorant perception and in photoreceptors.
00:05:0005 In the photoreceptors, the calcium-activated chloride channels formed by TMEM16B reside
00:05:0817 at the ribbon synapse.
00:05:1020 They bind to the PSD95 anchor protein, and they provide negative feedback regulation.
00:05:1910 In the odorant neurons, in the cilia where the odorant will activate G protein-coupled receptors,
00:05:2626 leading to the opening of cyclic nucleotide-gated ion channels that permeate
00:05:3310 both calcium and other positively charged ions like sodium, the calcium will then activate
00:05:4203 calcium-activated chloride channels.
00:05:4426 So, CaCC formed by TMEM16B provides the low-noise high-gain amplification of the odorant signal.
00:05:5720 In the nervous system, we see that TMEM16A is found in sensory neurons in the dorsal root ganglia.
00:06:0720 But TMEM16B is found in different brain regions, in central neurons.
00:06:1406 There's a curious correlation.
00:06:1617 The cells that express 16B tend to express potassium-chloride cotransporters,
00:06:2427 and these cells have low chloride concentration inside.
00:06:3004 And so chloride channels are inhibitory.
00:06:3224 But in the cells like the dorsal root ganglia, and also in immature neurons in the brain,
00:06:4108 the cells use a different transporter, the sodium-potassium-chloride cotransporter.
00:06:4828 And these cells have high chloride concentration, and chloride channels are excitatory.
00:06:5706 And this applies to many different cells in the periphery, and also cells in other organisms,
00:07:0411 including the green algae.
00:07:0601 We know from studies in the 1980s, calcium-activated chloride channels are present in the green algae.
00:07:1517 And actually, these are the channels that are responsible for generating action potentials.
00:07:2115 It's not sodium channels.
00:07:2326 And so we can see the action potentials in this green algae are slower.
00:07:2922 It takes seconds rather than milliseconds, as in the case of action potentials in the
00:07:3514 nerves and muscles.
00:07:3714 And these have been referred to as the calcium. as the chemical action potential because
00:07:4400 it requires the calcium rise to induce the action potential.
00:07:4917 And when the light is switched off, calcium becomes released from chloroplasts.
00:07:5826 And so we can see, then, there is a progressive shortening in the latency for the
00:08:0408 action potential generation.
00:08:0711 And during the action potential, there's further rise in the calcium.
00:08:1113 And that will result in a pause in the cytoplasmic streaming.
00:08:1928 And these are really large cells, as you can see, in the green algae.
00:08:2418 And the cytoplasmic streaming is one way to move, you know, the organelles and materials
00:08:3103 around in the cell.
00:08:3411 Now, back to the animal kingdom.
00:08:3808 In the airway epithelia, we see two different kinds of chloride channels on the apical side,
00:08:4516 the luminal side of the cell.
00:08:4805 One is calcium-activated chloride channel, formed by TMEM16A.
00:08:5314 And the other is CFTR.
00:08:5628 And that's the channel linked to the disease cystic fibrosis.
00:09:0203 And these chloride channels are responsible for controlling, or participate in the control,
00:09:0919 of the thickness of the airway surface liquid, the ASL.
00:09:1520 And that liquid, lining the luminal side of the epithelia, is very important for
00:09:2322 mucociliary clearance of pathogens in the airway.
00:09:3018 In the airway, these calcium-activated chloride channels formed by TMEM16A also facilitate
00:09:3900 the release of mucin into the lumen.
00:09:4222 And from the 1980s, we have learned from studies of different exocrine glands that
00:09:4926 calcium-activated chloride channels are important for controlling the secretion from,
00:09:5616 you know, salivary glands, sweat glands, and so on.
00:10:0005 And these glands express TMEM16A.
00:10:0628 In the smooth muscle, the calcium-activated chloride channels can be activated, for example,
00:10:1502 with the punctal release. a bolus of calcium from internal stores.
00:10:2201 And this would cause the nearby calcium-activated chloride channels on the cell membrane
00:10:2818 to open, leading to what's referred to as STIC: spontaneous transient inward current.
00:10:3700 That will cause depolarization.
00:10:3919 And that will further lead to opening of voltage-gated calcium channels.
