Introduction to Ion Channels

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

00:00:07;27 Hi, I'm Lily Jan.
00:00:10;14 And I'll be telling you about our studies of ion channels.
00:00:14;24 And that's been a long-term collaboration with Yuh-Nung Jan.
00:00:19;15 This is the first of a two-part talk on ion channel studies.
00:00:25;28 So for this part, I will start by giving some background on the ion channels and the way
00:00:31;22 we went about identifying the channel molecule.
00:00:35;20 And then I will tell you more about how we have learned about these ion channels.
00:00:42;05 For a neuron to receive signals that impinge on its dendrites, and then to generate
00:00:49;12 an action potential that propagates along the axon to the nerve terminals to trigger transmitter release,
00:00:56;16 it relies on the function of a number of different channels that are distributed
00:01:03;14 in different compartments of the cell, that then drives the axon initial segment,
00:01:10;15 the nodes of Ranvier, and then the nerve terminals.
00:01:16;00 Molecular identification of a channel of interest makes it possible to generate reagents
00:01:22;08 such as antibodies.
00:01:24;11 And it also makes it possible to generate mutants with altered channel functions
00:01:29;19 so that we can study the physiological contribution of this channel.
00:01:36;17 Knowing the molecule that forms the channel also makes it possible to express the channel
00:01:42;10 in Xenopus oocytes or other expression systems, so we can study these channels one at a time
00:01:49;23 and see how it works.
00:01:52;18 For the first attempt for molecular identification, the channel of interest was
00:01:58;20 voltage-gated potassium channels.
00:02:02;04 These channels control the waveform and the propagation of action potentials.
00:02:09;21 Action potentials were recorded nearly 70 years ago, for the first time,
00:02:15;20 from the squid giant axon.
00:02:20;04 Unlike mammalian axons that can have myelination to speed up the propagation of action potentials,
00:02:28;11 squid axons have no myelination.
00:02:31;24 And to speed up the propagation, some of the axons are very large,
00:02:37;09 nearly one millimeter in diameter.
00:02:39;23 In the 1950s, Hodgkin and Huxley recorded from these squid giant axons to provide
00:02:47;20 the insight that action potential generation relies on the voltage-gated ion channels,
00:02:55;15 as we can see from this schematic.
00:02:58;04 The resting potential of a cell or the axon is negative on the inside, typically about
00:03:07;24 -60 millivolts.
00:03:09;26 And that is because the resting cell is predominantly permeable to potassium ions.
00:03:17;15 And in this diagram, we have the blue channel to represent the potassium channel
00:03:23;06 that's active at rest.
00:03:25;22 And when the signal comes from presynaptic cells to trigger transmitter release,
00:03:32;25 this will activate the postsynaptic cell to generate an action potential.
00:03:38;24 And the first thing that happens is the depolarization will open sodium channels, shown here in red.
00:03:48;11 And the permeation of sodium ions will drive the membrane potential towards
00:03:53;19 a positive value, the sodium equilibrium potential.
00:03:59;15 And then the subsequent activation of, or opening of, voltage-gated potassium channels
00:04:06;17 -- that's shown in green in this diagram -- will drive the membrane potential back down
00:04:11;22 towards the potassium equilibrium potential.
00:04:16;25 Because the cell typically, actually, has many different kinds of potassium channels,
00:04:22;07 the heterogeneity and low abundance of these potassium channels has made it very difficult
00:04:29;09 to purify these potassium channels for the initial molecular studies.
00:04:35;14 And for this reason, we took the approach of positional cloning, that in the...
00:04:42;04 back in the 1980s, was possible in the fruit fly because of the polytene chromosomes.
00:04:49;12 These chromosomes in the larval salivary gland have very characteristic banding patterns.
00:04:56;03 So, if a gene can be mapped onto this chromosome, for example, by studying mutations that are
00:05:05;05 caused by chromosome translocations, and by analyzing the breakpoints on a chromosome squash
00:05:13;23 as shown here, one could in principle start with a piece of DNA that's near
00:05:22;17 the gene of interest, and by just isolating overlapping genomic DNA fragments eventually reach
00:05:32;27 the gene of interest.
00:05:34;05 So, that form of positional cloning has been referred to as a chromosome walk.
00:05:42;05 Knowing that Shaker mutations cause a symptom, or the phenotype, that is likely due to
00:05:50;10 a deficit in potassium channels, Diane Papazian, Tom Schwarz, and Bruce Tempel used the chromosome walk
00:06:00;20 as the approach to clone Shaker.
00:06:04;02 It took six years for the cloning and validation of Shaker as a potassium channel gene.
00:06:12;06 But then it took just a few months, using the Shaker DNA as the probe, to isolate
00:06:21;19 the orthologue in mammals.
00:06:23;18 That's known as Kv1.1.
00:06:26;15 The high evolutionary conservation of these potassium channels also allowed the identification
00:06:34;28 of the voltage-gated potassium channel in the squid axons.
00:06:39;24 It is the orthologue of Shaker.
00:06:42;11 And two different splice forms are expressed, one in the giant axon and the other one
00:06:50;05 in the small axons that are, you know, surrounding the giant axon.
00:06:56;02 The function of these voltage-gated potassium channels is evolutionarily conserved.
00:07:03;16 And we can see that from the phenotype.
00:07:05;28 So, starting with two control, wild type flies, put to sleep with ether anesthesia,
00:07:14;24 we see that the flies only begin to move their legs when they are about to wake up.
00:07:20;11 And that's very different from the Shaker mutants that shake their legs and wings
00:07:25;24 very vigorously under ether anesthesia.
00:07:31;17 This video was provided by John Adelman, my colleague at Vollum Institute in Oregon.
00:07:39;22 John also made video of a patient with episodic type... ataxia type 1.
00:07:48;05 And we see that moderate levels of physical activity will precipitate
00:07:56;07 the uncontrollable movement of the limbs of this patient.
00:07:59;00 The disease is caused by mutation of Kv1.1, or KCNA1, the human gene.
00:08:10;24 And the movement disorder can be recapitulated in mouse models as well by mutating Kv1.1.
