Normal vision and development in mice with low functional expression of Kir7.1 in heterozygosis for a blindness-producing mutation inactivating the channel.

K+ channel Kir7.1 expressed at the apical membrane of the retinal pigment epithelium (RPE) plays an essential role in retinal function. An isoleucine-to-threonine mutation at position 120 of the protein is responsible for blindness-causing vitreo-retinal dystrophy. We have studied the molecular mechanism of action of Kir7.1-I120T in vitro by heterologous expression and in vivo in CRISPR-generated knockin mice. Full-size Kir7.1-I120T reaches the plasma membrane but lacks any activity. Analysis of Kir7.1 and the I120T mutant in mixed transfection experiments, and that of tandem tetrameric constructs made by combining wild type (WT) and mutant protomers, leads us to conclude that they do not form heterotetramers in vitro. Homozygous I120T/I120T mice show cleft palate and tracheomalacia and do not survive beyond P0, whereas heterozygous WT/I120T develop normally. Membrane conductance of RPE cells isolated from WT/WT and heterozygous WT/I120T mice is dominated by Kir7.1 current. Using Rb+ as a charge carrier, we demonstrate that the Kir7.1 current of WT/I120T RPE cells corresponds to approximately 50% of that in cells from WT/WT animals, in direct proportion to WT gene dosage. This suggests a lack of compensatory effects or interference from the mutated allele product, an interpretation consistent with results obtained using WT/- hemizygous mouse. Electroretinography and behavioral tests also show normal vision in WT/I120T animals. The hypomorphic ion channel phenotype of heterozygous Kir7.1-I120T mutants is therefore compatible with normal development and retinal function. The lack of detrimental effect of this degree of functional deficit might explain the recessive nature of Kir7.1 mutations causing human eye disease.NEW & NOTEWORTHY Human retinal pigment epithelium K+ channel Kir7.1 is affected by generally recessive mutations leading to blindness. We investigate one such mutation, isoleucine-to-threonine at position 120, both in vitro and in vivo in knockin mice. The mutated channel is inactive and in heterozygosis gives a hypomorphic phenotype with normal retinal function. Mutant channels do not interfere with wild-type Kir7.1 channels which are expressed concomitantly without hindrance, providing an explanation for the recessive nature of the disease.

A new family of glutamate-gated chloride channels in parasitic sea louse Caligus rogercresseyi: A subunit refractory to activation by ivermectin is dominant in heteromeric assemblies.

Sea louse ectoparasitosis is a major threat to fish aquaculture. Avermectins such as ivermectin and emamectin have been effectively used against sea louse infestation, but the emergence of resistance has limited their use. A better understanding of the molecular targets of avermectins is essential to the development of novel treatment strategies or new, more effective drugs. Avermectins are known to act by inhibiting neurotransmission through allosteric activation of glutamate-gated chloride channels (GluCls). We have investigated the GluCl subunit present in Caligus rogercresseyi, a sea louse affecting aquaculture in the Southern hemisphere. We identify four new subunits, CrGluCl-B to CrGluCl-E, and characterise them functionally. CrGluCl-A (previously reported as CrGluClα), CrGluCl-B and CrGluCl-C all function as glutamate channel receptors with different sensitivities to the agonist, but in contrast to subunit -A and -C, CrGluCl-B is not activated by ivermectin but is rather antagonised by the drug. CrGluCl-D channel appears active in the absence of any stimulation by glutamate or ivermectin and CrGluCl-E does not exhibit any activity. Notably, the expression of CrGluCl-B with either -A or -C subunits gives rise to receptors unresponsive to ivermectin and showing altered response to glutamate, suggesting that coexpression has led to the preferential formation of heteromers to which the presence of CrGluCl-B confers the property of ivermectin-activation refractoriness. Furthermore, there was evidence for heteromer formation with novel properties only when coexpressing pairs E/C and D/B CrGluCl subtypes. Site-directed mutagenesis shows that three transmembrane domain residues contribute to the lack of activation by ivermectin, most crucially Gln 15' in M2, with mutation Q15'T (the residue present in ivermectin-activated subunits A and C) conferring ivermectin activation to CrGluCl-B. The differential response to avermectin of these Caligus rogercresseyi GluClsubunits, which are highly conserved in the Northern hemisphere sea louse Lepeophtheirus salmonis, could have an influence on the response of these parasites to treatment with macrocyclic lactones. They could serve as molecular markers to assess susceptibility to existing treatments and might be useful molecular targets in the search for novel antiparasitic drugs.