00:10:4321 So, it's a positive feedback to sustain the rising calcium and smooth muscle contraction.
00:10:5500 In the gut, you know, the gastrointestinal tract, there are cells referred to as
00:11:0116 interstitial cells of Cajal.
00:11:0416 And likewise, there are calcium-activated chloride channels.
00:11:0823 And when there's a puff of calcium from internal stores, that will generate a STIC,
00:11:1518 marked here, this little rising.
00:11:1821 And this spontaneous transient depolarization will then lead to opening of
00:11:2505 voltage-gated calcium channels, and generate these slow waves.
00:11:3103 The interstitial cells of Cajal are in gap junctions, electrically coupled, with smooth muscles.
00:11:3815 So, in the gut, there's actually a whole network of interstitial cells of Cajal
00:11:4514 electrically coupled to one another and also to smooth muscles.
00:11:5017 And the propagation of these slow waves controls the rhythmic movement of the stomach and the intestines.
00:12:0002 So, we see in the wildtype control the isolated stomach still goes through rhythmic contraction.
00:12:0828 But in the mutant mice, without TMEM16A, the mus. the stomach is not doing that.
00:12:1815 There's no rhythmic movement.
00:12:2125 So, in the interstitial cells of Cajal, TMEM16A is responsible or required for the formation
00:12:3220 of pacemaker activity, these slow waves that control the rhythmic movement of the GI tract.
00:12:4120 In the epithelial cells, at TMEM16A and CFTR, again, are on the luminal side,
00:12:5100 the apical side of the epithelial cells.
00:12:5415 And so having these two different chloride channels on the same side, the luminal side
00:13:0016 of epithelial cells in the intestine, and also in the airway,
00:13:0523 has raised the prospect that perhaps activating calcium-activated chloride channels may be one way to
00:13:1514 reduce or ameliorate some of the symptoms of patients with cystic fibrosis.
00:13:2418 As I mentioned, TMEM16A is very broadly expressed in different epithelial tissues.
00:13:3115 And in these epithelial cells, the channel protein is on the cell membrane and also
00:13:3720 on the surface of cilia, microvilli included.
00:13:4307 And to ask what these channels might be doing, or what functions these channels might have
00:13:4924 in epithelia, Mu He expressed a chloride sensor, a fluorescent protein, in the epithelial cells,
00:13:5724 and found that the fluorescence will change with the external chloride concentration.
00:14:0504 Reducing the chloride concentration will cause a rise in fluorescence.
00:14:0924 And restoring the higher chloride concentration will cause a fall, a drop in the fluorescence intensity.
00:14:1806 And so the fluor. the fluorescence intensity is inversely proportional to
00:14:2411 the chloride concentration in the cytosol.
00:14:2806 And in the mutant cells without 16A, the pink ones, or the control cells treated with
00:14:3814 a blocker of this channel, there's a reduction in the fluorescence intensity.
00:14:4417 So, we see that the channel in these cells controls chloride homeostasis.
00:14:5104 So, without the channel activity, the cytoplasmic chloride concentration is higher.
00:15:0008 And to look at the consequence of varying the chloride concentration,
00:15:0616 one thing Mu He noticed is that recycling endosome trafficking depends on the chloride concentration.
00:15:1515 So, reducing the chloride concentration will increase the appearance of E-cadherin
00:15:2217 in the recycling endosome.
00:15:2524 And the recycling of E-cadherin is a process that happens all the time.
00:15:3126 That allows the cells to rearrange the adherens junctions formed by E-cadherin.
00:15:3927 And this is particularly important when the cells are adjusting their arrangement
00:15:4621 with the neighbors, as in the case of embryogenesis, during development.
00:15:5125 So, in early stages, in these panels, we see that the epithelial cells are still at a stage
00:16:0200 of active proliferation.
00:16:0420 And they pack against each other, mainly as pentagons, with five edges.
00:16:1122 And later in development, then these epithelia stabilized and packed as hexagons, in a honeycomb form.
00:16:2318 And in the mutant mice without TMEM16A, this transition from. to the stable form of epithelia
00:16:3423 is deficient.
00:16:3615 We don't see this transition to hexagons.
00:16:3922 This most likely is the result of the alteration in the recycling of E-cadherin that's required
