Francisco V. Sepúlveda

Modulation of the two-pore domain acid-sensitive K^+ channel TASK-2 (KCNK5) by changes in cell volume

The molecular identity of K+channels involved in Ehrlich cell volume regulation is unknown. A background K+ conductance is activated by cell swelling and is also modulated by extracellular pH. These characteristics are most similar to those of newly emerging TASK (TWIK-related acid-sensitive K+ channels)-type of two pore-domain K+ channels. mTASK-2, but not TASK-1 or -3, is present in Ehrlich cells and mouse kidney tissue from where the full coding sequences were obtained. Heterologous expression of mTASK-2 cDNA in HEK-293 cells generated K+ currents in the absence intracellular Ca2+. Exposure to hypotonicity enhanced mTASK-2 currents and osmotic cell shrinkage led to inhibition. This occurred without altering voltage dependence and with only slight decrease in pK a in hypotonicity but no change in hypertonicity. Replacement with other cations yields a permselectivity sequence for mTASK-2 of K+ > Rb+ ≫ Cs+ > NH 4 + > Na+ ≅ Li+, similar to that for the native conductance (I K, vol). Clofilium, a quaternary ammonium blocker of I K, vol, blocked the mTASK-2-mediated K+ current with an IC50 of 25 μm. The presence of mTASK-2 in Ehrlich cells, its functional similarities with I K, vol, and its modulation by changes in cell volume suggest that this two-pore domain K+ channel participates in the regulatory volume decrease phenomenon.

The voltage‐dependent ClC‐2 chloride channel has a dual gating mechanism

Functional and structural studies demonstrate that Cl− channels of the ClC family have a dimeric double‐barrelled structure, with each monomer contributing an identical pore. Single protopore gating is a fast process dependent on Cl− interaction within the selectivity filter and in ClC‐0 has a low temperature coefficient over a 10°C range (Q10). A slow gating process closes both protopores simultaneously, has a high Q10, is facilitated by extracellular Zn2+ and Cd2+ and is abolished or markedly reduced by mutation of a cysteine conserved in ClC‐0, ‐1 and ‐2. In order to test the hypothesis that similar slow and fast gates exist in the widely expressed ClC‐2 Cl− channel we have investigated the effects of these manoeuvres on ClC‐2. We find that the time constants of both components of the double‐exponential hyperpolarization‐dependent activation (and deactivation) processes have a high temperature dependence, with Q10 values of about 4–5, suggesting important conformational changes of the channel. Mutating C256 (equivalent to C212 in ClC‐0) to A, led to a significant fraction of constitutively open channels at all potentials. Activation time constants were not affected but deactivation was slower and significantly less temperature dependent in the C256A mutant. Extracellular Cd2+, that inhibits wild‐type (WT) channels almost fully, inhibited C256A only by 50%. In the WT, the time constants for opening were not affected by Cd2+ but deactivation at positive potentials was accelerated by Cd2+. This effect was absent in the C256A mutant. The effect of intracellular Cl− on channel activation was unchanged in the C256A mutant. Collectively our results strongly support the hypothesis that ClC‐2 possesses a common gate and that part of the current increase induced by hyperpolarization represents an opening of the common gate. In contrast to the gating in ClC‐0, the protopore gate and the common gate of ClC‐2 do not appear to be independent.

Neutralization of a single arginine residue gates open a two-pore domain, alkali-activated K+ channel.

Potassium channels share a common selectivity filter that determines the conduction characteristics of the pore. Diversity in K+ channels is given by how they are gated open. TASK-2, TALK-1, and TALK-2 are two-pore region (2P) KCNK K+ channels gated open by extracellular alkalinization. We have explored the mechanism for this alkalinization-dependent gating using molecular simulation and site-directed mutagenesis followed by functional assay. We show that the side chain of a single arginine residue (R224) near the pore senses pH in TASK-2 with an unusual pKa of 8.0, a shift likely due to its hydrophobic environment. R224 would block the channel through an electrostatic effect on the pore, a situation relieved by its deprotonation by alkalinization. A lysine residue in TALK-2 fulfills the same role but with a largely unchanged pKa, which correlates with an environment that stabilizes its positive charge. In addition to suggesting unified alkaline pH-gating mechanisms within the TALK subfamily of channels, our results illustrate in a physiological context the principle that hydrophobic environment can drastically modulate the pKa of charged amino acids within a protein.