Altered phosphatidylinositol regulation of mutant inwardly rectifying K+ Kir7.1 channels associated with inherited retinal degeneration disease.

KEY POINTS: Kir7.1 K+ channel expressed in retinal pigment epithelium is mutated in inherited retinal degeneration diseases. We study Kir7.1 in heterologous expression to test the hypothesis that pathological R162 mutation to neutral amino acids results in loss of a crucial site that binds PI(4,5)P2 . Although R162W mutation inactivates Kir7.1, changes to smaller volume (e.g. Gln) amino acids are tolerated or even enhance function (Ala or Cys). Chemical modification of Kir7.1-R162C confirms that large residues of the size of Trp are incompatible with normal channel function even if positively charged. In addition to R162, K164 (and possibly K159) forms a binding site for the phosphoinositide and is essential for channel activity. R162 substitution with a large, neutral side chain like Trp exerts a dominant negative effect on Kir7.1 activity such that less than one fifth of the full activity is expected in a cell expressing the same amount of mutant and wild-type channels. ABSTRACT: Mutations in the Kir7.1 K+ channel, highly expressed in retinal pigment epithelium, have been linked to inherited retinal degeneration diseases. Examples are mutations changing Arg 162 to Trp in snowflake vitreoretinal degeneration (SVD) and Gln in retinitis pigmentosa. R162 is believed to be part of a site that binds PI(4,5)P2 and stabilises the open state. We have tested the hypothesis that R162 mutation to neutral amino acids will result in the loss of this crucial interaction to the detriment of channel function. Our findings indicate that although R612W mutation inactivates Kir7.1, changes to smaller volume (e.g. Gln) amino acids are tolerated or even enhance function (Ala or Cys). Cys chemical modification of Kir7.1-R162C confirms that large residues of the size of Trp are incompatible with normal channel function even if positively charged. Experiments titrating the levels of plasma membrane PI(4,5)P2 with voltage-dependent phosphatase DrVSP reveal that, in addition to R162, K164 (and possibly K159) forms a binding site for the phosphoinositide and ensures channel activity. Finally, the use of a concatemeric approach shows that substitution of R162 with a large, neutral side chain mimicking a Trp residue exerts a dominant negative effect on Kir7.1 activity such that less than one fifth of the full activity is expected in heterozygous cells carrying the SVD mutation. Our results suggest that if mutations in the human KCNJ13 gene resulting in the neutralisation of R162 and Kir7.1 malfunction led to retinal degeneration diseases, their severity might depend on the nature of the side chain of the replacing amino acid.

Kir7.1 inwardly rectifying K+ channel is expressed in ciliary body non pigment epithelial cells and might contribute to intraocular pressure regulation.

Inwardly rectifying K+ channel Kir7.1 is expressed in epithelia where it shares membrane localisation with the Na+/K+-pump. The ciliary body epithelium (CBE) of the eye is a determinant of intraocular pressure (IOP) through NaCl-driven fluid secretion of aqueous humour. In the present study we explored the presence Kir7.1 in this epithelium in the mouse and its possible functional role in the generation of IOP. Use heterozygous animals for total Kir7.1 knockout expressing ß-galactosidase under the control of Kir7.1 promoter, identified the expression of Kir7.1 in non-pigmented epithelial cells of CBE. Using conditional, floxed knockout Kir7.1 mice as negative controls, we found Kir7.1 at the basolateral membrane of the same CBE cell layer. This was confirmed using a knockin mouse expressing the Kir7.1 protein tagged with a haemagglutinin epitope. Measurements using the conditional knockout mouse show only a minor effect of Kir7.1 inactivation on steady-state IOP. Transient increases in IOP in response to general anaesthetics, or to water injection, are absent or markedly curtailed in Kir7.1-deficient mice. These results suggest a role for Kir7.1 in IOP regulation through a possible modulation of aqueous humour production by the CBE non-pigmented epithelial cells. The location of Kir7.1 in the CBE, together with the effect of its removal on dynamic changes in IOP, point to a possible role of the channel as a leak pathway preventing cellular overload of K+ during the secretion process. Kir7.1 could be used as a potential therapeutic target in pathological conditions leading to elevated intraocular pressure.

