Search

Chemical structures of selective blockers of TASK channels contain aromatic groups and amide bonds. Using this rationale, we designed and synthesized a series of compounds based on 3-benzamidobenzoic acid. These compounds block TASK-1 channels by binding to the central cavity. The most active compound is 3-benzoylamino

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 corroborates 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) form a binding site for the phosphoinositide and are 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 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 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 corroborates 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) form a binding site for the phosphoinositide and ensure 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 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. This article is protected by copyright. All rights reserved.

The 4TM family of K channels mediate many of the background potassium currents observed in native cells. They are open across the physiological voltage-range and are regulated by a wide array of neurotransmitters and biochemical mediators. The pore-forming α-subunit contains two pore loop (P) domains and two subunits assemble to form one ion conduction pathway lined by four P domains. It is important to note that single channels do not have two pores but that each subunit has two P domains in its primary sequence; hence the name two-pore domain, or K2P channels (and not two-pore channels). Some of the K2P subunits can form heterodimers across subfamilies (e.g. K2P3.1 with K2P9.1). The nomenclature of 4TM K channels in the literature is still a mixture of IUPHAR and common names. The suggested division into subfamilies, described in the More detailed introduction, is based on similarities in both structural and functional properties within subfamilies and this explains the "common abbreviation" nomenclature in the tables below.

The 4TM family of K channels mediate many of the background potassium currents observed in native cells. They are open across the physiological voltage-range and are regulated by a wide array of neurotransmitters and biochemical mediators. The pore-forming α-subunit contains two pore loop (P) domains and two subunits assemble to form one ion conduction pathway lined by four P domains. It is important to note that single channels do not have two pores but that each subunit has two P domains in its primary sequence; hence the name two P domain, or K2P channels (and not two-pore channels). Some of the K2P subunits can form heterodimers across subfamilies (e.g. K2P3.1 with K2P9.1). The nomenclature of 4TM K channels in the literature is still a mixture of IUPHAR and common names. The suggested division into subfamilies, described in the More detailed introduction, is based on similarities in both structural and functional properties within subfamilies and this explains the "common abbreviation" nomenclature in the tables below.

Kir7.1 KMutations in the Kir7.1 K

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 β-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.

The 4TM family of K channels mediate many of the background potassium currents observed in native cells. They are open across the physiological voltage-range and are regulated by a wide array of neurotransmitters and biochemical mediators. The pore-forming α-subunit contains two pore loop (P) domains and two subunits assemble to form one ion conduction pathway lined by four P domains. It is important to note that single channels do not have two pores but that each subunit has two P domains in its primary sequence; hence the name two P domain, or K2P channels (and not two-pore channels). Some of the K2P subunits can form heterodimers across subfamilies (e.g. K2P3.1 with K2P9.1). The nomenclature of 4TM K channels in the literature is still a mixture of IUPHAR and common names. The suggested division into subfamilies, described in the More detailed introduction, is based on similarities in both structural and functional properties within subfamilies and this explains the "common abbreviation" nomenclature in the tables below.

TASK-3 potassium (K+) channels are highly expressed in the central nervous system, regulating the membrane potential of excitable cells. TASK-3 is involved in neurotransmitter action and has been identified as an oncogenic K+ channel. For this reason, the understanding of the action mechanism of pharmacological modulators of these channels is essential to obtain new therapeutic strategies. In this study we describe the binding mode of the potent antagonist PK-THPP into the TASK-3 channel. PK-THPP blocks TASK-1, the closest relative channel of TASK-3, with almost nine-times less potency. Our results confirm that the binding is influenced by the fenestrations state of TASK-3 channels and occurs when they are open. The binding is mainly governed by hydrophobic contacts between the blocker and the residues of the binding site. These interactions occur not only for PK-THPP, but also for the antagonist series based on 5,6,7,8 tetrahydropyrido[4,3-d]pyrimidine scaffold (THPP series). However, the marked difference in the potency of THPP series compounds such as 20b, 21, 22 and 23 (PK-THPP) respect to compounds such as 17b, inhibiting TASK-3 channels in the micromolar range is due to the presence of a hydrogen bond acceptor group that can establish interactions with the threonines of the selectivity filter.