A conserved pore-lining glutamate as a voltage- and chloride-dependent gate in the ClC-2 chloride channel

ClC‐2 is a ubiquitously expressed, two‐pore homodimeric Cl− channel opened by hyperpolarisation. Little is known about its gating mechanisms. Crystallographic and functional studies in other ClC channels suggest that a conserved glutamate residue carboxylate side‐chain can close protopores by interacting with a Cl−‐binding site in the pore. Competition for this site is thought to provide the molecular basis for gating by extracellular Cl−. We now show that ClC‐2 gating depends upon intra‐ but not extracellular Cl− and that neutralisation of E217, the homologous pore glutamate, leads to loss of sensitivity to intracellular Cl− and voltage. Experiments testing for transient activation by extracellular protons demonstrate that E217 is not available for protonation in the closed channel state but becomes so after opening by hyperpolarisation. The results suggest that E217 is a hyperpolarisation‐dependent protopore gate in ClC‐2 and that access of intracellular Cl− to a site normally occupied by its side‐chain in the pore stabilises the open state. A remaining hyperpolarisation‐dependent gate might correspond to that closing both pores simultaneously in other ClC channels.

Abolition of Ca2+-mediated intestinal anion secretion and increased stool dehydration in mice lacking the intermediate conductance Ca2+-dependent K+ channel Kcnn4

Intestinal fluid secretion is driven by apical membrane, cystic fibrosis transmembrane conductance regulator (CFTR)‐mediated efflux of Cl– that is concentrated in cells by basolateral Na+−K+−2Cl– cotransporters (NKCC1). An absolute requirement for Cl– efflux is the parallel activation of K+ channels which maintain a membrane potential that sustains apical anion secretion. Both cAMP and Ca2+ are intracellular signals for intestinal Cl– secretion. The K+ channel involved in cAMP‐dependent secretion has been identified as the KCNQ1–KCNE3 complex, but the identity of the K+ channel driving Ca2+‐activated Cl– secretion is controversial. We have now used a Kcnn4 null mouse to show that the intermediate conductance IK1 K+ channel is necessary and sufficient to support Ca2+‐dependent Cl– secretion in large and small intestine. Ussing chambers were used to monitor transepithelial potential, resistance and equivalent short‐circuit current in colon and jejunum from control and Kcnn4 null mice. Na+, K+ and water content of stools was also measured. Distal colon and small intestinal epithelia from Kcnn4 null mice had normal cAMP‐dependent Cl– secretory responses. In contrast, they completely lacked Cl– secretion in response to Ca2+‐mobilizing agonists. Ca2+‐activated electrogenic K+ secretion was increased in colon epithelium of mice deficient in the IK1 channel. Na+ and water content of stools was diminished in IK1‐null animals. The use of Kcnn4 null mice has allowed us to demonstrate that IK1 K+ channels are solely responsible for driving intestinal Ca2+‐activated Cl– secretion. The absence of this channel leads to a marked reduction in water content in the stools, probably as a consequence of decreased electrolyte and water secretion.

Voltage-dependent and -independent titration of specific residues accounts for complex gating of a ClC chloride channel by extracellular protons

The ClC transport protein family comprises both Cl− ion channel and H+/Cl− and H+/NO3− exchanger members. Structural studies on a bacterial ClC transporter reveal a pore obstructed at its external opening by a glutamate side‐chain which acts as a gate for Cl− passage and in addition serves as a staging post for H+ exchange. This same conserved glutamate acts as a gate to regulate Cl− flow in ClC channels. The activity of ClC‐2, a genuine Cl− channel, has a biphasic response to extracellular pH with activation by moderate acidification followed by abrupt channel closure at pH values lower than ∼7. We have now investigated the molecular basis of this complex gating behaviour. First, we identify a sensor that couples extracellular acidification to complete closure of the channel. This is extracellularly‐facing histidine 532 at the N‐terminus of transmembrane helix Q whose neutralisation leads to channel closure in a cooperative manner. We go on to show that acidification‐dependent activation of ClC‐2 is voltage dependent and probably mediated by protonation of pore gate glutamate 207. Intracellular Cl− acts as a voltage‐independent modulator, as though regulating the pKa of the protonatable residue. Our results suggest that voltage dependence of ClC‐2 is given by hyperpolarisation‐dependent penetration of protons from the extracellular side to neutralise the glutamate gate deep within the channel, which allows Cl− efflux. This is reminiscent of a partial exchanger cycle, suggesting that the ClC‐2 channel evolved from its transporter counterparts.