00:08:21;07 The basis of this movement disorder is the ability of Kv1 to provide a brake to control
00:08:31;12 the propagation of action potentials.
00:08:35;15 By removing Kv1.1 with mutations, there are... reverberant action potentials in the axon.
00:08:44;06 And that results in hyperexcitability.
00:08:48;02 In the mouse model, we can see that stimulation of the motor axon just once will generate
00:08:56;18 reverberant action potentials that can be recorded with an extracellular electrode placed
00:09:03;23 near the muscle.
00:09:06;21 In the fruit fly larvae, we see the same thing, that the reverberant action potentials
00:09:13;12 can be recorded from the motor axon with extracellular recording.
00:09:18;16 And that can be seen either in the mutant fly, with mutation in the Shaker gene,
00:09:24;15 or in the wild type fly with the... in preparation treated with potassium channel blockers.
00:09:32;22 And so, we see that the physiological function of these potassium channels is
00:09:39;26 evolutionarily conserved in both vertebrates and invertebrates.
00:09:45;19 The high level of sequence similarity of different voltage-gated potassium channels made it possible
00:09:53;14 for our colleagues in the field to isolate a large number of the family members.
00:09:59;23 And some of the members are linked to diseases of the brain, like epilepsy or episodic ataxia.
00:10:08;17 And other members are linked to diseases of the heart, like arrhythmia or atrial fibrillation.
00:10:18;04 And still other types of voltage-gated potassium channels, when mutated, will cause deafness.
00:10:27;08 At the time, we already knew there are other types of potassium channels that are important
00:10:32;15 for controlling heart rate or release of insulin.
00:10:37;20 But it was not possible to use voltage-gated potassium channels as probes to isolate those channels.
00:10:44;19 So, it was necessary to try a different approach: expression cloning.
00:10:51;05 And so, in this approach, if the channel, or for that matter other transport
00:10:58;12 proteins of interest, can be identified in oocytes... so, if we start with a rich source and just
00:11:08;17 inject pools of RNA from this rich source into Xenopus oocytes, recording from the oocytes
00:11:19;13 can reveal the function of these channels.
00:11:23;11 We could keep on subdividing the cDNA library until we end up with single clones
00:11:30;06 to find that one clone that would generate the channel activity.
00:11:33;22 So, that was the approach used by Yoshihiro Kubo for expression cloning of Kir2.1 or IRK1.
00:11:43;16 That's one of the founding members for the Kir, or inward-rectifying potassium channel family.
00:11:52;20 And we see that the two different types of voltage... you know, the voltage-gated potassium channels
00:11:58;28 and the inward-rectifying potassium channels share a common pore design that includes
00:12:06;24 two transmembrane segments.
00:12:09;09 And what's different for the voltage-gated potassium channels is that these channels
00:12:15;16 have an additional voltage sensor domain with four transmembrane segments,
00:12:21;02 and multiple positively charged residues on the fourth transmembrane segment.
00:12:30;19 For the inward-rectifying potassium channel, once the different family members had been characterized,
00:12:37;04 we see that this family does include the channel that's important for controlling
00:12:42;26 insulin release.
00:12:44;08 So, a loss-of-function mutation will cause hypoglycemia of infancy.
00:12:51;17 And a gain-of-function mutation will cause neonatal diabetes.
00:12:56;06 This has been explained in detail by my colleague, Frances Ashcroft, in her biology talk.
00:13:05;22 Other members of this family can cause... when mutated, will cause periodic paralysis,
00:13:13;08 that's a disease of the skeletal muscle; hypertension, a disease of the kidney;
00:13:20;22 or arrhythmia, a disease of the heart.
00:13:27;06 And we see also, by now, that, besides the animal kingdom, potassium channels are present
00:13:35;26 in the plants, in bacteria, and also in viruses.
00:13:42;16 In the plants, depending on the cell types that express a potassium channel,
00:13:49;05 there are different functions that have been found to depend on potassium channel activity.
00:13:55;13 For example, in this stomatal pore, the potassium channel activities allow the pore
00:14:03;12 to open or close.
00:14:05;28 And that's by controlling the volume of the cell.
00:14:10;15 And back to the animal kingdom... well, before we get to the animal kingdom,
00:14:16;21 in bacteria potassium transporters and ion channels are important for maintaining the turgor pressure.
00:14:26;28 And from the structure of this potassium channel from bacteria, from E. coli, we can see that
00:14:35;15 the channel has a very similar design, but it's formed by one large polypeptide that
00:14:42;10 includes four repeats, each one looking like the 2TM, the inward-rectifier potassium channel,
00:14:50;01 the pore domain.
00:14:51;05 So now, in mammals we see two... three different designs.
00:14:56;17 The 2TM in the middle is the simplest design.
00:15:00;28 It's the channel subunit that has just the pore domain.
00:15:04;21 On the right, or closer to me, is what's called K2P channels.
00:15:14;19 This has two of the 2TM motif linked together.
00:15:19;00 And so... for the 2TM arrangement, there are four subunits coming together to form the channel.
00:15:29;04 And for the K2P, with four transmembrane segments, it takes two subunits to form the channel.
00:15:36;16 And the voltage-gated potassium channels that contain both the pore domain and the
00:15:41;19 voltage sensor domain also are tetramers.
00:15:47;03 And we see, for each of these three different types of potassium channels, in each family
00:15:55;17 there are multiple subfamilies.
00:15:59;03 And for each subfamily, there are several different channel proteins... proteins with
00:16:05;25 very similar sequences.
00:16:08;15 And the channel can be formed as homotetramers, with four identical subunits, or heterotetramers,
00:16:17;07 with similar but not identical subunits.
00:16:21;00 And this mix and match of subunits greatly increases the diversity of the channels that
00:16:26;26 are found in vivo.
00:16:29;12 So, take the voltage-gated potassium channels.
00:16:34;04 The pore is formed by the four pore domains of the four subunits.
00:16:41;04 There's a domain swap.
00:16:43;08 So, the voltage sensor of one subunit packs against the pore domain of its neighboring subunit.