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.

Kcnn4 is a modifier gene of intestinal cystic fibrosis preventing lethality in the Cftr-F508del mouse.

Nearly 70% of cystic fibrosis (CF) patients bear the phenylalanine-508 deletion but disease severity differs greatly, and is not explained by the existence of different mutations in compound heterozygous. Studies demonstrated that genes other than CFTR relate to intestinal disease in humans and CF-mouse. Kcnn4, the gene encoding the calcium-activated potassium channel KCa3.1, important for intestinal secretion, is present in a locus linked with occurrence of intestinal CF-disease in mice and humans. We reasoned that it might be a CF-modifier gene and bred a CF-mouse with Kcnn4 silencing, finding that lethality was almost abolished. Silencing of Kcnn4 did not improve intestinal secretory functions, but rather corrected increased circulating TNF-α level and reduced intestinal mast cell increase. Given the importance of mast cells in intestinal disease additional double mutant CF-animals were tested, one lacking mast cells (C-kitW-sh/W-sh) and Stat6-/- to block IgE production. While mast cell depletion had no effect, silencing Stat6 significantly reduced lethality. Our results show that Kcnn4 is an intestinal CF modifier gene partially acting through a STAT6-dependent mechanism.

K2P TASK-2 and KCNQ1-KCNE3 K+ channels are major players contributing to intestinal anion and fluid secretion.

KEY POINTS: K+ channels are important in intestinal epithelium as they ensure the ionic homeostasis and electrical potential of epithelial cells during anion and fluid secretion. Intestinal epithelium cAMP-activated anion secretion depends on the activity of the (also cAMP dependent) KCNQ1-KCNE3 K+ channel, but the secretory process survives after genetic inactivation of the K+ channel in the mouse. Here we use double mutant mice to investigate which alternative K+ channels come into action to compensate for the absence of KCNQ1-KCNE3 K+ channels. Our data establish that whilst Ca2+ -activated KCa 3.1 channels are not involved, K2P two-pore domain TASK-2 K+ channels are major players providing an alternative conductance to sustain the intestinal secretory process. Work with double mutant mice lacking both TASK-2 and KCNQ1-KCNE3 channels nevertheless points to yet-unidentified K+ channels that contribute to the robustness of the cAMP-activated anion secretion process. ABSTRACT: Anion and fluid secretion across the intestinal epithelium, a process altered in cystic fibrosis and secretory diarrhoea, is mediated by cAMP-activated CFTR Cl- channels and requires the simultaneous activity of basolateral K+ channels to maintain cellular ionic homeostasis and membrane potential. This function is fulfilled by the cAMP-activated K+ channel formed by the association of pore-forming KCNQ1 with its obligatory KCNE3 ß-subunit. Studies using mice show sizeable cAMP-activated intestinal anion secretion in the absence of either KCNQ1 or KCNE3 suggesting that an alternative K+ conductance must compensate for the loss of KCNQ1-KCNE3 activity. We used double mutant mouse and pharmacological approaches to identify such a conductance. Ca2+ -dependent anion secretion can also be supported by Ca2+ -dependent KCa 3.1 channels after independent CFTR activation, but cAMP-dependent anion secretion is not further decreased in the combined absence of KCa 3.1 and KCNQ1-KCNE3 K+ channel activity. We show that the K2P K+ channel TASK-2 is expressed in the epithelium of the small and large intestine. Tetrapentylammonium, a TASK-2 inhibitor, abolishes anion secretory current remaining in the absence of KCNQ1-KCNE3 activity. A double mutant mouse lacking both KCNQ1-KCNE3 and TASK-2 showed a much reduced cAMP-mediated anion secretion compared to that observed in the single KCNQ1-KCNE3 deficient mouse. We conclude that KCNQ1-KCNE3 and TASK-2 play major roles in the intestinal anion and fluid secretory phenotype. The persistence of an, admittedly reduced, secretory activity in the absence of these two conductances suggests that further additional K+ channel(s) as yet unidentified contribute to the robustness of the intestinal anion secretory process.