Inwardly rectifying K

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.

The many mechanisms governing NaCl absorption in the diverse parts of the renal tubule have been largely elucidated, although some of them, as neutral NaCl absorption across the cortical collecting duct or regulation through with-no-lysine (WNK) kinases have emerged only recently. Chloride channels, which are important players in these processes, at least in the distal nephron, are the focus of this review. Over the last 20-year period, experimental studies using molecular, electrophysiological, and physiological/functional approaches have deepened and renewed our views on chloride channels and their role in renal function. Two chloride channels of the ClC family, named as ClC-Ka and ClC-Kb in humans and ClC-K1 and ClC-K2 in other mammals, are preponderant and play complementary roles: ClC-K1/Ka is mainly involved in the building of the interstitial cortico-medullary concentration gradient, while ClC-K2/Kb participates in NaCl absorption in the thick ascending limb, distal convoluted tubule and the intercalated cells of the collecting duct. The two ClC-Ks might also be involved indirectly in proton secretion by type A intercalated cells. Other chloride channels in the kidneys include CFTR, TMEM16A, and probably volume-regulated LRRC8 chloride channels, whose function and molecular identity have not as yet been established. © 2019 American Physiological Society. Compr Physiol 9:301-342, 2019.

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.

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 K2Pchannels 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 K2Pand 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. K2Pchannels 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 K2Pchannels might explain their normal and pathological functions. Although a great deal has been learned about structure, molecular details of gating, and physiological functions of K2Pand Kir K+-transport channels, this has been only scratching at the surface. More molecular and animal studies are clearly needed to deepen our knowledge.

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 KCa3.1 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.

Kir7.1 encoded by the Kcnj13 gene in the mouse is an inwardly rectifying K+ channel present in epithelia where it shares membrane localization with the Na+/K+-pump. Further investigations of the localisation and function of Kir7.1 would benefit from the availability of a knockout mouse, but perinatal mortality attributed to cleft palate in the neonate has thwarted this research. To facilitate localisation studies we now use CRISPR/Cas9 technology to generate a knock-in mouse, the Kir7.1-HA that expresses the channel tagged with a haemagglutinin (HA) epitope. The availability of antibodies for the HA epitope allows for application of western blot and immunolocalisation methods using widely available anti-HA antibodies with WT tissues providing unambiguous negative control. We demonstrate that Kir7.1-HA cloned from the choroid plexus of the knock-in mouse has the electrophysiological properties of the native channel, including characteristically large Rb+ currents. These large Kir7.1-mediated currents are accompanied by abundant apical membrane Kir7.1-HA immunoreactivity. WT-controlled western blots demonstrate the presence of Kir7.1-HA in the eye and the choroid plexus, trachea and lung, and intestinal epithelium but exclusively in the ileum. In the kidney, and at variance with previous reports in the rat and guinea-pig, Kir7.1-HA is expressed in the inner medulla but not in the cortex or outer medulla. In isolated tubules immunoreactivity was associated with inner medulla collecting ducts but not thin limbs of the loop of Henle. Kir7.1-HA shows basolateral expression in the respiratory tract epithelium from trachea to bronchioli. The channel also appears basolateral in the epithelium of the nasal cavity and nasopharynx in newborn animals. We show that HA-tagged Kir7.1 channel introduced in the mouse by a knock-in procedure has functional properties similar to the native protein and the animal thus generated has clear advantages in localisation studies. It might therefore become a useful tool to unravel Kir7.1 function in the different organs where it is expressed.