00:16:52;15 And with the positive charges in the voltage sensor, it would detect a membrane potential change.
00:16:59;01 So, when the inside of the cell becomes more positive, the voltage sensor is moving outwards
00:17:06;11 by the... driven by the voltage.
00:17:08;25 And this triggers the opening of the channel.
00:17:11;24 And in the pore domain, in between the two transmembrane helices there's a pore loop
00:17:18;04 that comes close together in the tetrameric channel to form the selectivity filter.
00:17:25;13 This allows the channel to be highly selective, to allow potassium ions but not other ions
00:17:31;12 to go through.
00:17:34;15 And this... a very similar structural design of potassium channels with slight differences
00:17:43;21 in channel properties are found in different compartments of a neuron:
00:17:49;05 in the dendrites, the soma, and the axons.
00:17:53;09 And one striking example is at the nodes of Ranvier, where we see, at the node
00:18:00;14 that's marked in green in the micrograph, there are voltage-gated sodium channels and a subset
00:18:07;15 of voltage-gated potassium channels.
00:18:10;07 But on either side, at the juxtaparanodal region that's marked in red in the micrograph,
00:18:18;07 there's another set of voltage-gated potassium channels.
00:18:23;24 The different types of potassium channels are... that's marked in red, here,
00:18:33;05 are structurally related to channels like the cyclic nucleotide-gated channel, here,
00:18:40;06 and the TRP channel, here.
00:18:43;21 These have a similar design to the voltage-gated potassium channels.
00:18:47;18 They are not very dependent on voltage.
00:18:50;17 They are not selective for potassium.
00:18:52;15 They allow positively charged ions to go through.
00:18:56;18 And they quite often are found in sensory neurons to mediate sensory transduction.
00:19:03;07 And then we have the voltage-gated sodium channels and calcium channels,
00:19:08;15 that have four of the 6TM design, four pseudosubunits linked together in one large polypeptide.
00:19:18;13 And that's the alpha subunit, the pore-forming subunit for the sodium channel or calcium channel.
00:19:26;16 In prokaryotes, we can see already both types, the 2TM and the 6TM.
00:19:36;01 And we can see that with evolution there's a massive diversification of even the same design,
00:19:45;26 the 6TM, to have channels of different properties.
00:19:50;05 And then we have the 2TM strung together to form the K2P potassium channel.
00:19:58;12 And we have the 6TM strung together in two copies or four copies to form other channels.
00:20:07;20 And there's also examples of the pore domain used upside-down in an inverted version.
00:20:14;28 And that example is the glutamate receptor.
00:20:18;11 So, it's a ligand-gated ion channel.
00:20:22;04 And the ligand is the major transmitter, glutamate.
00:20:25;16 Binding of this channel to glutamate allows the channel to open.
00:20:31;08 And usually it's just positively charged ions that will go through these channels.
00:20:38;17 And we can see that the pore domain is upside down compared to the pore domain of the
00:20:44;13 voltage-gated channels.
00:20:46;05 So, we see that these ion channels serve a range of functions in sensory transduction,
00:20:53;28 in action potential generation, and in synaptic transmission.
00:20:59;16 And there are other functions, like volume regulation.
00:21:03;05 I've given you one example for channels in the plants.
00:21:07;24 And that's also true for animal cells.
00:21:11;01 And by having different channels placed in different parts of the cell, like the leading edge
00:21:16;05 and the... and the rear end of a migrating... migrating cell, it's also possible to have
00:21:23;06 local volume regulation that will allow the cell to move and not leave...
00:21:29;22 you know, be dragging its read end.
00:21:35;06 And Xi Huang, who studied medulloblastoma a few years ago, has found evidence for
00:21:44;17 a voltage-gated potassium channel, EAG2, to be involved in the control of volume regulation
00:21:53;15 that's important for proliferation and also migration of these brain cancer cells.
00:22:01;25 And after decades of study of these channels, we continue to run into surprises, of functions
00:22:10;01 that we didn't know anything about before.
00:22:13;10 This is one example.
00:22:16;23 The megencephaly mutant mice arose spontaneously in the Jackson Lab, actually, in the 1980s.
00:22:27;11 And by now we know that the mutation is in Kv1.1, to cause loss-of-function of
00:22:34;18 these voltage-gated potassium channels.
00:22:39;17 And we can also see that the brain is much larger.
00:22:43;09 That's why the name is megencephaly.
00:22:46;08 And this enlarged brain still keeps on growing into adulthood.
00:22:52;15 So, that's kind of heavy.
00:22:54;17 And so the mutant mice tend to take on a sitting posture, and holding up its tail to keep balance.
00:23:03;22 A recent study by Shi‐Bing Yang using mosaic analysis
00:23:10;16 has revealed the function of this channel in the neural progenitor cells.
00:23:15;25 So, Kv1.1 behaves as a brake to restrain, to control, the extent of adult neurogenesis.
00:23:25;25 Just how a voltage-gated potassium channel will control the division and formation of
00:23:34;20 adult neurons in the hippocampus of the adult brain is one of those open and intriguing questions
00:23:44;00 that still keeps us busy.
00:23:48;01 So, for the work I've described so far in this first part of my talk,
00:23:55;27 Diane Papazian, Tom Schwarz, and Bruce Tempel
00:24:02;02 worked on the chromosome walk, initially, to identify Shaker as the first voltage-gated potassium channel.
00:24:11;24 Yoshihiro Kubo relied on expression cloning to identify
00:24:18;04 a founding member of the inward-rectifier potassium channel.
00:24:22;10 More recently, Xi Huang did the study of medulloblastoma to identify the function of
00:24:30;22 another type of voltage-gated potassium channel in controlling proliferation and metastasis.
00:24:39;13 And Shi-Bing Yang did the mosaic analysis to find a role for Kv1.1 in the adult neural progenitors.
00:24:50;12 These studies of ion channels, spanning nearly four decades, has been a long collaboration
00:24:57;23 with Yuh-Nung Jan.
00:25:00;03 And it's supported by the Howard Hughes Medical Institute, NIH, and various postdoctoral fellowships,
00:25:09;15 from Human Frontier, Damon Runyon, and also American Diabetes Association.
00:25:16;05 Thank you.

