Temperature Dependence of Fast and Slow Gating Relaxations of ClC-0 Chloride Channels

The chloride channel from the Torpedo electric organ, ClC-0, is the best studied member of a large gene-family (Jentsch, T.J. 1996. Curr. Opin. Neurobiol. 6:303–310.). We investigate the temperature dependence of both the voltage- and chloride-dependent fast gate and of the slow gate of the “double-barreled” ClC-0 expressed in Xenopus oocytes. Kinetics of the fast gate exhibit only a moderate temperature dependence with a Q10 of 2.2. Steady-state popen of the fast gate is relatively independent of temperature. The slow gate, in contrast, is highly temperature sensitive. Deactivation kinetics at positive voltages are associated with a Q10 of ∼40. Steady-state open probability of the slow gate (popenslow(V)) can be described by a Boltzmann distribution with an apparent gating valence of ≈2 and a variable “offset” at positive voltages. We note a positive correlation of this offset (i.e., the fraction of channels that are not closed by the slow gate) with the amount of expression. This offset is also highly temperature sensitive, being drastically decreased at high temperatures. Paradoxically, the maximum degree of activation of the slow gate also decreases at higher temperatures. The strong temperature dependence of the slow gate was also observed at the single channel level in inside-out patches. The results imply that within a Markovian-type description at least two open and two closed states are needed to describe slow gating. The strong temperature dependence of the slow gate explains the phenotype of several ClC-0 point-mutants described recently by Ludewig et al. (Ludewig, U., T.J. Jentsch, and M. Pusch. 1996. J. Physiol. (Lond.). In press). The large Q10 of slow gating kinetics points to a complex rearrangement. This, together with the correlation of the fraction of noninactivating channels with the amount of expression and the fact that the slow gate closes both protochannels simultaneously suggests that the slow gate is coupled to subunit interaction of the multimeric ClC-0 channel

Determinants of anion-proton coupling in mammalian endosomal CLC proteins.

Many proteins of the CLC gene family are Cl(-) channels, whereas others, like the bacterial ecClC-1 or mammalian ClC-4 and -5, mediate Cl(-)/H(+) exchange. Mutating a "gating glutamate" (Glu-224 in ClC-4 and Glu-211 in ClC-5) converted these exchangers into anion conductances, as did the neutralization of another, intracellular "proton glutamate" in ecClC-1. We show here that neutralizing the proton glutamate of ClC-4 (Glu-281) and ClC-5 (Glu-268), but not replacing it with aspartate, histidine, or tyrosine, rather abolished Cl(-) and H(+) transport. Surface expression was unchanged by these mutations. Uncoupled Cl(-) transport could be restored in the ClC-4(E281A) and ClC-5(E268A) proton glutamate mutations by additionally neutralizing the gating glutamates, suggesting that wild type proteins transport anions only when protons are supplied through a cytoplasmic H(+) donor. Each monomeric unit of the dimeric protein was found to be able to carry out Cl(-)/H(+) exchange independently from the transport activity of the neighboring subunit. NO(3)(-) or SCN(-) transport was partially uncoupled from H(+) countertransport but still depended on the proton glutamate. Inserting proton glutamates into CLC channels altered their gating but failed to convert them into Cl(-)/H(+) exchangers. Noise analysis indicated that ClC-5 switches between silent and transporting states with an apparent unitary conductance of 0.5 picosiemens. Our results are consistent with the idea that Cl(-)/H(+) exchange of the endosomal ClC-4 and -5 proteins relies on proton delivery from an intracellular titratable residue at position 268 (numbering of ClC-5) and that the strong rectification of currents arises from the voltage-dependent proton transfer from Glu-268 to Glu-211

Disruption of Kcc2-dependent inhibition of olfactory bulb output neurons suggests its importance in odour discrimination