00:16:4905 for the repacking of epithelial cells.
00:16:5311 And the other effect or control mediated by chloride concentration in the cytoplasm is
00:17:0213 the trafficking of recycling endosomes to the pericentriolar region.
00:17:0828 And the recycling endosomes in this region are actually the membrane supply,
00:17:1511 the source of membrane for ciliogenesis, for the formation of primary cilia.
00:17:2115 And this explains why in the mutants, in multiple tissues, we see much shorter primary cilia.
00:17:3424 And now that we have gone through some of the physiological functions,
00:17:3909 I will switch gears and talk about how these channels work.
00:17:4416 In our recent study in collaboration with my UCSF colleague, Yifan Cheng, we have seen
00:17:5200 the channel. in the structure, with cryo-EM analysis.
00:17:5807 We see that the protein forms a dimer.
00:18:0202 And there are actually very well organized lipids, marked in red, at the interface.
00:18:0927 And we see two calcium ions in each monomer.
00:18:1411 And they are fairly close to where the pore is.
00:18:1802 So, the two calcium ions are coordinated by five acidic residues plus an asparagine.
00:18:2825 And right next to the calcium binding site is the pore.
00:18:3320 That's formed by six of the ten transmembrane segments.
00:18:3927 And three of the six are the transmembrane segments that include the calcium binding sites,
00:18:4606 the acidic residues and asparagine.
00:18:5109 When we mutated residues lining the pore, we found a cluster of residues near
00:19:0024 the constriction of the pore that play a role in gating of the channel, and then, also, pore-lining residues
00:19:0815 all along the pore that are important for anion permeation.
00:19:1320 So, replacement of any one of these pore-lining residues, all ten of them, with alanine,
00:19:2301 one at a time, we see that the permeability to iodide versus the permeability to chloride is altered,
00:19:3020 indicating that these residues along the pore in interact with anions in the pore to control their permeation.
00:19:4305 And the cluster of residues that are near the constriction site appear to influence
00:19:5211 the stability of the protein in the open state versus the closed state of the channel.
00:19:5808 So that alanine mutations of these residues will altered the apparent calcium sensitivity
00:20:0525 of the channel for activation.
00:20:1122 A hallmark feature that has been known ever since the '80s is indicated by the blue triangles
00:20:2126 and the red diamonds in the current-voltage relationship.
00:20:2719 And that is when the calcium concentration in the cytosol is low, the channel shows
00:20:3412 very strong voltage dependence.
00:20:3701 But when the calcium concentration is much higher, there is a linear current-voltage relationship.
00:20:4313 There's very little voltage dependence.
00:20:4526 Our recent study, reported this year in Nature. in Neuron, gives further insight to the way
00:20:5520 the channel works.
00:20:5802 We see that most likely the channel has actually two different open states.
00:21:0408 When the channel, or each monomer, has one calcium bound, it's highly voltage-dependent,
00:21:1313 so the channel is closed unless there is depolarization.
00:21:1928 And so when the membrane is depolarized to a more positive value, we see an instantaneous current.
00:21:2705 That's reflecting this open state.
00:21:3122 And physiologically, the significance of this single. singly-occupied channel is that
00:21:4400 these channels will not really affect the resting membrane potential, but they will
00:21:4925 modulate the excitatory synaptic potential and also the action potential.
00:21:5520 Because, during those synaptic potentials or action potentials, there will be depolarization.
00:22:0311 Now, if we look at the green curve and the blue curve, we see that just having
00:22:1022 different anions going through the pore, the channel activity is different.
00:22:1722 And so the iodide will have a greater effect in potentiating the channel activity compared
00:22:2508 to chloride.
00:22:2713 And so this is one form of positive feedback.
00:22:3104 Once the channel is opened and the anions are going through the pore,
00:22:3517 it will actually potentiate the channel activity.
00:22:4013 And then we see in this voltage clamp experiment with prolonged depolarization,
00:22:4800 there's a gradual rise in the channel activity.