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.

Gating, Regulation, and Structure in K2P K+ Channels: In Varietate Concordia?

K2P K(+) channels with two pore domains in tandem associate as dimers to produce so-called background conductances that are regulated by a variety of stimuli. Whereas gating in K2P channels has been poorly understood, recent developments have provided important clues regarding the gating mechanism for this family of proteins. Two modes of gating present in other K(+) channels have been considered. The first is the so-called activation gating that occurs by bundle crossing and the splaying apart of pore-lining helices commanding ion passage. The second mode involves a change in conformation at the selectivity filter (SF), which impedes ion flow at this narrow portion of the conduction pathway and accounts for extracellular pH modulation of several K2P channels. Although some evidence supports the existence of an activation gate in K2P channels, recent results suggest that perhaps all stimuli, even those sensed at a distant location in the protein, are also mediated by SF gating. Recently resolved crystal structures of K2P channels in conductive and nonconductive conformations revealed that the nonconductive state is reached by blockade by a lipid acyl chain that gains access to the channel cavity through intramembrane fenestrations. Here we discuss whether this novel type of gating, proposed so far only for membrane tension gating, might mediate gating in response to other stimuli or whether SF gating is the only type of opening/closing mechanism present in K2P channels.

Cleft Palate, Moderate Lung Developmental Retardation and Early Postnatal Lethality in Mice Deficient in the Kir7.1 Inwardly Rectifying K+ Channel.

Kir7.1 is an inwardly rectifying K+ channel of the Kir superfamily encoded by the kcnj13 gene. Kir7.1 is present in epithelial tissues where it colocalizes with the Na+/K+-pump probably serving to recycle K+ taken up by the pump. Human mutations affecting Kir7.1 are associated with retinal degeneration diseases. We generated a mouse lacking Kir7.1 by ablation of the Kcnj13 gene. Homozygous mutant null mice die hours after birth and show cleft palate and moderate retardation in lung development. Kir7.1 is expressed in the epithelium covering the palatal processes at the time at which palate sealing takes place and our results suggest it might play an essential role in late palatogenesis. Our work also reveals a second unexpected role in the development and the physiology of the respiratory system, where Kir7.1 is expressed in epithelial cells all along the respiratory tree.

TASK-2 K2p K⁺ channel: thoughts about gating and its fitness to physiological function.

TASK-2 (K2P5) was one of the earliest members of the K2P two-pore, four transmembrane domain K(+) channels to be identified. TASK-2 gating is controlled by changes in both extra- and intracellular pH through separate sensors: arginine 224 and lysine 245, located at the extra- and intracellular ends of transmembrane domain 4. TASK-2 is inhibited by a direct effect of CO2 and is regulated by and interacts with G protein subunits. TASK-2 takes part in regulatory adjustments and is a mediator in the chemoreception process in neurons of the retrotrapezoid nucleus where its pHi sensitivity could be important in regulating excitability and therefore signalling of the O2/CO2 status. Extracellular pH increases brought about by HCO3 (-) efflux from proximal tubule epithelial cells have been proposed to couple to TASK-2 activation to maintain electrochemical gradients favourable to HCO3 (-) reabsorption. We demonstrate that, as suspected previously, TASK-2 is expressed at the basolateral membrane of the same proximal tubule cells that express apical membrane Na(+)-H(+)-exchanger NHE-3 and basolateral membrane Na(+)-HCO3 (-) cotransporter NBCe1-A, the main components of the HCO3 (-) transport machinery. We also discuss critically the mechanism by which TASK-2 is modulated and impacts the process of HCO3 (-) reclaim by the proximal tubule epithelium, concluding that more than a mere shift in extracellular pH is probably involved.

