Electrophysiological and molecular identification of hepatocellular volume-activated K channels

Although K⁺ channels are essential for hepatocellular function, it is not known which channels are involved in the regulatory volume decrease (RVD) in these cells. We have used a combination of electrophysiological and molecular approaches to describe the potential candidates for these channels. The dialysis of short-term cultured rat hepatocytes with a hypotonic solution containing high K⁺ and low Cl⁻ concentration caused the slow activation of an outward, time-independent current under whole-cell configuration of the patch electrode voltage clamp. The reversal potential of this current suggested that K⁺ was the primary charge carrier. The swelling-induced K⁺ current (I_Kvol) occurred in the absence of Ca²⁺ and was inhibited with 1 μM Ca²⁺ in the pipette solution. The activation of I_Kvol required both Mg²⁺ and ATP and an increasing concentration of Mg-ATP from 0.25 through 0.5 to 0.9 mM activated I_Kvol increasingly faster and to a larger extent. The KCNQ1 inhibitor chromanol 293B reversibly depressed I_Kvol with an IC₅₀ of 26 μM. RT-PCR detected the expression of members of the KCNQ family from KCNQ1 to KCNQ5 and of the accessory proteins KCNE1 to KCNE3 in the rat hepatocytes, but not KCNQ2 and KCNE2 in human liver. Western blotting showed KCNE3 expression in a plasma membrane-enriched fraction from rat hepatocytes. The results suggest that KCNQ1, probably with KCNE2 or KCNE3 as its accessory unit, provides a significant fraction of I_Kvol in rat hepatocytes.     

The Oxidant Thimerosal Modulates Gating Behavior of KCNQ1 by Interaction with the Channel Outer Shell

Thimerosal (o-Ethylmercurithio)benzoic acid, TMS), a membrane-impermeable, sulfhydryl-oxidizing agent, has been described to increase the K+ current IKs in KCNE1-injected Xenopus laevis oocytes. Since there are no cysteine residues in the extracellular domain of KCNE1, it has been proposed that TMS interacts with its partner protein KCNQ1. The aim of this study was therefore to investigate the interaction of TMS with KCNQ1 and the respective K+current IK. In CHO cells stably transfected with KCNQ1/KCNE1, TMS increased IKs, whereas in CHO cells expressing KCNQ1 alone, TMS initially decreased IK. TMS also affected the cytosolic pH (pHi) and the cytosolic Ca2+ activity ([Ca2+]i) in these cells. TMS slowly decreased pHi. With a short delay, TMS increased [Ca2+]i by store depletion and capacitative influx. The time course of the effects of TMS on pHi and [Ca2+]i did not correlate with the effect of TMS on IK. We therefore anticipated a different mode of action by TMS and investigated the influence of TMS on cysteine residues of KCNQ1. For this purpose, KCNQ1wt and two mutants lacking a cysteine residue in the S6 or the S3 segment (KCNQ1C331A and KCNQ1C214A, respectively) were expressed in Xenopus laevis oocytes. A sustained current decrease was observed in KCNQ1wt and KCNQ1C331A, but not in KCNQ1C214A-injected oocytes. The analysis of tail currents, I/V curves and activation kinetics revealed a complex effect of TMS on the gating of KCNQ1wt and KCNQ1C331A. In another series we investigated the effect of TMS on IKs. TMS increased IKs of KCNQ1C214A/KCNE1-injected oocytes significantly less than IKs in KCNQ1wt/KCNE1- or KCNQ1C331A/KCNE1-injected cells. These results suggest that thimerosal interacts with the cysteine residue C214 in the S3 segment of KCNQ1, leading to a change of its gating properties. Our results support the idea that not only the inner shell, but also the outer shell of the channel is important for the gating behavior of voltage dependent K+ channels.     

