A novel Kir7.1 splice variant expressed in various mouse tissues shares organisational and functional properties with human Leber amaurosis-causing mutations of this K+ channel.

Kir7.1 is an inwardly rectifying K+ channel present in epithelia where it shares membrane localization with the Na+/K+-pump. In the present communication we report the presence of a novel splice variant of Kir7.1 in mouse tissues including kidney, lung, choroid plexus and retinal pigment epithelium (RPE). The variant named mKir7.1-SV2 lacks most of the C-terminus domain but is predicted to have the two transmembrane domains and permeation pathway unaffected. Similarly truncated predicted proteins, Kir7.1-R166X and Kir7.1-Q219X, would arise from mutations associated with Leber Congenital Amaurosis, a rare recessive hereditary retinal disease that results in vision loss at early age. We found that mKir7.1-SV2 and the pathological variants do not produce any channel activity when expressed alone in HEK-293 cells due to their scarce presence in the plasma membrane. Simultaneous expression with the full length Kir7.1 however leads to a reduction in activity of the wild-type channel that might be due to partial proteasome degradation of WT-mutant channel heteromers.

Phosphatidylinositol (4,5)-bisphosphate dynamically regulates the K2P background K+ channel TASK-2.

Two-pore domain K2P K+ channels responsible for the background K+ conductance and the resting membrane potential, are also finely regulated by a variety of chemical, physical and physiological stimuli. Hormones and transmitters acting through Gq protein-coupled receptors (GqPCRs) modulate the activity of various K2P channels but the signalling involved has remained elusive, in particular whether dynamic regulation by membrane PI(4,5)P2, common among other classes of K+ channels, affects K2P channels is controversial. Here we show that K2P K+ channel TASK-2 requires PI(4,5)P2 for activity, a dependence that accounts for its run down in the absence of intracellular ATP and its full recovery by addition of exogenous PI(4,5)P2, its inhibition by low concentrations of polycation PI scavengers, and inhibition by PI(4,5)P2 depletion from the membrane. Comprehensive mutagenesis suggests that PI(4,5)P2 interaction with TASK-2 takes place at C-terminus where three basic aminoacids are identified as being part of a putative binding site.

Two splice variants derived from a Drosophila melanogaster candidate ClC gene generate ClC-2-type Cl- channels.

Members of the ClC family of membrane proteins have been found in a variety of species and they can function as Cl- channels or Cl-/H+ antiporters. Three potential ClC genes are present in the Drosophila melanogaster genome. Only one of them shows homology with a branch of the mammalian ClC genes that encode plasma membrane Cl- channels. The remaining two are close to mammalian homologues coding for intracellular ClC proteins. Using RT-PCR we have identified two splice variants showing highest homology (41% residue identity) to the mammalian ClC-2 chloride channel. One splice variant (DmClC-2S) is expressed in the fly head and body and an additional, larger variant (DmClC-2L) is only present in the head. Both putative Drosophila channels conserve key features of the ClC channels cloned so far, including residues conforming the selectivity filter and C-terminus CBS domains. The splice variants differ in a stretch of 127 aa at the intracellular C-terminal portion separating cystathionate beta synthase (CBS) domains. Expression of either Drosophila ClC-2 variant in HEK-293 cells generated inwardly rectifying Cl- currents with similar activation and deactivation characteristics. There was great similarity in functional characteristics between DmClC-2 variants and their mammalian counterpart, save for slower opening kinetics and faster closing rate. As CBS domains are believed to be sites of regulation of channel gating and trafficking, it is suggested that the extra amino acids present between CBS domains in DmClC-2L might endow the channel with a differential response to signals present in the fly cells where it is expressed.

Functional evaluation of human ClC-2 chloride channel mutations associated with idiopathic generalized epilepsies.

The ClC-2 Cl- channel has been postulated to play a role in the inhibitory GABA response in neurons or to participate in astrocyte-dependent extracellular electrolyte homeostasis. Three different mutations in the CLCN2 gene, encoding the voltage-dependent homodimeric ClC-2 channel, have been associated with idiopathic generalized epilepsy (IGE). We study their function in vitro by patch clamp and confocal microscopy in transiently transfected HEK-293 cells. A first mutation predicts a premature stop codon (M200fsX231). An altered splicing, due to an 11-bp deletion in intron 2 (IVS2-14del11), predicts exon 3 skipping (Delta74-117). A third is a missense mutation (G715E). M200fsX231 and Delta74-117 are nonfunctional and do not affect the function of the normal (wild type, WT) channel. Neither M200fsX231 nor Delta74-117 reach the plasma membrane. Concerning the IVS2-14del11 mutation, we find no difference in the proportion of exon-skipped to normally spliced mRNA using a minigene approach and, on this basis, predict no alteration in channel expression in affected individuals. G715E has voltage dependence and intracellular Cl- dependence indistinguishable from WT channels. ClC-2 channels are shown to be sensitive to intracellular replacement of ATP by AMP, which accelerates the opening and closing kinetics. This effect is diminished in the G715E mutant and not significant in WT+G715E coexpression. We do not know whether, in a situation of cellular ATP depletion, this might become pathological in individuals carrying the mutation. We postulate that loss of function mutation M200fsX231 of ClC-2 might contribute to the IGE phenotype through a haploinsufficiency mechanism.

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 degrees C range (Q(10)). A slow gating process closes both protopores simultaneously, has a high Q(10), is facilitated by extracellular Zn(2+) and Cd(2+) 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 Q(10) 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 Cd(2+), 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 Cd(2+) but deactivation at positive potentials was accelerated by Cd(2+). 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.

Extracellular conserved cysteine forms an intersubunit disulphide bridge in the KCNK5 (TASK-2) K+ channel without having an essential effect upon activity.

The functional channel unit of K(+) channels with two pore regions in tandem is thought to be a homodimer and it has been suggested that this dimeric structure occurs by interaction of an extracellular domain, the self-interacting domain. Interaction and functional assembly have been studied in some detail for KCNK1. It is proposed that a disulphide bond between highly conserved C69 residues of the self-interacting domain is formed which is essential for channel activity. We mutated C51, the equivalent residue in the pH-dependent KCNK5, to study its effect on channel function. Western analysis of proteins from cells expressing epitope-tagged KCNK5 and KCNK5-C51S was consistent with reduction-sensitive self-association of monomers dependent upon the presence of C51. Patch-clamp analysis of heterologously expressed KCNK5-C51S, however, revealed it was functional and indistinguishable in rectification properties and pH dependence from the non-mutated channel. The same result was found with KCNK5-C115S. It is concluded that the proposed disulphide bond between cysteine 51 residues of KCNK5 subunits does occur and preserves a dimeric structure in the detergent solubilized complex. Functional assays, on the other hand, suggest that such a disulphide bridge is not essential for correct functional expression.

A voltage-independent K+ conductance activated by cell swelling in Ehrlich cells is modulated by a G-protein-mediated process.

Cell swelling following hypoosmotic stress leads to the activation of volume-sensitive ion channels that allow a K+ and Cl- efflux accompanied by water loss. A Ca2+-insensitive K+ channel (I(K,vol)) has been described in Ehrlich cells that can be activated by hypotonicity and leukotriene D4 and is inhibited by clofilium. We have studied the activation and deactivation by osmotic stimuli of this channel. A G-protein appears to be involved in these processes since GTP-gamma-S accelerates deactivation, while GDP-beta-S blocks the channel in the open state, a result mimicked by pertussis toxin (PTX). In addition, PTX accelerates the onset of I(K,vol). We propose that I(K,vol) is tonically inhibited by a PTX-sensitive G-protein.