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

00:00:07;23 Hi.
00:00:08;23 I am Lily Jan.
00:00:10;23 In this second of the two-part series on our studies of ion channels, I will tell you about
00:00:19;24 calcium-activated chloride channels.
00:00:22;25 This is part of a long-term collaboration I have had with Yuh-Nung Jan.
00:00:28;20 Calcium-activated chloride channels have only been molecularly identified in this millennium,
00:00:36;20 about a decade ago, even though these channels have been studied ever since the 1980s
00:00:43;18 and they have been associated with a number of different functions that are important.
00:00:53;17 In this talk, I will first go over the ways we went about identifying the channel molecule,
00:01:00;16 and then tell you what we have learned about the function of these channels.
00:01:07;06 For a channel of interest, where we know about the function but not the molecules that form these channels,
00:01:17;15 one general approach is to identify a rich source for this channel and
00:01:25;09 inject pools of RNA into Xenopus oocytes so that the channel activity can be detected
00:01:34;02 with recording from the oocytes.
00:01:37;05 And we can then subdivide these pools of cDNAs until we end up with a single clone for the channel.
00:01:46;13 For this approach to work, however, the Xenopus oocytes used as the expression system
00:01:55;03 cannot be expressing the channel of interest.
00:01:57;27 So, if we inject water into the Xenopus oocytes, we should see no channel activity.
00:02:07;01 This approach of expression cloning was initially pioneered by Julius and Nakanishi.
00:02:15;21 And in their early studies using this approach, they cloned a G protein-coupled receptor
00:02:24;13 that activates a signaling pathway including the activation of phospholipase C and
00:02:32;23 the release of calcium from internal stores.
00:02:36;07 And they relied on the calcium-activated chloride channels that are endogenous to the
00:02:42;17 Xenopus oocytes to report the activation of this whole signaling pathway.
00:02:51;08 And we know that these calcium-activated chloride channels in the Xenopus oocytes
00:02:57;03 serve an important function, to prevent polyspermia.
00:03:01;24 And these channels actually have been studied in the oocyte ever since the 1980s.
00:03:08;06 And for this reason, we know Xenopus oocytes cannot be used as the expression system
00:03:15;05 for expression cloning of CaCC.
00:03:18;12 And instead, Bjorn Schroeder went to the axolotl oocytes that are physiologically polyspermic.
00:03:29;02 And after finding very little endogenous CaCC expression in the axolotl ooctyes, Bjorn used
00:03:37;16 these oocytes as the expression system, and Xenopus oocytes as the source of RNA for CaCC
00:03:47;08 to clone a calcium-activated chloride channel.
00:03:51;25 And that led to the identification of Xenopus TMEM16A as a CaCC.
00:03:58;16 And he then tested the mammalian homologues and found that of... in the family with ten members,
00:04:05;09 TMEM16A and 16B formed calcium-activated chloride channels.
00:04:11;22 And around the same time, Oh's group in Korea and Galietta's group in Italy independently
00:04:21;17 came to the conclusion that TMEM16A forms calcium-activated chloride channels,
00:04:28;16 but using very different approaches.
00:04:33;06 From recent studies, we see that TMEM16A is very broadly expressed in the periphery,
00:04:41;25 including epithelial cells and smooth muscle cells.
00:04:46;04 And TMEM16B is expressed in multiple brain regions, and also in sensory neurons for
00:04:54;20 odorant perception and in photoreceptors.
00:05:00;05 In the photoreceptors, the calcium-activated chloride channels formed by TMEM16B reside
00:05:08;17 at the ribbon synapse.
00:05:10;20 They bind to the PSD95 anchor protein, and they provide negative feedback regulation.
00:05:19;10 In the odorant neurons, in the cilia where the odorant will activate G protein-coupled receptors,
00:05:26;26 leading to the opening of cyclic nucleotide-gated ion channels that permeate
00:05:33;10 both calcium and other positively charged ions like sodium, the calcium will then activate
00:05:42;03 calcium-activated chloride channels.
00:05:44;26 So, CaCC formed by TMEM16B provides the low-noise high-gain amplification of the odorant signal.
00:05:57;20 In the nervous system, we see that TMEM16A is found in sensory neurons in the dorsal root ganglia.
00:06:07;20 But TMEM16B is found in different brain regions, in central neurons.
00:06:14;06 There's a curious correlation.
00:06:16;17 The cells that express 16B tend to express potassium-chloride cotransporters,
00:06:24;27 and these cells have low chloride concentration inside.
00:06:30;04 And so chloride channels are inhibitory.
00:06:32;24 But in the cells like the dorsal root ganglia, and also in immature neurons in the brain,
00:06:41;08 the cells use a different transporter, the sodium-potassium-chloride cotransporter.
00:06:48;28 And these cells have high chloride concentration, and chloride channels are excitatory.
00:06:57;06 And this applies to many different cells in the periphery, and also cells in other organisms,
00:07:04;11 including the green algae.
00:07:06;01 We know from studies in the 1980s, calcium-activated chloride channels are present in the green algae.
00:07:15;17 And actually, these are the channels that are responsible for generating action potentials.