KCNE4 juxtamembrane region is required for interaction with calmodulin and for functional suppression of KCNQ1

Voltage-gated potassium (K(V)) channels, such as KCNQ1 (K(V)7.1), are modulated by accessory subunits and regulated by intracellular second messengers. Accessory subunits belonging to the KCNE family exert diverse functional effects on KCNQ1, have been implicated in the pathogenesis of various genetic disorders of heart rhythm, and contribute to transducing intracellular signaling events into changes in K(V) channel activity. We investigated the interactions between calmodulin (CaM), the ubiquitous Ca(2+)-transducing protein that binds and confers Ca(2+) sensitivity to the biophysical properties of KCNQ1, and KCNE4. These studies were motivated by the observed similarities between the suppression of KCNQ1 function by pharmacological disruption of KCNQ1-CaM interactions and the effects of KCNE4 co-expression on the channel. We determined that KCNE4, but not KCNE1, can biochemically interact with CaM and that this interaction is Ca(2+)-dependent and requires a tetraleucine motif in the juxtamembrane region of the KCNE4 C terminus. Furthermore, disruption of the KCNE4-CaM interaction either by mutagenesis of the tetraleucine motif or by acute Ca(2+) chelation impairs the ability of KCNE4 to inhibit KCNQ1. Our findings have potential relevance to KCNQ1 regulation both by KCNE accessory subunits and by an important intracellular signaling molecule.     

Modulation of functional properties of KCNQ1 channel by association of KCNE1 and KCNE2

The KCNE proteins (KCNE1 through KCNE5) function as beta-subunits of several voltage-gated K(+) channels. Assembly of KCNQ1 K(+) channel alpha-subunits and KCNE1 underlies cardiac I(Ks), while KCNQ1 interacts with all other members of KCNE forming complexes with different properties. Here we investigated synergic actions of KCNE1 and KCNE2 on functional properties of KCNQ1 heterologously expressed in COS7 cells. Patch-clamp recordings from cells expressing KCNQ1 and KCNE1 exhibited the slowly activating current, while co-expression of KCNQ1 with KCNE2 produced a practically time-independent current. When KCNQ1 was co-expressed with both of KCNE1 and KCNE2, the membrane current exhibited a voltage- and time-dependent current whose characteristics differed substantially from those of the KCNQ1/KCNE1 current. The KCNQ1/KCNE1/KCNE2 current had a more depolarized activation voltage, a faster deactivation kinetics, and a less sensitivity to activation by mefenamic acid. These results suggest that KCNE2 can functionally couple to KCNQ1 even in the presence of KCNE1.     

Structural requirements for differential sensitivity of KCNQ K+ channels to modulation by Ca2+/calmodulin

Calmodulin modulation of ion channels has emerged as a prominent theme in biology. The sensitivity of KCNQ1-5 K+ channels to modulation by Ca2+/calmodulin (CaM) was studied using patch-clamp, Ca2+ imaging, and biochemical and pharmacological approaches. Coexpression of CaM in Chinese hamster ovary (CHO) cells strongly reduced currents of KCNQ2, KCNQ4, and KCNQ5, but not KCNQ1 or KCNQ3. In simultaneous current recording/Ca2+ imaging experiments, CaM conferred Ca2+ sensitivity to KCNQ4 and KCNQ5, but not to KCNQ1, KCNQ3, or KCNQ1/KCNE1 channels. A chimera constructed from the carboxy terminus of KCNQ4 and the rest KCNQ1 displayed Ca2+ sensitivity similar to KCNQ4. Chimeras constructed from different lengths of the KCNQ4 carboxy terminal and the rest KCNQ3 localized a region that confers sensitivity to Ca2+/CaM. Lobe-specific mutations of CaM revealed that its amino-terminal lobe mediates the Ca2+ sensitivity of the KCNQ/CaM complex. The site of CaM action within the channel carboxy terminus overlaps with that of the KCNQ opener N-ethylmaleimide (NEM). We found that CaM overexpression reduced NEM augmentation of KCNQ2, KCNQ4, and KCNQ5, and NEM pretreatment reduced Ca2+/CaM-mediated suppression of M current in sympathetic neurons by bradykinin. We propose that two functionally distinct types of carboxy termini underlie the observed differences among this channel family.     