Synaptic inhibition in the olfactory bulb (OB), the first relay station of olfactory information, is believed to be important for odour discrimination. We interfered with GABAergic inhibition of mitral and tufted cells (M/T cells), the principal neurons of the OB, by disrupting their potassium- chloride cotransporter 2 (Kcc2). Roughly, 70% of mice died around 3 weeks, but surviving mice appeared normal. In these mice, the resulting increase in the intracellular Cl− concentration nearly abolished GABA-induced hyperpolarization of mitral cells (MCs) and unexpectedly increased the number of perisomatic synapses on MCs. In vivo analysis of odorant-induced OB electrical activity revealed increased M/T cell firing rate, altered phasing of action potentials in the breath cycle and disrupted separation of odour- induced M/T cell activity patterns. Mice also demonstrated a severely impaired ability to discriminate chemically similar odorants or odorant mixtures. Our work suggests that precisely tuned GABAergic inhibition onto M/T cells is crucial for M/T cell spike pattern separation needed to distinguish closely similar odours

A cation counterflux supports lysosomal acidification

Lysosomes lose cations to balance the influx of protons as the organelle acidifies

Impaired Autophagic Clearance with a Gain-of-Function Variant of the Lysosomal Cl−/H+ Exchanger ClC-7

ClC-7 is a ubiquitously expressed voltage-gated Cl−/H+ exchanger that critically contributes to lysosomal ion homeostasis. Together with its β-subunit Ostm1, ClC-7 localizes to lysosomes and to the ruffled border of osteoclasts, where it supports the acidification of the resorption lacuna. Loss of ClC-7 or Ostm1 leads to osteopetrosis accompanied by accumulation of storage material in lysosomes and neurodegeneration. Interestingly, not all osteopetrosis-causing CLCN7 mutations from patients are associated with a loss of ion transport. Some rather result in an acceleration of voltage-dependent ClC-7 activation. Recently, a gain-of-function variant, ClC-7Y715C, that yields larger ion currents upon heterologous expression, was identified in two patients with neurodegeneration, organomegaly and albinism. However, neither the patients nor a mouse model that carried the equivalent mutation developed osteopetrosis, although expression of ClC-7Y715C induced the formation of enlarged intracellular vacuoles. Here, we investigated how, in transfected cells with mutant ClC-7, the substitution of this tyrosine impinged on the morphology and function of lysosomes. Combinations of the tyrosine mutation with mutations that either uncouple Cl− from H+ counter-transport or strongly diminish overall ion currents were used to show that increased ClC-7 Cl−/H+ exchange activity is required for the formation of enlarged vacuoles by membrane fusion. Degradation of endocytosed material was reduced in these compartments and resulted in an accumulation of lysosomal storage material. In cells expressing the ClC-7 gain-of-function mutant, autophagic clearance was largely impaired, resulting in a build-up of autophagic material