Identification and functional expression of a glutamate- and avermectin-gated chloride channel from Caligus rogercresseyi, a southern Hemisphere sea louse affecting farmed fish.

Parasitic sea lice represent a major sanitary threat to marine salmonid aquaculture, an industry accounting for 7% of world fish production. Caligus rogercresseyi is the principal sea louse species infesting farmed salmon and trout in the southern hemisphere. Most effective control of Caligus has been obtained with macrocyclic lactones (MLs) ivermectin and emamectin. These drugs target glutamate-gated chloride channels (GluCl) and act as irreversible non-competitive agonists causing neuronal inhibition, paralysis and death of the parasite. Here we report the cloning of a full-length CrGluClα receptor from Caligus rogercresseyi. Expression in Xenopus oocytes and electrophysiological assays show that CrGluClα is activated by glutamate and mediates chloride currents blocked by the ligand-gated anion channel inhibitor picrotoxin. Both ivermectin and emamectin activate CrGluClα in the absence of glutamate. The effects are irreversible and occur with an EC(50) value of around 200 nM, being cooperative (n(H) = 2) for ivermectin but not for emamectin. Using the three-dimensional structure of a GluClα from Caenorabditis elegans, the only available for any eukaryotic ligand-gated anion channel, we have constructed a homology model for CrGluClα. Docking and molecular dynamics calculations reveal the way in which ivermectin and emamectin interact with CrGluClα. Both drugs intercalate between transmembrane domains M1 and M3 of neighbouring subunits of a pentameric structure. The structure displays three H-bonds involved in this interaction, but despite similarity in structure only of two these are conserved from the C. elegans crystal binding site. Our data strongly suggest that CrGluClα is an important target for avermectins used in the treatment of sea louse infestation in farmed salmonids and open the way for ascertaining a possible mechanism of increasing resistance to MLs in aquaculture industry. Molecular modeling could help in the design of new, more efficient drugs whilst functional expression of the receptor allows a first stage of testing of their efficacy.

Zebrafish and mouse TASK-2 K(+) channels are inhibited by increased CO2 and intracellular acidification.

TASK-2 is a K2P K(+) channel considered as a candidate to mediate CO2 sensing in central chemosensory neurons in mouse. Neuroepithelial cells in zebrafish gills sense CO2 levels through an unidentified K2P K(+) channel. We have now obtained zfTASK-2 from zebrafish gill tissue that is 49 % identical to mTASK-2. Like its mouse equivalent, it is gated both by extra- and intracellular pH being activated by alkalinization and inhibited by acidification. The pHi dependence of zfTASK-2 is similar to that of mTASK-2, with pK 1/2 values of 7.9 and 8.0, respectively, but pHo dependence occurs with a pK 1/2 of 8.8 (8.0 for mTASK-2) in line with the relatively alkaline plasma pH found in fish. Increasing CO2 led to a rapid, concentration-dependent (IC50 ~1.5 % CO2) inhibition of mouse and zfTASK-2 that could be resolved into an inhibition by intracellular acidification and a CO2 effect independent of pHi change. Indeed a CO2 effect persisted despite using strongly buffered intracellular solutions abolishing any change in pHi, was present in TASK-2-K245A mutant insensitive to pHi, and also under carbonic anhydrase inhibition. The mechanism by which TASK-2 senses CO2 is unknown but requires the presence of the 245-273 stretch of amino acids in the C terminus that comprises numerous basic amino acids and is important in TASK-2 G protein subunit binding and regulation of the channel. The described CO2 effect might be of importance in the eventual roles played by TASK-2 in chemoreception in mouse and zebrafish.

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.

G protein modulation of K2P potassium channel TASK-2 : a role of basic residues in the C terminus domain.