00:22:5119 And that reflects the occupation of the second calcium binding site.
00:22:5703 And when the channel has both calcium binding sites occupied, it transitions into a different
00:23:0414 open conformation that shows no voltage dependence.
00:23:0804 And this increased activity is also physiologically important.
00:23:1402 So, we see in recent studies, in this case recording of neurons from the inferior olive,
00:23:2322 removing the calcium-activated chloride channel formed by TMEM16B will alter the action potential waveform,
00:23:3122 the duration, and also the afterhyperpolarization.
00:23:3620 And in this other example, it's recording from thalamocortical neurons,
00:23:4403 it makes the point that with prolonged depolarization and a whole series of action potentials being generated,
00:23:5314 this prolonged depolarization and calcium entry during the action potential
00:24:0006 will lead to a progressively larger fraction, or a larger number, of the calcium-activated chloride channels
00:24:1104 getting both calcium binding sites occupied and entering into a more active state.
00:24:2126 And that will lead to a progressive decrease in the firing rate.
00:24:2716 And this is the phenomenon known as spike frequency adaptation.
00:24:3621 This makes the point that in mammals the family of TMEM16
00:24:4426 -- TMEM stands for transmembrane protein with unknown function --
00:24:5101 we know that 16A and 16B form calcium-activated chloride channels.
00:24:5608 It was quite surprising to see that the functions of other family members are really very diverse.
00:25:0406 They are not all calcium-activated chloride channels.
00:25:0921 When we just go down the list, we found that TMEM16C behaves as an auxiliary subunit of
00:25:1715 a potassium channel, a sodium-activated potassium channel.
00:25:2205 So, having. the channel has both the alpha subunit and the beta subunit, TMEM16C,
00:25:3019 and will have greater sodium sensitivity and also greater stability.
00:25:3502 So, in the sensory neurons of the dorsal root ganglia, in the wild type there are
00:25:4309 many more of these channels and greater sodium-activated potassium currents then in the TMEM.
00:25:5010 in the animals without TMEM16C.
00:25:5513 And the end result is knocking out TMEM16C will increase the excitability of these sensory neurons
00:26:0419 and also increase the pain sensitivity of the animal.
00:26:1203 And another example is TMEM16F.
00:26:1424 That turns out to be associated, linked, to a human disease that's a bleeding disorder
00:26:2225 known as Scott syndrome.
00:26:2516 And the function of TMEM16F is required for calcium-activated lipid scramblase activity
00:26:3502 in platelet cells and other cell types.
00:26:3904 And the scrambling of lipids in the lipid bilayer allows the lipids marked in red,
00:26:4619 the phosphatidyl serine, to be exposed to the cell surface.
00:26:5112 And that serves as a landing pad for the tissue factors.
00:26:5611 And that eventually leads to the production of thrombin and blood coagulation.
00:27:0515 And for the other members, likely the functions are going to be intriguing but quite different.
00:27:1208 So, those are all still open questions.
00:27:1512 So, for this study of the TMEM16 family, Bjorn Schroeder used those axolotl oocytes for
00:27:2600 expression cloning of the channel.
00:27:2920 And so TMEM16A and B are the calcium-activated chloride channels.
00:27:3515 Fen Huang did the study of TMEM16C that turned out to be an auxiliary subunit of a potassium channel.
00:27:4516 Andrew Kim and Huanghe Yang did the initial study from our lab on TMEM16F that's linked
00:27:5328 to the bleeding disorder.
00:27:5514 Jason Tien, John Gilchrist, Mu He, Shengjie Feng, and Chris Peters have done
00:28:0409 the more recent biophysical and physiological studies, including the cryo EM study in collaboration
00:28:1207 with Yifan Cheng.
00:28:1404 And several other UCSF colleagues, including Dan Minor, Charly Craik, and Michael Grabe.
00:28:2319 The pain study was done together with Allan Basbaum.
00:28:2811 And the bleeding disorder. you know, the blood coagulation study was done in collaboration
00:28:3614 with Shawn Coughlin.
00:28:3821 And all of this is a long-term collaboration with Yuh-Nung Jan.
00:28:4227 And the study was supported by Howard Hughes Medical Institute, NIH,
00:28:4927 and a number of postdoctoral fellowships.
00:28:5300 Thank you.