Two-pore domain potassium (K2P) channels maintain the cell&rsquo, s background conductance by stabilizing the resting membrane potential. They assemble as dimers possessing four transmembrane helices in each subunit. K2P channels were crystallized in &ldquo, up&rdquo, and &ldquo, down&rdquo, states. The movements of the pore-lining transmembrane TM4 helix produce the aperture or closure of side fenestrations that connect the lipid membrane with the central cavity. When the TM4 helix is in the up-state, the fenestrations are closed, while they are open in the down-state. It is thought that the fenestration states are related to the activity of K2P channels and the opening of the channels preferentially occurs from the up-state. TASK-2, a member of the TALK subfamily of K2P channels, is opened by intracellular alkalization leading the deprotonation of the K245 residue at the end of the TM4 helix. This charge neutralization of K245 could be sensitive or coupled to the fenestration state. Here, we describe the relationship between the states of the intramembrane fenestrations and K245 residue in TASK-2 channel. By using molecular modeling and simulations, we show that the protonated state of K245 (K245+) favors the open fenestration state and, symmetrically, that the open fenestration state favors the protonated state of the lysine residue. We show that the channel can be completely blocked by Prozac, which is known to induce fenestration opening in TREK-2. K245 protonation and fenestration aperture have an additive effect on the conductance of the channel. The opening of the fenestrations with K245+ increases the entrance of lipids into the selectivity filter, blocking the channel. At the same time, the protonation of K245 introduces electrostatic potential energy barriers to ion entrance. We computed the free energy profiles of ion penetration into the channel in different fenestration and K245 protonation states, to show that the effects of the two transformations are summed up, leading to maximum channel blocking. Estimated rates of ion transport are in qualitative agreement with experimental results and support the hypothesis that the most important barrier for ion transport under K245+ and open fenestration conditions is the entrance of the ions into the channel.

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 β-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.

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 Gβγ 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-β-S instead. Purified Gβγ introduced intracellularly also inhibited TASK-2 independently of whether GTP or GDP-β-S was present. The effects of GTP-γ-S and Gβγ 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 Gβ1 and Gβ2 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-β-S. Our data are compatible with the concept that TASK-2 channels are modulated by Gβγ 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.

A1899 is a potent and selective inhibitor of the two-pore domain potassium (K2P) channel TASK-1. It was previously reported that A1899 acts as an open-channel blocker and binds to residues of the P1 and P2 regions, the M2 and M4 segments, and the halothane response element. The recently described crystal structures of K2P channels together with the newly identified side fenestrations indicate that residues relevant for TASK-1 inhibition are not purely facing the central cavity as initially proposed. Accordingly, the TASK-1 binding site and the mechanism of inhibition might need a re-evaluation. We have used TASK-1 homology models based on recently crystallized K2P channels and molecular dynamics simulation to demonstrate that the highly potent TASK-1 blocker A1899 requires binding to residues located in the side fenestrations. Unexpectedly, most of the previously described residues that interfere with TASK-1 blockade by A1899 project their side chains toward the fenestration lumina, underlining the releva...

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.

Background & Aims The fluid secretion model predicts that intestinal obstruction disorders can be alleviated by promoting epithelial Cl − secretion. The adenosine 3′,5′-cyclic monophosphate (cAMP)-activated anion channel CFTR mediates Cl − -dependent fluid secretion in the intestine. Although the role of the ClC-2 channel has not been determined in the intestine, this voltage-gated Cl − channel might compensate for the secretory defects observed in patients with cystic fibrosis and other chronic constipation disorders. We investigated whether mice that lack ClC-2 channels ( Clcn2 −/− ) have defects in intestinal ion transport. Methods Immunolocalization and immunoblot analyses were used to determine the cellular localization and the amount of ClC-2 expressed in mouse early distal colon (EDC) and late distal colon (LDC). Colon sheets from wild-type and Clcn2 −/− littermates were mounted in Ussing chambers to determine transepithelial bioelectrical parameters and Na + , K + , and Cl − fluxes. Results Expression of ClC-2 was higher in the basolateral membrane of surface cells in the EDC compared with the LDC, with little expression in crypts. Neither cAMP nor Ca 2+ -induced secretion of Cl − was affected in the EDC or LDC of Clcn2 −/− mice, whereas the amiloride-sensitive short-circuit current was increased approximately 3-fold in Clcn2 −/− EDC compared with control littermates. Conversely, electroneutral Na + , K + , and Cl − absorption was dramatically reduced in colons of Clcn2 −/− mice. Conclusions Basolateral ClC-2 channels are required for colonic electroneutral absorption of NaCl and KCl. The increase in the amiloride-sensitive short-circuit current in Clcn2 −/− mice revealed a compensatory mechanism that is activated in the colons of mice that lack the ClC-2 channel.