TASK-2 (K2P5.1) is a background K(+) channel opened by extra- or intracellular alkalinisation that plays a role in renal bicarbonate handling, central chemoreception and cell volume regulation. Here, we present results that suggest that TASK-2 is also modulated by Gßγ subunits of heterotrimeric G protein. TASK-2 was strongly inhibited when GTP-γ-S was used as a replacement for intracellular GTP. No inhibition was present using GDP-ß-S instead. Purified Gßγ introduced intracellularly also inhibited TASK-2 independently of whether GTP or GDP-ß-S was present. The effects of GTP-γ-S and Gßγ subunits were abolished by neutralisation of TASK-2 C terminus double lysine residues K257-K258 or K296-K297. Use of membrane yeast two hybrid (MYTH) experiments and immunoprecipitation assays using tagged proteins gave evidence for a physical interaction between Gß1 and Gß2 subunits and TASK-2, in agreement with expression of these subunits in proximal tubule cells. Co-immunoprecipitation was impeded by mutating C terminus K257-K258 (but not K296-K297) to alanines. Gating by extra- or intracellular pH was unaltered in GTP-γ-S-insensitive TASK-2-K257A-K258A mutant. Shrinking TASK-2-expressing cells in hypertonic solution decreased the current to 36 % of its initial value. The same manoeuvre had a significantly diminished effect on TASK-2-K257A-K258A- or TASK-2-K296-K297-expressing cells, or in cells containing intracellular GDP-ß-S. Our data are compatible with the concept that TASK-2 channels are modulated by Gßγ subunits of heterotrimeric G protein. We propose that this modulation is a novel way in which TASK-2 can be tuned to its physiological functions.

Novel CLCNKB mutations causing Bartter syndrome affect channel surface expression.

Mutations in the CLCNKB gene encoding the ClC-Kb Cl(-) channel cause Bartter syndrome, which is a salt-losing renal tubulopathy. Here, we investigate the functional consequences of seven mutations. When expressed in Xenopus laevis oocytes, four mutants carried no current (c.736G>C, p.Gly246Arg; c.1271G>A, p.Gly424Glu; c.1313G>A, p.Arg438His; c.1316T>C, p.Leu439Pro), whereas others displayed a 30%-60% reduction in conductance as compared with wild-type ClC-Kb (c.242T>C, p.Leu81Pro; c.274C>T, p.Arg92Trp; c.1052G>C, p.Arg351Pro). Anion selectivity and sensitivity to external Ca(2+) and H(+), typical of the ClC-Kb channel, were not modified in the partially active mutants. In oocytes, we found that all the mutations reduced surface expression with a profile similar to that observed for currents. In HEK293 cells, the currents in the mutants had similar profiles to those obtained in oocytes, except for p.Leu81Pro, which produced no current. Furthermore, p.Arg92Trp and p.Arg351Pro mutations did not modify the unit-conductance of closely related ClC-K1. Western blot analysis in HEK293 cells showed that ClC-Kb protein abundance was lower for the nonconducting mutants but similar to wild-type for other mutants. Overall, two classes of mutants can be distinguished: nonconducting mutants associated with low total protein expression, and partially conducting mutants with unaltered channel properties and ClC-Kb protein abundance.

An extracellular ion pathway plays a central role in the cooperative gating of a K(2P) K+ channel by extracellular pH.

Proton-gated TASK-3 K(+) channel belongs to the K(2P) family of proteins that underlie the K(+) leak setting the membrane potential in all cells. TASK-3 is under cooperative gating control by extracellular [H(+)]. Use of recently solved K(2P) structures allows us to explore the molecular mechanism of TASK-3 cooperative pH gating. Tunnel-like side portals define an extracellular ion pathway to the selectivity filter. We use a combination of molecular modeling and functional assays to show that pH-sensing histidine residues and K(+) ions mutually interact electrostatically in the confines of the extracellular ion pathway. K(+) ions modulate the pK(a) of sensing histidine side chains whose charge states in turn determine the open/closed transition of the channel pore. Cooperativity, and therefore steep dependence of TASK-3 K(+) channel activity on extracellular pH, is dependent on an effect of the permeant ion on the channel pH(o) sensors.