00:07:21;15 It's not sodium channels.
00:07:23;26 And so we can see the action potentials in this green algae are slower.
00:07:29;22 It takes seconds rather than milliseconds, as in the case of action potentials in the
00:07:35;14 nerves and muscles.
00:07:37;14 And these have been referred to as the calcium... as the chemical action potential because
00:07:44;00 it requires the calcium rise to induce the action potential.
00:07:49;17 And when the light is switched off, calcium becomes released from chloroplasts.
00:07:58;26 And so we can see, then, there is a progressive shortening in the latency for the
00:08:04;08 action potential generation.
00:08:07;11 And during the action potential, there's further rise in the calcium.
00:08:11;13 And that will result in a pause in the cytoplasmic streaming.
00:08:19;28 And these are really large cells, as you can see, in the green algae.
00:08:24;18 And the cytoplasmic streaming is one way to move, you know, the organelles and materials
00:08:31;03 around in the cell.
00:08:34;11 Now, back to the animal kingdom.
00:08:38;08 In the airway epithelia, we see two different kinds of chloride channels on the apical side,
00:08:45;16 the luminal side of the cell.
00:08:48;05 One is calcium-activated chloride channel, formed by TMEM16A.
00:08:53;14 And the other is CFTR.
00:08:56;28 And that's the channel linked to the disease cystic fibrosis.
00:09:02;03 And these chloride channels are responsible for controlling, or participate in the control,
00:09:09;19 of the thickness of the airway surface liquid, the ASL.
00:09:15;20 And that liquid, lining the luminal side of the epithelia, is very important for
00:09:23;22 mucociliary clearance of pathogens in the airway.
00:09:30;18 In the airway, these calcium-activated chloride channels formed by TMEM16A also facilitate
00:09:39;00 the release of mucin into the lumen.
00:09:42;22 And from the 1980s, we have learned from studies of different exocrine glands that
00:09:49;26 calcium-activated chloride channels are important for controlling the secretion from,
00:09:56;16 you know, salivary glands, sweat glands, and so on.
00:10:00;05 And these glands express TMEM16A.
00:10:06;28 In the smooth muscle, the calcium-activated chloride channels can be activated, for example,
00:10:15;02 with the punctal release... a bolus of calcium from internal stores.
00:10:22;01 And this would cause the nearby calcium-activated chloride channels on the cell membrane
00:10:28;18 to open, leading to what's referred to as STIC: spontaneous transient inward current.
00:10:37;00 That will cause depolarization.
00:10:39;19 And that will further lead to opening of voltage-gated calcium channels.
00:10:43;21 So, it's a positive feedback to sustain the rising calcium and smooth muscle contraction.
00:10:55;00 In the gut, you know, the gastrointestinal tract, there are cells referred to as
00:11:01;16 interstitial cells of Cajal.
00:11:04;16 And likewise, there are calcium-activated chloride channels.
00:11:08;23 And when there's a puff of calcium from internal stores, that will generate a STIC,
00:11:15;18 marked here, this little rising.
00:11:18;21 And this spontaneous transient depolarization will then lead to opening of
00:11:25;05 voltage-gated calcium channels, and generate these slow waves.
00:11:31;03 The interstitial cells of Cajal are in gap junctions, electrically coupled, with smooth muscles.
00:11:38;15 So, in the gut, there's actually a whole network of interstitial cells of Cajal
00:11:45;14 electrically coupled to one another and also to smooth muscles.
00:11:50;17 And the propagation of these slow waves controls the rhythmic movement of the stomach and the intestines.
00:12:00;02 So, we see in the wildtype control the isolated stomach still goes through rhythmic contraction.
00:12:08;28 But in the mutant mice, without TMEM16A, the mus... the stomach is not doing that.
00:12:18;15 There's no rhythmic movement.
00:12:21;25 So, in the interstitial cells of Cajal, TMEM16A is responsible or required for the formation
00:12:32;20 of pacemaker activity, these slow waves that control the rhythmic movement of the GI tract.
00:12:41;20 In the epithelial cells, at TMEM16A and CFTR, again, are on the luminal side,
00:12:51;00 the apical side of the epithelial cells.
00:12:54;15 And so having these two different chloride channels on the same side, the luminal side
00:13:00;16 of epithelial cells in the intestine, and also in the airway,
00:13:05;23 has raised the prospect that perhaps activating calcium-activated chloride channels may be one way to
00:13:15;14 reduce or ameliorate some of the symptoms of patients with cystic fibrosis.
00:13:24;18 As I mentioned, TMEM16A is very broadly expressed in different epithelial tissues.
00:13:31;15 And in these epithelial cells, the channel protein is on the cell membrane and also
00:13:37;20 on the surface of cilia, microvilli included.
00:13:43;07 And to ask what these channels might be doing, or what functions these channels might have
00:13:49;24 in epithelia, Mu He expressed a chloride sensor, a fluorescent protein, in the epithelial cells,
00:13:57;24 and found that the fluorescence will change with the external chloride concentration.
00:14:05;04 Reducing the chloride concentration will cause a rise in fluorescence.
00:14:09;24 And restoring the higher chloride concentration will cause a fall, a drop in the fluorescence intensity.