  • Part 1: Introduction to Ion Channels: A Close Look at the Role and Function of Potassium Channels

Receptor subunits and components

To date, seventeen nAChR subunits and five 5-HT3R subunits have been identified. The nAChR subunits include multiple α (α1−α10) and β subunits (β1−β4) as well as δ, γ, and ɛ subunits, and the 5-HT3R subunits include A, B, C, D, and E subtypes 17 . These subunits have been highly conserved through evolution and each single subunit has more than 80% amino acid identity across vertebrate species. The nAChR subunits can be divided into four subfamilies (I–IV) based on similarities in protein sequence, and the classification of 5-HT3R subunits is relatively simple 18 (Figure 3).

Subunits of the nAChRs and 5-HT3Rs.

The diversity in subunit composition may influence the characteristics of nAChRs and 5-HT3Rs, including their agonist sensitivity, channel kinetics, Ca 2+ permeability, assembly, interactions with chaperones, trafficking and cell localization 19,20,21,22,23 . Muscle-type nAChRs are composed of α1, β1, γ, and δ subunits in a 2:1:1:1 ratio or composed of α1, β1, δ and ɛ subunits in a 2:1:1:1 ratio. Neuronal-type receptors are homomeric or heteromeric combinations of twelve different nicotinic receptor subunits, α2−α10 and β2−β4, such as (α4)3(β2)2, (α4)2(β2)3, or (α7)5 24 . A functional 5-HT3 receptor may be composed of five identical 5-HT3A subunits (homopentameric) or a mixture of 5-HT3A and one of the other four 5-HT3B, 5-HT3C, 5-HT3D, and 5-HT3E subunits (heteropentameric) 25 .

The homomeric nAChR and 5-HT3Rs have five identical ligand binding sites located at the interface between two adjacent subunits 26 . Each heteromeric nAChR contains two agonist binding sites with different affinities. Although the subunit stoichiometry of the heteromeric 5-HT3Rs is not clearly studied, it was demonstrated that agonists bind to an interface between two adjacent 5-HT3A subunits in the heteromeric 5-HT3AB receptor 27 , which may explain why the 5-HT3A subunit is essential to form functional 5-HT3 receptors. Because the binding sites cooperate, all sites need to be occupied with agonist to fully activate the ion channel. Elucidation of the influence of subunit composition on ligand binding and channel function will be an important topic of future research on these two receptors.

Plant Physiology and Development

Ion Channels

Ion channels are integral membrane proteins. They span the phospholipid bilayer to form an aqueous pore through which ions cross the membrane. They can be distinguished from uniport carriers by their turnover rate. When a single ion channel is open, it can transport up to 10 million ions per second. This is several orders of magnitude greater than the turnover rate of a carrier protein. Ion channels are selective for the ions they pass, this selectivity being generated by a structure termed the selectivity filter situated at the mouth of the pore. In addition to the pore-forming subunit, some ion channels also have beta (regulatory) subunits. The activity of ion channels is regulated by gating mechanisms. These are generally controlled by changes in membrane potential, the binding of ligands, covalent modification or block by cytoplasmic solutes. Ion channels serve four basic functions: (1) to transport nutrients across membranes, (2) to accommodate osmotically significant fluxes over short periods, for example, during the closing of stomata or the movements of touch-sensitive plants, (3) to propagate signals along or across membranes, for example, the propagation of electrical signals via action potentials or of chemical signals (such as elicitors) via ligand-gated channels, (4) to maintain an optimal membrane potential or constant electrochemical gradient, which is important, for example, in controlling nutrient uptake by roots.

Voltage Gated

The majority of ion channels fall into two broad categories: voltage-gated ion channels (VGIC) and ligand-gated ion channels (LGIC). Members of the VGIC superfamily are usually closed at the resting potential of the cell. A change in the membrane potential causes conformational changes that result in the opening of the pore (voltage-dependent activation), which may be followed by a transitional conformational change (inactivation) to an inactivated state. 1 The VGIC superfamily includes calcium channels, chloride channels, potassium channels and sodium channels. Other, smaller categories exist, such as the vanilloid (TRP) receptors, the ATP-gated channels, the cyclic nucleotide-gated channels (CNG) and aquaporins (water channels).

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Aquaporin (Water)
Antibodies to Water Channels

Antibodies to Calcium Channels
Calcium Channel Modulators

Antibodies to Chloride Channels
Chloride Channel Modulators

Antibodies to Potassium Channels
Potassium Channel Modulators

Antibodies to Sodium Channels
Sodium Channel Modulators

Other Ion Channels
Antibodies to Other Ion Channels

Ion Exchangers and Co-Transporters
Ion Probes
Antibodies to Ion Pumps
Ion Pump Inhibitors

Meaning and definition of chemically-gated ion channels :

Specialized ion channels that open or close in response to a chemical stimulus.