Dent's disease is an X-linked recessive disorder affecting the proximal tubules. Mutations in the 2Cl(-)/H(+) exchanger ClC-5 gene CLCN5 are frequently associated with Dent's disease. Functional characterization of mutations of CLCN5 have helped to elucidate the physiopathology of Dent's disease and provided evidence that several different mechanisms underlie the ClC-5 dysfunction in Dent's disease. Modeling studies indicate that many CLCN5 mutations are located at the interface between the monomers of ClC-5, demonstrating that this protein region plays an important role in Dent's disease. On the basis of functional data, CLCN5 mutations can be divided into three different classes. Class 1 mutations impair processing and folding, and as a result, the ClC-5 mutants are retained within the endoplasmic reticulum and targeted for degradation by quality control mechanisms. Class 2 mutations induce a delay in protein processing and reduce the stability of ClC-5. As a consequence, the cell surface expression and currents of the ClC-5 mutants are lower. Class 3 mutations do not alter the trafficking of ClC-5 to the cell surface and early endosomes but induce altered electrical activity. Here, we discuss the functional consequences of the three classes of CLCN5 mutations on ClC-5 structure and function.

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 17β-oestradiol (E2 10 nM) produced a rapid (

The application of genetic modification offers considerable opportunities for more efficient and more effective fish aquaculture. Susceptibility to infectious diseases and limitations in growth rates could be successfully tackled by the application of transgenic technology. Acceptance of transgenic technology applied to commercial fish has to surmount technical difficulties, regulatory barriers and public acceptance concerns. It seems desirable to implement transgenesis strategies that cause the least qualitative and quantitative alterations in the genetic composition of the resulting enhanced fish. With Atlantic salmon being the most important farmed fish worldwide, it seemed of interest to obtain genomic sequences from this species capable of directing autogenic expression of selected transgenes. We now report the isolation of the β-actin promoter from genomic DNA of Atlantic salmon locally produced in Chile. The activity of the promoter was demonstrated ex vivo in cultured salmon cells, as well as in vivo in transgenic fish. Transiently transfected CHSE-214 cells exhibited constitutive expression of reporter constructs driven by the cloned salmon β-actin genomic region. The suitability of the Atlantic salmon β-actin promoter to drive expression of the fluorescent protein EGFP in transgenic zebrafish was demonstrated. Germline transmission of the transgenes suggests that this promoter could be safely applied into fish transgenesis without health concerns. The isolation of functionally competent β-actin promoter from Atlantic salmon opens the way for its use in autogenic transgenesis in this commercially important salmonid species.