Effect of an N-terminus deletion on voltage-dependent gating of the ClC-2 chloride channel

ClC‐2, a chloride channel widely expressed in mammalian tissues, is activated by hyperpolarisation and extracellular acidification. Deletion of amino acids 16‐61 in rat ClC‐2 abolishes voltage and pH dependence in two‐electrode voltage‐clamp experiments in amphibian oocytes. These results have been interpreted in terms of a ball‐and‐chain type of mechanism in which the N‐terminus would behave as a ball that is removed from an inactivating site upon hyperpolarisation. We now report whole‐cell patch‐clamp measurements in mammalian cells showing hyperpolarization‐activation of rClC‐2Δ16‐61 differing only in presenting faster opening and closing kinetics than rClC‐2. The lack of time and voltage dependence observed previously was reproduced, however, in nystatin‐perforated patch experiments. The behaviour of wild‐type rClC‐2 did not differ between conventional and nystatin‐perforated patches. Similar results were obtained with ClC‐2 from guinea‐pig. One possible explanation of the results is that some diffusible component is able to lock the channel in an open state but does so only to the mutated channel. Alternative explanations involving the osmotic state of the cell and cytoskeleton structure are also considered. Low extracellular pH activates the wild‐type channel but not rClC‐2Δ16‐61 when expressed in oocytes, a result that had been interpreted to suggest that protons affect the ball‐and‐chain mechanism. In our experiments no difference was seen in the effect of extracellular pH upon rClC‐2 and rClC‐2Δ16‐61 in either recording configuration, suggesting that protons act independently from possible effects of the N‐terminus on gating. Our observations of voltage‐dependent gating of the N‐terminal deleted ClC‐2 are an argument against a ball‐and‐chain mechanism for this channel.

TASK-2: a K2P K+ channel with complex regulation and diverse physiological functions

TASK-2 (K2P5.1) is a two-pore domain K+ channel belonging to the TALK subgroup of the K2P family of proteins. TASK-2 has been shown to be activated by extra- and intracellular alkalinization. Extra- and intracellular pH-sensors reside at arginine 224 and lysine 245 and might affect separate selectivity filter and inner gates respectively. TASK-2 is modulated by changes in cell volume and a regulation by direct G-protein interaction has also been proposed. Activation by extracellular alkalinization has been associated with a role of TASK-2 in kidney proximal tubule bicarbonate reabsorption, whilst intracellular pH-sensitivity might be the mechanism for its participation in central chemosensitive neurons. In addition to these functions TASK-2 has been proposed to play a part in apoptotic volume decrease in kidney cells and in volume regulation of glial cells and T-lymphocytes. TASK-2 is present in chondrocytes of hyaline cartilage, where it is proposed to play a central role in stabilizing the membrane potential. Additional sites of expression are dorsal root ganglion neurons, endocrine and exocrine pancreas and intestinal smooth muscle cells. TASK-2 has been associated with the regulation of proliferation of breast cancer cells and could become target for breast cancer therapeutics. Further work in native tissues and cells together with genetic modification will no doubt reveal the details of TASK-2 functions that we are only starting to suspect.

Removal of gating in voltage-dependent ClC-2 chloride channel by point mutations affecting the pore and C-terminus CBS-2 domain

Functional and structural studies demonstrate that Cl− channels of the ClC family have a dimeric double‐barrelled structure, with each monomer contributing an identical pore. Studies with ClC‐0, the prototype ClC channel, show the presence of independent mechanisms gating the individual pores or both pores simultaneously. A single‐point mutation in the CBS‐2 domain of ClC‐0 has been shown to abolish slow gating. We have taken advantage of the high conservation of CBS domains in ClC channels to test for the presence of a slow gate in ClC‐2 by reproducing this mutation (H811A). ClC‐2‐H811A showed faster opening kinetics and opened at more positive potentials than ClC‐2. There was no difference in [Cl−]i dependence. Additional neutralization of a putative pore gate glutamate side chain (E207V) abolished all gating. Resolving slow and fast gating relaxations, however, revealed that the H811A mutation affected both fast and slow gating processes in ClC‐2. This suggests that slow and fast gating in ClC‐2 are coupled, perhaps with slow gating contributing to the operation of the pore E207 as a protopore gate.