KCNE4 can co-associate with the I(Ks) (KCNQ1-KCNE1) channel complex

Voltage-gated potassium (K(V)) channels can form heteromultimeric complexes with a variety of accessory subunits, including KCNE proteins. Heterologous expression studies have demonstrated diverse functional effects of KCNE subunits on several K(V) channels, including KCNQ1 (K(V)7.1) that, together with KCNE1, generates the slow-delayed rectifier current (I(Ks)) important for cardiac repolarization. In particular, KCNE4 exerts a strong inhibitory effect on KCNQ1 and other K(V) channels, raising the possibility that this accessory subunit is an important potassium current modulator. A polyclonal KCNE4 antibody was developed to determine the human tissue expression pattern and to investigate the biochemical associations of this protein with KCNQ1. We found that KCNE4 is widely and variably expressed in several human tissues, with greatest abundance in brain, liver and testis. In heterologous expression experiments, immunoprecipitation followed by immunoblotting was used to establish that KCNE4 directly associates with KCNQ1, and can co-associate together with KCNE1 in the same KCNQ1 complex to form a 'triple subunit' complex (KCNE1-KCNQ1-KCNE4). We also used cell surface biotinylation to demonstrate that KCNE4 does not impair plasma membrane expression of either KCNQ1 or the triple subunit complex, indicating that biophysical mechanisms probably underlie the inhibitory effects of KCNE4. The observation that multiple KCNE proteins can co-associate with and modulate KCNQ1 channels to produce biochemically diverse channel complexes has important implications for understanding K(V) channel regulation in human physiology.     

Regulation and Properties of KCNQ1 (KVLQT1) and Impact of the Cystic Fibrosis Transmembrane Conductance Regulator

The K⁺ channel KCNQ1 (K_VLQT1) is a voltage-gated K⁺ channel, coexpressed with regulatory subunits such as KCNE1 (IsK, mink) or KCNE3, depending on the tissue examined. Here, we investigate regulation and properties of human and rat KCNQ1 and the impact of regulators such as KCNE1 and KCNE3. Because the cystic fibrosis transmembrane conductance regulator (CFTR) has also been suggested to regulate KCNQ1 channels we studied the effects of CFTR on KCNQ1 in Xenopus oocytes. Expression of both human and rat KCNQ1 induced time dependent K⁺ currents that were sensitive to Ba²⁺ and 293B. Coexpression with KCNE1 delayed voltage activation, while coexpression with KCNE3 accelerated current activation. KCNQ1 currents were activated by an increase in intracellular cAMP, independent of coexpression with KCNE1 or KCNE3. cAMP dependent activation was abolished in N-terminal truncated hKCNQ1 but was still detectable after deletion of a single PKA phosphorylation motif. In the presence but not in the absence of KCNE1 or KCNE3, K⁺ currents were activated by the Ca²⁺ ionophore ionomycin. Coexpression of CFTR with either human or rat KCNQ1 had no impact on regulation of KCNQ1 K⁺ currents by cAMP but slightly shifted the concentration response curve for 293B. Thus, KCNQ1 expressed in Xenopus oocytes is regulated by cAMP and Ca²⁺ but is not affected by CFTR. Received: 13 December 2000/Revised: 30 March 2001     

KCNE5 induces time- and voltage-dependent modulation of the KCNQ1 current

The function of the KCNE5 (KCNE1-like) protein has not previously been described. Here we show that KCNE5 induces both a time- and voltage-dependent modulation of the KCNQ1 current. Interaction of the KCNQ1 channel with KCNE5 shifted the voltage activation curve of KCNQ1 by more than 140 mV in the positive direction. The activation threshold of the KCNQ1+KCNE5 complex was +40 mV and the midpoint of activation was +116 mV. The KCNQ1+KCNE5 current activated slowly and deactivated rapidly as compared to the KCNQ1+KCNE1 at 22 degrees C; however, at physiological temperature, the activation time constant of the KCNQ1+KCNE5 current decreased fivefold, thus exceeding the activation rate of the KCNQ1+KCNE1 current. The KCNE5 subunit is specific for the KCNQ1 channel, as none of other members of the KCNQ-family or the human ether a-go-go related channel (hERG1) was affected by KCNE5. Four residues in the transmembrane domain of the KCNE5 protein were found to be important for the control of the voltage-dependent activation of the KCNQ1 current. We speculate that since KCNE5 is expressed in cardiac tissue it may here along with the KCNE1 beta-subunit regulate KCNQ1 channels. It is possible that KCNE5 shapes the I(Ks) current in certain parts of the mammalian heart.     