Vestibular role of KCNQ4 and KCNQ5 K+ channels revealed by mouse models

The function of sensory hair cells of the cochlea and vestibular organs depends on an influx of K+ through apical mechanosensitive ion channels and its subsequent removal over their basolateral membrane. The KCNQ4 (Kv7.4) K+ channel, which is mutated in DFNA2 human hearing loss, is expressed in the basal membrane of cochlear outer hair cells (OHCs) where it may mediate K+ efflux. Like the related K+ channel KCNQ5 (Kv7.5), KCNQ4 is also found at calyx terminals ensheathing type I vestibular hair cells where it may be localized pre- or postsynaptically. Making use of Kcnq4-/- mice lacking KCNQ4, as well as Kcnq4dn/dn and Kcnq5dn/dn mice expressing dominant negative channel mutants, we now show unambiguously that in adult mice both channels reside in postsynaptic calyx-forming neurons, but cannot be detected in the innervated hair cells. Accordingly whole-cell currents of vestibular hair cells did not differ between genotypes. Neither Kcnq4-/-, Kcnq5dn/dn nor Kcnq4-/-/Kcnq5dn/dn double mutant mice displayed circling behavior found with severe vestibular impairment. However, a milder form of vestibular dysfunction was apparent from altered vestibulo-ocular reflexes in Kcnq4-/-/Kcnq5dn/dn and Kcnq4-/- mice. The larger impact of KCNQ4 may result from its preferential expression in central zones of maculae and cristae, which are innervated by phasic neurons that are more sensitive than the tonic neurons predominantly present in the surrounding peripheral zones where KCNQ5 is found. The impact of postsynaptic KCNQ4 on vestibular function may be related to K+ removal and modulation of synaptic transmission.Fil: Spitzmaul, Guillermo Federico. Leibniz Institut Fur Molekulare Pharmakologie; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Bahía Blanca. Instituto de Investigaciones Bioquímicas Bahía Blanca (i); ArgentinaFil: Tolosa, Leonardo. Netherlands Institute For Neuroscience; Países BajosFil: Winkelman, Beerend H. J.. Netherlands Institute For Neuroscience; Países BajosFil: Heidenreich, Matthias. Leibniz_Institut Fur Molekulare Pharmakologie (Fmp) ; AlemaniaFil: Frens, Maartens. Department Of Neurosciences, Erasmus; Países BajosFil: Chabbert, Christian. Institut Des Neurosciences De Montpellier; FranciaFil: de Zeeuw, Chris I.. Netherlands Institute For Neuroscience; Países BajosFil: Jentsch, Thomas J.. Charité-Universitätsmedizin. Cluster of Excellence NeuroCure; Alemani

Vestibular role of KCNQ4 and KCNQ5 K+ channels revealed by mouse models

The function of sensory hair cells of the cochlea and vestibular organs depends on an influx of K+ through apical mechanosensitive ion channels and its subsequent removal over their basolateral membrane. The KCNQ4 (Kv7.4) K+ channel, which is mutated in DFNA2 human hearing loss, is expressed in the basal membrane of cochlear outer hair cells (OHCs) where it may mediate K+ efflux. Like the related K+ channel KCNQ5 (Kv7.5), KCNQ4 is also found at calyx terminals ensheathing type I vestibular hair cells where it may be localized pre- or postsynaptically. Making use of Kcnq4-/- mice lacking KCNQ4, as well as Kcnq4dn/dn and Kcnq5dn/dn mice expressing dominant negative channel mutants, we now show unambiguously that in adult mice both channels reside in postsynaptic calyx-forming neurons, but cannot be detected in the innervated hair cells. Accordingly whole-cell currents of vestibular hair cells did not differ between genotypes. Neither Kcnq4-/-, Kcnq5dn/dn nor Kcnq4-/-/Kcnq5dn/dn double mutant mice displayed circling behavior found with severe vestibular impairment. However, a milder form of vestibular dysfunction was apparent from altered vestibulo-ocular reflexes in Kcnq4-/-/Kcnq5dn/dn and Kcnq4-/- mice. The larger impact of KCNQ4 may result from its preferential expression in central zones of maculae and cristae, which are innervated by phasic neurons that are more sensitive than the tonic neurons predominantly present in the surrounding peripheral zones where KCNQ5 is found. The impact of postsynaptic KCNQ4 on vestibular function may be related to K+ removal and modulation of synaptic transmission.Fil: Spitzmaul, Guillermo Federico. Leibniz Institut Fur Molekulare Pharmakologie; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Bahía Blanca. Instituto de Investigaciones Bioquímicas Bahía Blanca (i); ArgentinaFil: Tolosa, Leonardo. Netherlands Institute For Neuroscience; Países BajosFil: Winkelman, Beerend H. J.. Netherlands Institute For Neuroscience; Países BajosFil: Heidenreich, Matthias. Leibniz_Institut Fur Molekulare Pharmakologie (Fmp) ; AlemaniaFil: Frens, Maartens. Department Of Neurosciences, Erasmus; Países BajosFil: Chabbert, Christian. Institut Des Neurosciences De Montpellier; FranciaFil: de Zeeuw, Chris I.. Netherlands Institute For Neuroscience; Países BajosFil: Jentsch, Thomas J.. Charité-Universitätsmedizin. Cluster of Excellence NeuroCure; Alemani