00:14:18;06 And so the fluor... the fluorescence intensity is inversely proportional to
00:14:24;11 the chloride concentration in the cytosol.
00:14:28;06 And in the mutant cells without 16A, the pink ones, or the control cells treated with
00:14:38;14 a blocker of this channel, there's a reduction in the fluorescence intensity.
00:14:44;17 So, we see that the channel in these cells controls chloride homeostasis.
00:14:51;04 So, without the channel activity, the cytoplasmic chloride concentration is higher.
00:15:00;08 And to look at the consequence of varying the chloride concentration,
00:15:06;16 one thing Mu He noticed is that recycling endosome trafficking depends on the chloride concentration.
00:15:15;15 So, reducing the chloride concentration will increase the appearance of E-cadherin
00:15:22;17 in the recycling endosome.
00:15:25;24 And the recycling of E-cadherin is a process that happens all the time.
00:15:31;26 That allows the cells to rearrange the adherens junctions formed by E-cadherin.
00:15:39;27 And this is particularly important when the cells are adjusting their arrangement
00:15:46;21 with the neighbors, as in the case of embryogenesis, during development.
00:15:51;25 So, in early stages, in these panels, we see that the epithelial cells are still at a stage
00:16:02;00 of active proliferation.
00:16:04;20 And they pack against each other, mainly as pentagons, with five edges.
00:16:11;22 And later in development, then these epithelia stabilized and packed as hexagons, in a honeycomb form.
00:16:23;18 And in the mutant mice without TMEM16A, this transition from... to the stable form of epithelia
00:16:34;23 is deficient.
00:16:36;15 We don't see this transition to hexagons.
00:16:39;22 This most likely is the result of the alteration in the recycling of E-cadherin that's required
00:16:49;05 for the repacking of epithelial cells.
00:16:53;11 And the other effect or control mediated by chloride concentration in the cytoplasm is
00:17:02;13 the trafficking of recycling endosomes to the pericentriolar region.
00:17:08;28 And the recycling endosomes in this region are actually the membrane supply,
00:17:15;11 the source of membrane for ciliogenesis, for the formation of primary cilia.
00:17:21;15 And this explains why in the mutants, in multiple tissues, we see much shorter primary cilia.
00:17:34;24 And now that we have gone through some of the physiological functions,
00:17:39;09 I will switch gears and talk about how these channels work.
00:17:44;16 In our recent study in collaboration with my UCSF colleague, Yifan Cheng, we have seen
00:17:52;00 the channel... in the structure, with cryo-EM analysis.
00:17:58;07 We see that the protein forms a dimer.
00:18:02;02 And there are actually very well organized lipids, marked in red, at the interface.
00:18:09;27 And we see two calcium ions in each monomer.
00:18:14;11 And they are fairly close to where the pore is.
00:18:18;02 So, the two calcium ions are coordinated by five acidic residues plus an asparagine.
00:18:28;25 And right next to the calcium binding site is the pore.
00:18:33;20 That's formed by six of the ten transmembrane segments.
00:18:39;27 And three of the six are the transmembrane segments that include the calcium binding sites,
00:18:46;06 the acidic residues and asparagine.
00:18:51;09 When we mutated residues lining the pore, we found a cluster of residues near
00:19:00;24 the constriction of the pore that play a role in gating of the channel, and then, also, pore-lining residues
00:19:08;15 all along the pore that are important for anion permeation.
00:19:13;20 So, replacement of any one of these pore-lining residues, all ten of them, with alanine,
00:19:23;01 one at a time, we see that the permeability to iodide versus the permeability to chloride is altered,
00:19:30;20 indicating that these residues along the pore in interact with anions in the pore to control their permeation.
00:19:43;05 And the cluster of residues that are near the constriction site appear to influence
00:19:52;11 the stability of the protein in the open state versus the closed state of the channel.
00:19:58;08 So that alanine mutations of these residues will altered the apparent calcium sensitivity
00:20:05;25 of the channel for activation.
00:20:11;22 A hallmark feature that has been known ever since the '80s is indicated by the blue triangles
00:20:21;26 and the red diamonds in the current-voltage relationship.
00:20:27;19 And that is when the calcium concentration in the cytosol is low, the channel shows
00:20:34;12 very strong voltage dependence.
00:20:37;01 But when the calcium concentration is much higher, there is a linear current-voltage relationship.
00:20:43;13 There's very little voltage dependence.
00:20:45;26 Our recent study, reported this year in Nature... in Neuron, gives further insight to the way
00:20:55;20 the channel works.
00:20:58;02 We see that most likely the channel has actually two different open states.
00:21:04;08 When the channel, or each monomer, has one calcium bound, it's highly voltage-dependent,
00:21:13;13 so the channel is closed unless there is depolarization.
00:21:19;28 And so when the membrane is depolarized to a more positive value, we see an instantaneous current.
00:21:27;05 That's reflecting this open state.
00:21:31;22 And physiologically, the significance of this single... singly-occupied channel is that