Mice have proven to be powerful models for the study of human physiology and pathophysiology. With the advent of techniques for genomic manipulation, the possibilities for studying inherited diseases in this convenient laboratory mammal are increasing by the day. It has been reported that when knocking out or otherwise modifying genes of interest in mice, the phenotype obtained can vary markedly depending on the genetic background of the animals used in the study. The aim of this work was to study whether the genetic background can influence the characteristics of fluid and electrolyte transepithelial transport in the distal colon of three mouse strains most in use in our and other laboratories. Ussing chamber recordings revealed that the colons of C57Bl/6J, Sv 129 and Black Swiss animals have distinctive responses to the calcium agonists carbachol and histamine that are not explained by the presence of different types of muscarinic and histaminergic receptors in these tissues. We have also found differences in the cAMP-activated, KCNMA1-channel-dependent potassium secretion between the strains. We interpret this to indicate a unique distribution of KCNMA1 channels in lower parts of the crypt of Sv 129 colonic epithelium compared with that of C57Bl/6J and Black Swiss animals. The reported differences should be taken into account when choosing the genetic background of animals to be used for genetic modification.

Currents mediated by calcium-activated chloride channels (CaCCs), observed for the first time in Xenopus oocytes, have been recorded in many cells and tissues ranging from different types of neurons to epithelial and muscle cells. CaCCs play a role in the regulation of excitability in neurons including sensory receptors. In addition, they are crucial mediators of chloride movements in epithelial cells where their activity regulates electrolyte and fluid transport. The roles of CaCCs, particularly in epithelia, are briefly reviewed with emphasis on their function in secretory epithelia. The recent identification by three independent groups, using different strategies, of TMEM16A as the molecular counterpart of the CaCC is discussed. TMEM16A is part of a family that has 10 other members in mice. The discovery of the potential TMEM16 anion channel activity opens the way for the molecular investigation of the role of these anion channels in specific cells and in organ physiology and pathophysiology. The identification of TMEM16A protein as a CaCC chloride channel molecule represents a great triumph of scientific perseverance and ingenuity. The varied approaches used by the three independent research groups also augur well for the solidity of the discovery.

The ClC transport protein family comprises both Cl(-) ion channel and H(+)/Cl(-) and H(+)/NO(3)(-) 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 approximately 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 pK(a) 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.

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.

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.

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 pK a 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 pK a , 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 pK a of charged amino acids within a protein.

Two-pore domain potassium (K2P) channels are membrane proteins widely identified in mammals, plants, and other organisms. A functional channel is a dimer with each subunit comprising two pore-forming loops and four transmembrane domains. The genome of the model plant Arabidopsis thaliana harbors five genes coding for K2P channels. Homologs of Arabidopsis K2P channels have been found in all higher plants sequenced so far. As with the K2P channels in mammals, plant K2P channels are targets of external and internal stimuli, which fine-tune the electrical properties of the membrane for specialized transport and/or signaling tasks. Plant K2P channels are modulated by signaling molecules such as intracellular H+ and calcium and physical factors like temperature and pressure. In this review, we ask the following: What are the similarities and differences between K2P channels in plants and animals in terms of their physiology? What is the nature of the last common ancestor (LCA) of these two groups of proteins? To answer these questions, we present physiological, structural, and phylogenetic evidence that discards the hypothesis proposing that the duplication and fusion that gave rise to the K2P channels occurred in a prokaryote LCA. Conversely, we argue that the K2P LCA was most likely a eukaryote organism. Consideration of plant and animal K2P channels in the same study is novel and likely to stimulate further exchange of ideas between students of these fields.

Excitatory synaptic transmission is accompanied by a local surge in interstitial lactate that occurs despite adequate oxygen availability, a puzzling phenomenon termed aerobic glycolysis. In addition to its role as an energy substrate, recent studies have shown that lactate modulates neuronal excitability acting through various targets, including NMDA receptors and G-protein-coupled receptors specific for lactate, but little is known about the cellular and molecular mechanisms responsible for the increase in interstitial lactate. Using a panel of genetically encoded fluorescence nanosensors for energy metabolites, we show here that mouse astrocytes in culture, in cortical slices, andin vivomaintain a steady-state reservoir of lactate. The reservoir was released to the extracellular space immediately after exposure of astrocytes to a physiological rise in extracellular K+or cell depolarization. Cell-attached patch-clamp analysis of cultured astrocytes revealed a 37 pS lactate-permeable ion channel activated by cell depolarization. The channel was modulated by lactate itself, resulting in a positive feedback loop for lactate release. A rapid fall in intracellular lactate levels was also observed in cortical astrocytes of anesthetized mice in response to local field stimulation. The existence of an astrocytic lactate reservoir and its quick mobilization via an ion channel in response to a neuronal cue provides fresh support to lactate roles in neuronal fueling and in gliotransmission.