VRAC: molecular identification as LRRC8 heteromers with differential functions

Abstract A major player of vertebrate cell volume regulation is the volume-regulated anion channel (VRAC), which conducts halide ions and organic osmolytes to counteract osmotic imbalances. The molecular entity of this channel was unknown until very recently, although its biophysical characteristics and diverse physiological roles have been extensively studied over the last 30 years. On the road to the molecular identification of VRAC, experimental difficulties led to the proposal of a variety of false candidates. In 2014, in a final breakthrough, two groups independently identified LRRC8A as indispensable component of VRAC. LRRC8A is part of the leucine-rich repeat containing 8 family, which is comprised of five members (LRRC8A-E). Of those, LRRC8A is an obligatory subunit of VRAC but it needs at least one of the other family members to mediate the swelling-induced Cl − current I Cl,vol . This review discusses the remarkable journey which led to the molecular identification of VRAC, evidence for LRRC8 proteins forming the VRAC pore and their heteromeric assembly. Furthermore, first major insights on the role of LRRC8 proteins in cancer drug resistance and apoptosis and the role of LRRC8D in cisplatin and taurine transport will be summarized

Uncoupling endosomal CLC chloride/proton exchange causes severe neurodegeneration

CLC chloride/proton exchangers may support acidification of endolysosomes and raise their luminal Cl− concentration. Disruption of endosomal ClC‐3 causes severe neurodegeneration. To assess the importance of ClC‐3 Cl−/H+ exchange, we now generate Clcn3unc/unc mice in which ClC‐3 is converted into a Cl− channel. Unlike Clcn3−/− mice, Clcn3unc/unc mice appear normal owing to compensation by ClC‐4 with which ClC‐3 forms heteromers. ClC‐4 protein levels are strongly reduced in Clcn3−/−, but not in Clcn3unc/unc mice because ClC‐3unc binds and stabilizes ClC‐4 like wild‐type ClC‐3. Although mice lacking ClC‐4 appear healthy, its absence in Clcn3unc/unc/Clcn4−/− mice entails even stronger neurodegeneration than observed in Clcn3−/− mice. A fraction of ClC‐3 is found on synaptic vesicles, but miniature postsynaptic currents and synaptic vesicle acidification are not affected in Clcn3unc/unc or Clcn3−/− mice before neurodegeneration sets in. Both, Cl−/H+‐exchange activity and the stabilizing effect on ClC‐4, are central to the biological function of ClC‐3

Disrupting MLC1 and GlialCAM and ClC-2interactions in leukodystrophy entails glial chloridechannel dysfunction

Defects in the astrocytic membrane protein MLC1, the adhesion molecule GlialCAM or the chloride channel ClC-2 underlie human leukoencephalopathies. Whereas GlialCAM binds ClC-2 and MLC1, and modifies ClC-2 currents in vitro, no functional connections between MLC1 and ClC-2 are known. Here we investigate this by generating loss-of-function Glialcam and Mlc1 mouse models manifesting myelin vacuolization. We find that ClC-2 is unnecessary for MLC1 and GlialCAM localization in brain, whereas GlialCAM is important for targeting MLC1 and ClC-2 to specialized glial domains in vivo and for modifying ClC-2's biophysical properties specifically in oligodendrocytes (OLs), the cells chiefly affected by vacuolization. Unexpectedly, MLC1 is crucial for proper localization of GlialCAM and ClC-2, and for changing ClC-2 currents. Our data unmask an unforeseen functional relationship between MLC1 and ClC-2 in vivo, which is probably mediated by GlialCAM, and suggest that ClC-2 participates in the pathogenesis of megalencephalic leukoencephalopathy with subcortical cysts