00:21:44;00 these channels will not really affect the resting membrane potential, but they will
00:21:49;25 modulate the excitatory synaptic potential and also the action potential.
00:21:55;20 Because, during those synaptic potentials or action potentials, there will be depolarization.
00:22:03;11 Now, if we look at the green curve and the blue curve, we see that just having
00:22:10;22 different anions going through the pore, the channel activity is different.
00:22:17;22 And so the iodide will have a greater effect in potentiating the channel activity compared
00:22:25;08 to chloride.
00:22:27;13 And so this is one form of positive feedback.
00:22:31;04 Once the channel is opened and the anions are going through the pore,
00:22:35;17 it will actually potentiate the channel activity.
00:22:40;13 And then we see in this voltage clamp experiment with prolonged depolarization,
00:22:48;00 there's a gradual rise in the channel activity.
00:22:51;19 And that reflects the occupation of the second calcium binding site.
00:22:57;03 And when the channel has both calcium binding sites occupied, it transitions into a different
00:23:04;14 open conformation that shows no voltage dependence.
00:23:08;04 And this increased activity is also physiologically important.
00:23:14;02 So, we see in recent studies, in this case recording of neurons from the inferior olive,
00:23:23;22 removing the calcium-activated chloride channel formed by TMEM16B will alter the action potential waveform,
00:23:31;22 the duration, and also the afterhyperpolarization.
00:23:36;20 And in this other example, it's recording from thalamocortical neurons,
00:23:44;03 it makes the point that with prolonged depolarization and a whole series of action potentials being generated,
00:23:53;14 this prolonged depolarization and calcium entry during the action potential
00:24:00;06 will lead to a progressively larger fraction, or a larger number, of the calcium-activated chloride channels
00:24:11;04 getting both calcium binding sites occupied and entering into a more active state.
00:24:21;26 And that will lead to a progressive decrease in the firing rate.
00:24:27;16 And this is the phenomenon known as spike frequency adaptation.
00:24:36;21 This makes the point that in mammals the family of TMEM16
00:24:44;26 -- TMEM stands for transmembrane protein with unknown function --
00:24:51;01 we know that 16A and 16B form calcium-activated chloride channels.
00:24:56;08 It was quite surprising to see that the functions of other family members are really very diverse.
00:25:04;06 They are not all calcium-activated chloride channels.
00:25:09;21 When we just go down the list, we found that TMEM16C behaves as an auxiliary subunit of
00:25:17;15 a potassium channel, a sodium-activated potassium channel.
00:25:22;05 So, having... the channel has both the alpha subunit and the beta subunit, TMEM16C,
00:25:30;19 and will have greater sodium sensitivity and also greater stability.
00:25:35;02 So, in the sensory neurons of the dorsal root ganglia, in the wild type there are
00:25:43;09 many more of these channels and greater sodium-activated potassium currents then in the TMEM...
00:25:50;10 in the animals without TMEM16C.
00:25:55;13 And the end result is knocking out TMEM16C will increase the excitability of these sensory neurons
00:26:04;19 and also increase the pain sensitivity of the animal.
00:26:12;03 And another example is TMEM16F.
00:26:14;24 That turns out to be associated, linked, to a human disease that's a bleeding disorder
00:26:22;25 known as Scott syndrome.
00:26:25;16 And the function of TMEM16F is required for calcium-activated lipid scramblase activity
00:26:35;02 in platelet cells and other cell types.
00:26:39;04 And the scrambling of lipids in the lipid bilayer allows the lipids marked in red,
00:26:46;19 the phosphatidyl serine, to be exposed to the cell surface.
00:26:51;12 And that serves as a landing pad for the tissue factors.
00:26:56;11 And that eventually leads to the production of thrombin and blood coagulation.
00:27:05;15 And for the other members, likely the functions are going to be intriguing but quite different.
00:27:12;08 So, those are all still open questions.
00:27:15;12 So, for this study of the TMEM16 family, Bjorn Schroeder used those axolotl oocytes for
00:27:26;00 expression cloning of the channel.
00:27:29;20 And so TMEM16A and B are the calcium-activated chloride channels.
00:27:35;15 Fen Huang did the study of TMEM16C that turned out to be an auxiliary subunit of a potassium channel.
00:27:45;16 Andrew Kim and Huanghe Yang did the initial study from our lab on TMEM16F that's linked
00:27:53;28 to the bleeding disorder.
00:27:55;14 Jason Tien, John Gilchrist, Mu He, Shengjie Feng, and Chris Peters have done
00:28:04;09 the more recent biophysical and physiological studies, including the cryo EM study in collaboration
00:28:12;07 with Yifan Cheng.
00:28:14;04 And several other UCSF colleagues, including Dan Minor, Charly Craik, and Michael Grabe.
00:28:23;19 The pain study was done together with Allan Basbaum.
00:28:28;11 And the bleeding disorder... you know, the blood coagulation study was done in collaboration
00:28:36;14 with Shawn Coughlin.
00:28:38;21 And all of this is a long-term collaboration with Yuh-Nung Jan.
00:28:42;27 And the study was supported by Howard Hughes Medical Institute, NIH,
00:28:49;27 and a number of postdoctoral fellowships.
00:28:53;00 Thank you.