Sexual dimorphism and oestrogen regulation of KCNE3 expression modulates the functional properties of KCNQ1 K⁺ channels.

The KCNQ1 potassium channel associates with various KCNE ancillary subunits that drastically affect channel gating and pharmacology. Co-assembly with KCNE3 produces a current with nearly instantaneous activation, some time-dependent activation at very positive potentials, a linear current-voltage relationship and a 10-fold higher sensitivity to chromanol 293B. KCNQ1:KCNE3 channels are expressed in colonic crypts and mediate basolateral K(+) recycling required for Cl(-) secretion. We have previously reported the female-specific anti-secretory effects of oestrogen via KCNQ1:KCNE3 channel inhibition in colonic crypts. This study was designed to determine whether sex and oestrogen regulate the expression and function of KCNQ1 and KCNE3 in rat distal colon. Colonic crypts were isolated from Sprague-Dawley rats and used for whole-cell patch-clamp and to extract total RNA and protein. Sheets of epithelium were used for short-circuit current recordings. KCNE1 and KCNE3 mRNA and protein abundance were significantly higher in male than female crypts. No expression of KCNE2 was found and no difference was observed in KCNQ1 expression between male and female (at oestrus) colonic crypts. Male crypts showed a 2.2-fold higher level of association of KCNQ1 and KCNE3 compared to female cells. In female colonic crypts, KCNQ1 and KCNE3 protein expression fluctuated throughout the oestrous cycle and 17ß-oestradiol (E2 10 nM) produced a rapid (<15 min) dissociation of KCNQ1 and KCNE3 in female crypts only. Whole-cell K(+) currents showed a linear current-voltage relationship in male crypts, while K(+) currents in colonic crypts isolated from females displayed voltage-dependent outward rectification. Currents in isolated male crypts and epithelial sheets were 10-fold more sensitive to specific KCNQ1 inhibitors, such as chromanol 293B and HMR-1556, than in female. The effect of E2 on K(+) currents mediated by KCNQ1 with or without different ß-subunits was assayed from current-voltage relations elicited in CHO cells transfected with KCNQ1 and KCNE3 or KCNE1 cDNA. E2 (100 nM) reduced the currents mediated by the KCNQ1:KCNE3 potassium channel and had no effect on currents via KCNQ1:KCNE1 or KCNQ1 alone. Currents mediated by the complex formed by KCNQ1 and the mutant KCNE3-S82A ß-subunit (mutation of the site for PKCδ-promoted phosphorylation and modulation of the activity of KCNE3) showed rapid run-down and insensitivity to E2. Together, these data suggest that oestrogen regulates the expression of the KCNE1 and KCNE3 and with it the gating and pharmacological properties of the K(+) conductance required for Cl(-) secretion. The decreased association of the KCNQ1:KCNE3 channel complex promoted by oestrogen exposure underlies the molecular mechanism for the sexual dimorphism and oestrous cycle dependence of the anti-secretory actions of oestrogen in the intestine.

Extracellular pH in restricted domains as a gating signal for ion channels involved in transepithelial transport.

The importance of intracellular pH (pH(i)) in the regulation of diverse cellular activities ranging from cell proliferation and differentiation to cell cycle, migration and apoptosis has long been recognised. More recently, extracellular pH (pH0), in particular that of relatively inaccessible compartments or domains that occur between cells in tissues, has begun to be acknowledged as a relevant signal in cell regulation. This should not be surprising given the abundant reports highlighting the pH0-dependence of the activity of membrane proteins facing the extracellular space such as receptors, transporters, ion channels and enzymes. Changes in pH affect the ionisation state of proteins through the effect on their titratable groups. There are proteins, however, which respond to pH shifts with conformational changes that are crucial for catalysis or transport activity. In such cases protons act as signalling molecules capable of eliciting fast and localised responses. We provide examples of ion channels that appear fastidiously designed to respond to extracellular pH in a manner that suggests specific functions in transporting epithelia. We shall also present ideas as to how these channels participate in complex transepithelial transport processes and provide preliminary experiments illustrating a new way to gauge pH0 in confined spaces of native epithelial tissue.