KCNE1 binds to the KCNQ1 pore to regulate potassium channel activity

Potassium channels control the resting membrane potential and excitability of biological tissues. Many voltage-gated potassium channels are controlled through interactions with accessory subunits of the KCNE family through mechanisms still not known. Gating of mammalian channel KCNQ1 is dramatically regulated by KCNE subunits. We have found that multiple segments of the channel pore structure bind to the accessory protein KCNE1. The sites that confer KCNE1 binding are necessary for the functional interaction, and all sites must be present in the channel together for proper regulation by the accessory subunit. Specific gating control is localized to a single site of interaction between the ion channel and accessory subunit. Thus, direct physical interaction with the ion channel pore is the basis of KCNE1 regulation of K+ channels.     

KCNQ1/KCNE1 assembly,co-translation not required

Voltage-gated potassium channels are often assembled with accessory proteins which increases their functional diversity. KCNE proteins are small accessory proteins that modulate voltage-gated potassium (K_V) channels. Although the functional effects of various KCNE proteins have been described, many questions remain regarding their assembly with the pore-forming subunits. For example, while previous experiments with some K_V channels suggest that the association of the pore-subunit with the accessory subunits occurs co-translationally in the endoplasmic reticulum, it is not known whether KCNQ1 assembly with KCNE1 occurs in a similar manner to generate the medically important cardiac slow delayed rectifier current (IKs). In this study we used a novel approach to demonstrate that purified recombinant human KCNE1 protein (prKCNE1) modulates KCNQ1 channels heterologously expressed in Xenopus oocytes resulting in generation of IKs. Incubation of KCNQ1-expressing oocytes with cycloheximide did not prevent IKs expression following prKCNE1 injection. By contrast, incubation with brefeldin A prevented KCNQ1 modulation by prKCNE1. Moreover, injection of the trafficking-deficient KCNE1-L51H reduced KCNQ1 currents. Together, these observations indicate that while assembly of KCNE1 with KCNQ1 does not require co-translation, functional KCNQ1-prKCNE1 channels assemble early in the secretory pathway and reach the plasma membrane via vesicular trafficking.     

KCNQ1/KCNE1 channels during germ-cell differentiation in the rat: expression associated with testis pathologies

KCNQ1/KCNE1 channels are responsible for the Jervell-Lange-Nielsen cardiac syndrome, which is also characterized by congenital deafness. KCNQ1/KCNE1 is crucial for K+ transport in the inner ear. We show that KCNQ1 and KCNE1 are associated in testis and that their expression is closely regulated during development. Both genes were expressed in undifferentiated germ cells in 21-day-old rats and mostly confined to basal immature germ cells in adulthood. Leydig and Sertoli cells were negative. KCNQ1 and KCNE1 were also studied in various germ-cell pathologies. First, in spontaneous unilateral rat testis atrophy, hematoxylin-eosin analysis revealed massive germ-cell aplasia with only Sertoli cells and groups of interstitial Leydig cells. In these samples, KCNQ1 and KCNE1 were not expressed. In human seminoma samples characterized by a proliferation of undifferentiated germ cells, KCNQ1/KCNE1 protein levels were higher than in healthy samples. Our results demonstrate that the expression of KCNQ1 and KCNE1 is associated with early stages of spermatogenesis and with the presence of undifferentiated healthy or neoplastic germ cells. The presence of a K+ rich-fluid in the seminiferous tubule suggests that KCNQ1/KCNE1 is involved in K+ transport, probably during germ-cell development.     