Videos in this Talk

Part 1: Introduction to Ion Channels: A Close Look at the Role and Function of Potassium Channels
Audience:
- Student
- Researcher
- Educators of H. School / Intro Undergrad
- Educators of Adv. Undergrad / Grad
Part 2: Calcium-Activated Chloride Channel (CaCC) in the Enigmatic TMEM16 Family
Audience:
- Researcher
- Educators of H. School / Intro Undergrad
- Educators of Adv. Undergrad / Grad

Speaker: Lily Jan

Total Duration: 0:54:47

Recorded: March 2018

All Talks in Neuroscience

Talk Overview

Ion channels are crucial for proper neuronal communication and cellular homeostasis. But, how do ion channels perform their work? In this seminar, Dr. Lily Jan defines the physiological role of potassium channels in the generation of an action potential and regulation of excitability in neurons. She outlines the role of voltage-gated and inward-rectifier potassium channels and provides evidence of the evolutionary importance of these proteins.

In her second talk, Jan characterizes the Calcium-Activated Chloride Channels (CaCC) in the TMEM16 family of transmembrane proteins with unknown functions and explains experiments from her lab that aided in the understanding of the molecular role of these channels. As she explains, one family member, TMEM16A, is typically expressed in peripheral cells and is involved in the rhythmical contraction of smooth muscles in the gastrointestinal tract. On the other hand, the TMEM16B CaCC is expressed in multiple brain regions as well as sensory neurons, like photoreceptors, and they have an inhibitory role in these cells.

Speaker Bio

Lily Jan

Dr. Lily Jan is a Jack and DeLoris Lange Professor of Physiology and Biophysics at the University of California, San Francisco, and a Howard Hughes Medical Institute Investigator. Jan completed her undergraduate studies in physics at the National Taiwan University in 1968. She had her graduate training at California Institute of Technology (Caltech), where she… Continue Reading