For the term chemically-gated ion channels may also exist other definitions and meanings, the meaning and definition indicated above are indicative not be used for medical and legal or special purposes.

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Chemically-Gated Ion Channels

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Voltage-gated ion channels

Voltage-gated ion channels (VGICs) are responsive to changes in the local electrical membrane potential, and are critical for the function of excitable cells, such as neurons and muscle cells. VGICs are ion-selective, with separate channels identified for each of the major physiological ions- Na + , K + , Ca 2+ , Cl - . Each type of channel is a multimeric complex of subunits encoded by a number of genes. Subunit combinations vary in different tissues, with each combination having distinctive voltage dependence and cellular localization. Some VGICs are highly localized, such as the CatSper Ca channels, whose expression is restricted only to the principal piece of the sperm tail.

There are many drugs whose mechanism of action involves perturbation of VGIC activity. Some of the main classes of drugs are discussed below:

Calcium channel blockers (CCBs) -see also the topic 'Calcium channel blocking drugs' in the Cardiovascular system section of the Drugs module

Calcium-channel blockers are smooth-muscle relaxors having a negative inotropic effect on the working myocardial cells of the atria and ventricles, with inhibition of Ca 2+ entry blunting the ability of Ca 2+ to serve as an intracellular messenger.

The dihydropyridine class of CCB drugs block activity of L-type calcium channels. Examples of this drug class are amlodipine, felodipine, isradipine, lacidipine, nicardipine, and nimodipine which are used in the treatment of hypertension. In comparison to phenylalkylamine class CCBs such as verapamil, the dihydropyridines are relatively vascular selective in their mechanism of action in lowering blood pressure.

Sodium channel blockers

Class III antiarrhythmics are primarily sodium channel blocking agents, and include the prescription medicines dronedarone and amiodarone hydrochloride.

Many local anaesthetic agents are also sodium channel blockers, and include lidocaine, bupivacaine, prilocaine, mepivacaine, tetracaine and ropivacaine. Mechanistically these drugs bind to an intracellular portion of voltage-gated sodium channels blocking sodium influx into nerve cells, which prevents depolarization. Without depolarization, no initiation or conduction of a pain signal can occur.

Some anticonvulsants (antiepileptic drugs or AEDs) work at least in part, by blocking sodium channels. By inhibiting sodium (and/or calcium) channel activity, AEDs act to reduce the release of excitatory glutamate which is elevated in epilepsy and may also reduce γ-aminobutyric acid (GABA) secretion.

Whilst not strictly ion channel inhibitors, proton-pump inhibitors also block ion transport across the membrane. In this case by irreversibly blocking the H + /K + ATPase transporter activity of the proton pump on the surface of gastric parietal cells. This action produces a pronounced and long-lasting reduction of gastric acid production. PPIs are the most potent inhibitors of acid secretion available, and have largely superseded histamine H2 receptor antagonists which have similar effects, but a different mode of action. Prescription PPIs include omeprazole, esomeprazole, pantoprazole, lansoprazole, and rabeprazole.

The Transient Receptor Potential (TRP) superfamily of channels are found in sensory receptor cells that are involved in heat sensation, taste, smell, touch, and osmotic and volume regulation.

8.2: Ligand-gated Ion Channel Receptors

  • Contributed by Kevin Ahern & Indira Rajagopal
  • Professor (Biochemistry and Biophysics) at Oregon State University

This type of swift response is seen, for example, in neuromuscular junctions, where muscle cells respond to a message from the neighboring nerve cell. The nerve cell releases a neurotransmitter signal into the synaptic cleft, which is the space between the nerve cell and the muscle cell it is "talking to". Examples of neurotransmitter signal molecules are acetylcholine and serotonin, shown in Figure 8.2.2.

Figure 8.2.2: Neurotransmitter

When acetylcholine molecules are released into the synaptic cleft (the space between the pre- and post-synaptic cells) they diffuse rapidly till they reach their receptors on the membrane of the muscle cell. The binding of the acetylcholine to its receptor, an ion channel on the membrane of the muscle cell, causes the gate in the ion channel to open. The resulting ion flow through the channel can immediately change the membrane potential. This, in turn, can trigger other changes in the cell. The speed with which changes are brought about in neurotransmitter signaling is evident when you think about how quickly you remove your hand from a hot surface. Sensory neurons carry information to the brain from your hand on the hot surface and motor neurons signal to your muscles to move the hand, in less time than it took you to read this sentence!