KCNE variants reveal a critical role of the beta subunit carboxyl terminus in PKA-dependent regulation of the IKs potassium channel

Co-assembly of KCNQ1 with different accessory, or beta, subunits that are members of the KCNE family results in potassium (K⁺) channels that conduct functionally distinct currents. The alpha subunit KCNQ1 conducts a slowly-activated delayed rectifier K⁺ current (I_Ks), a major contributor to cardiac repolarization, when co-assembled with KCNE1 and channels that favor the open state when co-assembled with either KCNE2 or KCNE3. In the heart, stimulation of the sympathetic nervous system enhances I_Ks. A macromolecular signaling complex of the I_Ks channel including the targeting protein Yotiao coordinates up- or down- regulation of channel activity by protein kinase A (PKA) phosphorylation and dephosphorylation of molecules in the complex. β-adrenergic receptor mediated I_Ks up-regulation, a functional consequence of PKA phosphorylation of the KCNQ1 amino terminus (N-T), requires co-expression of KCNQ1/Yotiao with KCNE1. Here, we report that co-expression of KCNE2, like KCNE1, confers a functional channel response to KCNQ1 phosphorylation, but co-expression of KCNE3 does not. Amino acid sequence comparison among the KCNE peptides, and KCNE1 truncation experiments, reveal a segment of the predicted intracellular KCNE1 carboxyl terminus (C-T) that is necessary for functional transduction of PKA phosphorylated KCNQ1. Moreover, chimera analysis reveals a region of KCNE1 sufficient to confer cAMP-dependent functional regulation upon the KCNQ1_KCNE3_Yotiao channel. The property of specific beta subunits to transduce post-translational regulation of alpha subunits of ion channels adds another dimension to our understanding molecular mechanisms underlying the diversity of regulation of native K⁺ channels.     

KCNE beta subunits determine pH sensitivity of KCNQ1 potassium channels.

BACKGROUND/AIMS: Heteromeric KCNEx/KCNQ1 (=KvLQT1, Kv7.1) K(+) channels are important for repolarization of cardiac myocytes, endolymph secretion in the inner ear, gastric acid secretion, and transport across epithelia. They are modulated by pH in a complex way: homomeric KCNQ1 is inhibited by external acidification (low pH(e)); KCNE2/KCNQ1 is activated; and for KCNE1/KCNQ1, variable effects have been reported. Methods: The role of KCNE subunits for the effect of pH(e) on KCNQ1 was analyzed in transfected COS cells and cardiac myocytes by the patch-clamp technique. RESULTS: In outside-out patches of transfected cells, hKCNE2/hKCNQ1 current was increased by acidification down to pH 4.5. Chimeras with the acid-insensitive hKCNE3 revealed that the extracellular N-terminus and at least part of the transmembrane domain of hKCNE2 are needed for activation by low pH(e). hKCNE1/hKCNQ1 heteromeric channels exhibited marked changes of biophysical properties at low pH(e): The slowly activating hKCNE1/hKCNQ1 channels were converted into constitutively open, non-deactivating channels. Experiments on guinea pig and mouse cardiac myocytes pointed to an important role of KCNQ1 during acidosis implicating a significant contribution to cardiac repolarization under acidic conditions. CONCLUSION: External pH can modify current amplitude and biophysical properties of KCNQ1. KCNE subunits work as molecular switches by modulating the pH sensitivity of human KCNQ1.     

KCNQ1 subdomains involved in KCNE modulation revealed by an invertebrate KCNQ1 orthologue