No evidence for a role of CLCN2 variants in idiopathic generalized epilepsy

To the Editor: We note the retraction of a paper published in Nature Genetics in 2003, which had reported that mutations in CLCN2 (NCBI Reference Sequence NC_000003.11), the gene encoding the chloride channel ClC-2, were associated with several subtypes of idiopathic generalized epilepsy1. Despite the retraction, Kleefuß-Lie et al.2 recently asserted that “other major aspects of the work remain unaltered” and that their electrophysiological studies are supported by further work published subsequently2. We believe that their logical argument is flawed and that, in addition, the assertion misrepresents the work of others, including our own. First, the authors maintain that they “still believe that the reported genetic variations may contribute to the epileptic phenotypes”2. Without the link between the reported genetic variations and epilepsy, there is no rational basis for such a belief. Second, concerning the functional consequences of the mutations, Kleefuß-Lie et al.2 state that “studies in other laboratories... supported some of the functional changes that were originally reported.” This statement is untrue. The two papers cited as “studies in other laboratories” come from our respective groups3,4. The first of these papers (Niemeyer et al.3) in fact contradicts every one of the functional findings of Haug et al.1. The first mutant, 3792_3793insG (M200fsX231), corresponding to family 1 in the retracted publication, predicts a truncated protein lacking 13 out of 18 expected membrane helices including most putative pore-forming regions. The second consists of an 11-bp deletion (2776_2788del11) in intron 2 close to the splice acceptor site, which was suggested to lead preferentially to an alternatively spliced mRNA and a protein, V74_Q117del, lacking most of transmembrane α-helix B, the largest α-helix predicted to lie at the interface between the ClC-2 channel and the membrane. Our results showed that, in contrast to the claims in the retracted paper, these altered proteins did not reach the plasma membrane and did not exert any dominant negative effect on the function of normal ClC-2 (ref. 3). Also, in regard to the 2776_2788del11 mutation, using a minigene approach, we could find no difference in the proportion of exon-skipped to normally spliced mRNA as a consequence of the mutation and, on this basis, predicted no alteration in ClC-2–channel expression in affected individuals. A third mutation, G8794A, produces an amino acid replacement (G715E) purportedly associated with a gain of function1, allowing the channel to be conductive at reduced intracellular Cl– concentration. We could not reproduce this result of the retracted paper either3. The contrast between our results and those in the retracted paper was reflected in the first paragraph of our Discussion section, which reads: “Our results are in marked contrast to those reported previously by Haug et al. and suggest that the pathophysiological mechanisms proposed by these authors to account for the phenotype need to be revised” 3. The other paper cited as supporting the functional results of the retracted paper is that by Blanz et al.4, in which we in fact confirm the failure to reproduce the dominant negative effect of mutation 3792_3793insG (M200fsX231) reported by Haug et al.1. We concluded that “our electrophysiological analysis of CLCN2 sequence abnormalities described in patients with epilepsy (Haug et al.) did not provide evidence for them being epileptogenic”4. In the same paper4, we showed that other CLCN2 sequence variants identified more recently in patients with epilepsy5 did not alter the biophysical properties of ClC-2 and were also found in humans not displaying epilepsy. We have also reported that the ClC-2–null genotype in mice failed to induce spontaneous seizures or to alter the seizure threshold for the response of the animals to proconvulsants4,6. We discussed these points in recent reviews7,8 and had concluded before the retraction of the paper by Haug et al.1 that “the sum of these observations... warrants skepticism toward the proposed causative role of ClC-2 in epilepsy”7. These observations both suggest that even the functional results of the retracted paper cannot be relied upon, and they also support the view that there is no basis to claim that CLCN2 plays a role in epilepsy.