Figure 8.2.3: Signaling across nerve cells

Phases of action potential & role of gated ion channels

1. All cells have a membrane potential however, only certain kinds of cells, including neurons and muscle cells, have the ability to generate changes in their membrane potentials. Collectively these cells are called excitable cells. The membrane potential of an excitable cell in a resting (unexcited) state is called the resting potential, and a change in the resting potential may result in an active electrical impulse.

2 Neurons have special ion channels, called the gated ion channels, that

ahoy the cell to change its membrane potential in response to stimuli the cell receives. If the stimulus opens a potassium channel, an increase in efflux of potassium will occur, and the membrane potential will become more negative. Such an increase in the electrical gradient across the membrane is called a hyperpolarization. If the channel opened by the stimulus is a sodium channel, an increased influx of sodium will occur, and the membrane potential will become .less negative. Such a reduction in the electrical gradient is called a depolarization. Voltage changes produced by stimulation of this type are called graded potentials because the magnitude of change (either hyperpolarization or depolarization) depends on the strength of the stimulus: A larger stimuls will open more channels and will produce a larger change in permeability.

  1. In an excitable cell, such as a neuron, the response to a depolarizing
    stimulus is graded with stimulus intensity only up to, a particular level of depolarization, called the threshold potential. If a depolarization reaches the threshold, a different type of response, called an action potential, will be triggered.
  2. The action potential is the nerve impulse. It is a nongraded all-or-none event, meaning that the magnitude of the action potential is independent of the strength of the depolarizing stimulus that produced it, provided the depolarization is sufficiently large to reach threshold. Once an action potential is triggered, the membrane potential goes through a stereotypical sequence of changes.
  3. During the depolarizing phase, the membrane polarity briefly reverses, with the interior of the cell becoming positive with respect to the outside. This is followed rapidly by a steep repolarizing phase, during which the membrane potential returns to its resting level. Fig. 2.5.
  4. There may also be a phase, called the undershoot, during which the membrane potential is more negative than the normal resting potential. The whole event is typically over within a few milliseconds.

Role of gated ion channelgein the action potential:

The action potential arises because the plasma membranes of excitable cells have special voltage-gated channels. These ion channels have gates that open and close in response to changes in membrane potential. Fig. 2.4, 2.5

Two types of voltage-gated channels contribute to the action potential: potassium channels and sodium channels.

Each potassium channel has , a single gate that is voltage-sensitive it is closed when resting and opens slowly in response to depolarization.

By contrast, each sodium channel has two voltage-sensitive gates

(i) an ‘activation gate, that is closed when resting and responds to depolarization by opening rapidly, and

(ii) an inactivation gate, that is open when resting and responds to depolarization by closing slowly.

In the membrane’s resting state, the inactivation gate is open but the activation gate is closed, so the channel does not allow Na + to enter the neuron. Upon

depolarization the activation gate opens quickly, causing an influx of Na, which depolarizes the membrane further, opening more voltage-gated sodium channels and causing still more depolarization. This inherently explosive process. example of positive feed back, continues until all the sodium channels at the stimulated site of the membrane are open.

Two factors underlie the rapid repolarizing phase of the action potential as membrane potential is returned to rest. First, the sodium channel inactivation gate, which is slow to respond to changes in voltage, has time to respond to depolarization by closing, returning sodium permeability to its low resting level. Second, potassium channels whose voltage-sensitive gates respond relatively slowly to depolarization, have had time to open. As a result, during repolarization, K + flows rapidly out of the cell, helping restore the internal negativity of the resting neuron. The potassium channel gates are also the main cause of the undershoot, or hyperpolarization, which follows the repolarizing phase. Instead of returning immediately to their resting position, these relatively slow-moving gates remain open during the undershoot, allowing potassium to keep flowing out of the neuron. The continued potassium outflow makes the membrane potential more negative. During the undershoot, both the activation gate and the inactivation gate of the sodium channel are closed. If a second depolarizing stimulus arrives during this period, it will be unable to trigger an action potential because the inacthiation gates have not had time to reopen after the preceding action potential. This period when the neuron is insensitive to depolarization is called the refractory period, and it sets the limit on the maximum rates at which action potentials can be generated. Fig. 2.6

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