Potassium channels have a conserved selectivity filter that is important in determining which ions are conducted and at what rate. Although K+ channels of different conductance characteristics are known, they differ more widely in the way their opening and closing, the gating, is governed. TASK and TALK subfamily proteins are two-pore region KCNK K+ channels gated open by extracellular pH. We discuss the mechanism for this gating in terms of electrostatic effects on the pore changing the occupancy and open probability of the channels in a way reminiscent of C-type inactivation gating at the selectivity filter. Essential to this proposed mechanism is the replacement of two highly conserved aspartate residues at the pore mouth by asparagine or histidine residues in the TALK and TASK channels.

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.

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 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.

The principal function of the colon in fluid homeostasis is the absorption of NaCl and water. Apical membrane Na+ channels, Na+/H+ and Cl−/HCO[Formula: see text] exchangers, have all been postulated to mediate NaCl entry into colonocytes. The identity of the basolateral exit pathway for Cl− is unknown. We have previously demonstrated the presence of the ClC-2 transcript in the guinea pig intestine. Now we explore in more detail, the tissue and cellular distribution of chloride channel ClC-2 in the distal colon by in situ hybridization and immunohistochemistry. The patch-clamp technique was used to characterize Cl− currents in isolated surface epithelial cells from guinea pig distal colon and these were compared with those mediated by recombinant guinea pig (gp)ClC-2. ClC-2 mRNA and protein were found in the surface epithelium of the distal colon. Immunolocalization revealed that, in addition to some intracellular labeling, ClC-2 was present in the basolateral membranes but absent from the apical pole of colonocytes. Isolated surface epithelial cells exhibited hyperpolarization-activated chloride currents showing a Cl− > I− permeability and Cd2+ sensitivity. These characteristics, as well as some details of the kinetics of activation and deactivation, were very similar to those of recombinant gpClC-2 measured in parallel experiments. The presence of active ClC-2 type currents in surface colonic epithelium, coupled to a basolateral location for ClC-2 in the distal colon, suggests a role for ClC-2 channel in mediating basolateral membrane exit of Cl− as an essential step in a NaCl absorption process.

Two-pore domain potassium (K2P) channels are membrane proteins widely identified in mammals, plants, and other organisms. A functional channel is a dimer with each subunit comprising two pore-forming loops and four transmembrane domains. The genome of the model plant Arabidopsis thaliana harbors five genes coding for K2P channels. Homologs of Arabidopsis K2P channels have been found in all higher plants sequenced so far. As with the K2P channels in mammals, plant K2P channels are targets of external and internal stimuli, which fine-tune the electrical properties of the membrane for specialized transport and/or signaling tasks. Plant K2P channels are modulated by signaling molecules such as intracellular H(+) and calcium and physical factors like temperature and pressure. In this review, we ask the following: What are the similarities and differences between K2P channels in plants and animals in terms of their physiology? What is the nature of the last common ancestor (LCA) of these two groups of proteins? To answer these questions, we present physiological, structural, and phylogenetic evidence that discards the hypothesis proposing that the duplication and fusion that gave rise to the K2P channels occurred in a prokaryote LCA. Conversely, we argue that the K2P LCA was most likely a eukaryote organism. Consideration of plant and animal K2P channels in the same study is novel and likely to stimulate further exchange of ideas between students of these fields.

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.

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 Ca(2+). 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(+) congruent with 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 IC(50) of 25 microm. 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.