L. Pablo Cid

Hereditary parkinsonism with dementia is caused by mutations in ATP13A2, encoding a lysosomal type 5 P-type ATPase

Neurodegenerative disorders such as Parkinson and Alzheimer disease cause motor and cognitive dysfunction and belong to a heterogeneous group of common and disabling disorders. Although the complex molecular pathophysiology of neurodegeneration is largely unknown, major advances have been achieved by elucidating the genetic defects underlying mendelian forms of these diseases. This has led to the discovery of common pathophysiological pathways such as enhanced oxidative stress, protein misfolding and aggregation and dysfunction of the ubiquitin-proteasome system. Here, we describe loss-of-function mutations in a previously uncharacterized, predominantly neuronal P-type ATPase gene, ATP13A2, underlying an autosomal recessive form of early-onset parkinsonism with pyramidal degeneration and dementia (PARK9, Kufor-Rakeb syndrome). Whereas the wild-type protein was located in the lysosome of transiently transfected cells, the unstable truncated mutants were retained in the endoplasmic reticulum and degraded by the proteasome. Our findings link a class of proteins with unknown function and substrate specificity to the protein networks implicated in neurodegeneration and parkinsonism.

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.

Separate taurine and chloride efflux pathways activated during regulatory volume decrease

Organic osmolyte and halide permeability pathways activated in epithelial HeLa cells by cell swelling were studied by radiotracer efflux techniques and single-cell volume measurements. The replacement of extracellular Cl- by anions that are more permeant through the volume-activated Cl- channel, as indicated by electrophysiological measurements, significantly decreased taurine efflux. In the presence of less-permeant anions, an increase in taurine efflux was observed. Simultaneous measurement of the 125I, used as a tracer for Cl-, and [3H]taurine efflux showed that the time courses for the two effluxes differed. In Cl--rich medium the increase in I- efflux was transient, whereas that for taurine was sustained. Osmosensitive Cl- conductance, assessed by measuring changes in cell volume, increased rapidly after hypotonic shock. The influx of taurine was able to counteract Cl- conductance-dependent cell shrinkage but only approximately 4 min after triggering cell swelling. This taurine-induced effect was blocked by DIDS. Differences in anion sensitivity, the time course of activation, and sensitivity to DIDS suggest that the main cell swelling-activated permeability pathways for taurine and Cl- are separate.Organic osmolyte and halide permeability pathways activated in epithelial HeLa cells by cell swelling were studied by radiotracer efflux techniques and single-cell volume measurements. The replacement of extracellular Cl- by anions that are more permeant through the volume-activated Cl- channel, as indicated by electrophysiological measurements, significantly decreased taurine efflux. In the presence of less-permeant anions, an increase in taurine efflux was observed. Simultaneous measurement of the125I, used as a tracer for Cl-, and [3H]taurine efflux showed that the time courses for the two effluxes differed. In Cl--rich medium the increase in I- efflux was transient, whereas that for taurine was sustained. Osmosensitive Cl- conductance, assessed by measuring changes in cell volume, increased rapidly after hypotonic shock. The influx of taurine was able to counteract Cl- conductance-dependent cell shrinkage but only ∼4 min after triggering cell swelling. This taurine-induced effect was blocked by DIDS. Differences in anion sensitivity, the time course of activation, and sensitivity to DIDS suggest that the main cell swelling-activated permeability pathways for taurine and Cl- are separate.

Basolateral localization of native ClC-2 chloride channels in absorptive intestinal epithelial cells and basolateral sorting encoded by a CBS-2 domain di-leucine motif

The Cl– channel ClC-2 is expressed in transporting epithelia and has been proposed as an alternative route for Cl– efflux that might compensate for the malfunction of CFTR in cystic fibrosis. There is controversy concerning the cellular and membrane location of ClC-2, particularly in intestinal tissue. The aim of this paper is to resolve this controversy by immunolocalization studies using tissues from ClC-2 knockout animals as control, ascertaining the sorting of ClC-2 in model epithelial cells and exploring the possible molecular signals involved in ClC-2 targeting. ClC-2 was exclusively localized at the basolateral membranes of surface colonic cells or villus duodenal enterocytes. ClC-2 was sorted to the basolateral membranes in MDCK, Caco-2 and LLC-PK1-μ1B, but not in LLC-PK1-μ1A cells. Mutating a di-leucine motif (L812L813) to a di-alanine changed the basolateral targeting of ClC-2 to an apical location. The basolateral membrane localization of ClC-2 in absorptive cells of the duodenum and the colon is compatible with an absorptive function for this Cl– channel. Basolateral targeting information is contained in a di-leucine motif (L812L813) within CBS-2 domain at the C-terminus of ClC-2. It is speculated that ClC-2 also contains an apical sorting signal masked by L812L813. The proposal that CBS domains in ClC channels might behave as regulatory sites sensing intracellular signals opens an opportunity for pharmacological modulation of ClC-2 targeting.

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.

Separate gating mechanisms mediate the regulation of K2P potassium channel TASK-2 by intra- and extracellular pH

TASK-2 (KCNK5 or K2P5.1) is a background K+ channel that is opened by extracellular alkalinization and plays a role in renal bicarbonate reabsorption and central chemoreception. Here, we demonstrate that in addition to its regulation by extracellular protons (pHo) TASK-2 is gated open by intracellular alkalinization. The following pieces of evidence suggest that the gating process controlled by intracellular pH (pHi) is independent from that under the command of pHo. It was not possible to overcome closure by extracellular acidification by means of intracellular alkalinization. The mutant TASK-2-R224A that lacks sensitivity to pHo had normal pHi-dependent gating. Increasing extracellular K+ concentration acid shifts pHo activity curve of TASK-2 yet did not affect pHi gating of TASK-2. pHo modulation of TASK-2 is voltage-dependent, whereas pHi gating was not altered by membrane potential. These results suggest that pHo, which controls a selectivity filter external gate, and pHi act at different gating processes to open and close TASK-2 channels. We speculate that pHi regulates an inner gate. We demonstrate that neutralization of a lysine residue (Lys245) located at the C-terminal end of transmembrane domain 4 by mutation to alanine abolishes gating by pHi. We postulate that this lysine acts as an intracellular pH sensor as its mutation to histidine acid-shifts the pHi-dependence curve of TASK-2 as expected from its lower pKa. We conclude that intracellular pH, together with pHo, is a critical determinant of TASK-2 activity and therefore of its physiological function.

Molecular Aspects of Structure, Gating, and Physiology of pH-Sensitive Background K2P and Kir K+-Transport Channels

K(+) channels fulfill roles spanning from the control of excitability to the regulation of transepithelial transport. Here we review two groups of K(+) channels, pH-regulated K2P channels and the transport group of Kir channels. After considering advances in the molecular aspects of their gating based on structural and functional studies, we examine their participation in certain chosen physiological and pathophysiological scenarios. Crystal structures of K2P and Kir channels reveal rather unique features with important consequences for the gating mechanisms. Important tasks of these channels are discussed in kidney physiology and disease, K(+) homeostasis in the brain by Kir channel-equipped glia, and central functions in the hearing mechanism in the inner ear and in acid secretion by parietal cells in the stomach. K2P channels fulfill a crucial part in central chemoreception probably by virtue of their pH sensitivity and are central to adrenal secretion of aldosterone. Finally, some unorthodox behaviors of the selectivity filters of K2P channels might explain their normal and pathological functions. Although a great deal has been learned about structure, molecular details of gating, and physiological functions of K2P and Kir K(+)-transport channels, this has been only scratching at the surface. More molecular and animal studies are clearly needed to deepen our knowledge.

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.