KCNQ1 channels are voltage-gated potassium channels that are widely expressed in various non-neuronal tissues, such as the heart, pancreas, and intestine. KCNE proteins are known as the auxiliary subunits for KCNQ1 channels. The effects and functions of the different KCNE proteins on KCNQ1 modulation are various; the KCNQ1-KCNE1 ion channel complex produces a slowly activating potassium channel that is crucial for heartbeat regulation, while the KCNE3 protein makes KCNQ1 channels constitutively active, which is important for K(+) and Cl(-) transport in the intestine. The mechanisms by which KCNE proteins modulate KCNQ1 channels have long been studied and discussed; however, it is not well understood how different KCNE proteins exert considerably different effects on KCNQ1 channels. Here, we approached this point by taking advantage of the recently isolated Ci-KCNQ1, a KCNQ1 homologue from marine invertebrate Ciona intestinalis. We found that Ci-KCNQ1 alone could be expressed in Xenopus laevis oocytes and produced a voltage-dependent potassium current, but that Ci-KCNQ1 was not properly modulated by KCNE1 and totally unaffected by coexpression of KCNE3. By making chimeras of Ci-KCNQ1 and human KCNQ1, we determined several amino acid residues located in the pore region of human KCNQ1 involved in KCNE1 modulation. Interestingly, though, these amino acid residues of the pore region are not important for KCNE3 modulation, and we subsequently found that the S1 segment plays an important role in making KCNQ1 channels constitutively active by KCNE3. Our findings indicate that different KCNE proteins use different domains of KCNQ1 channels, and that may explain why different KCNE proteins give quite different outcomes by forming a complex with KCNQ1 channels.     

Distinct subdomains of the KCNQ1 S6 segment determine channel modulation by different KCNE subunits

Modulation of voltage-gated potassium (K_V) channels by the KCNE family of single transmembrane proteins has physiological and pathophysiological importance. All five KCNE proteins (KCNE1–KCNE5) have been demonstrated to modulate heterologously expressed KCNQ1 (K_V7.1) with diverse effects, making this channel a valuable experimental platform for elucidating structure–function relationships and mechanistic differences among members of this intriguing group of accessory subunits. Here, we specifically investigated the determinants of KCNQ1 inhibition by KCNE4, the least well-studied KCNE protein. In CHO-K1 cells, KCNQ1, but not KCNQ4, is strongly inhibited by coexpression with KCNE4. By studying KCNQ1-KCNQ4 chimeras, we identified two adjacent residues (K326 and T327) within the extracellular end of the KCNQ1 S6 segment that determine inhibition of KCNQ1 by KCNE4. This dipeptide motif is distinct from neighboring S6 sequences that enable modulation by KCNE1 and KCNE3. Conversely, S6 mutations (S338C and F340C) that alter KCNE1 and KCNE3 effects on KCNQ1 do not abrogate KCNE4 inhibition. Further, KCNQ1-KCNQ4 chimeras that exhibited resistance to the inhibitory effects of KCNE4 still interact biochemically with this protein, implying that accessory subunit binding alone is not sufficient for channel modulation. These observations indicate that the diverse functional effects observed for KCNE proteins depend, in part, on structures intrinsic to the pore-forming subunit, and that distinct S6 subdomains determine KCNQ1 responses to KCNE1, KCNE3, and KCNE4.     

KCNE3 truncation mutants reveal a bipartite modulation of KCNQ1 K+ channels

The five KCNE genes encode a family of type I transmembrane peptides that assemble with KCNQ1 and other voltage-gated K(+) channels, resulting in potassium conducting complexes with varied channel-gating properties. It has been recently proposed that a triplet of amino acids within the transmembrane domain of KCNE1 and KCNE3 confers modulation specificity to the peptide, since swapping of these three residues essentially converts the recipient KCNE into the donor (Melman, Y.F., A. Domenech, S. de la Luna, and T.V. McDonald. 2001. J. Biol. Chem. 276:6439-6444). However, these results are in stark contrast with earlier KCNE1 deletion studies, which demonstrated that a COOH-terminal region, highly conserved between KCNE1 and KCNE3, was responsible for KCNE1 modulation of KCNQ1 (Tapper, A.R., and A.L. George. 2000 J. Gen. Physiol. 116:379-389.). To ascertain whether KCNE3 peptides behave similarly to KCNE1, we examined a panel of NH(2)- and COOH-terminal KCNE3 truncation mutants to directly determine the regions required for assembly with and modulation of KCNQ1 channels. Truncations lacking the majority of their NH(2) terminus, COOH terminus, or mutants harboring both truncations gave rise to KCNQ1 channel complexes with basal activation, a hallmark of KCNE3 modulation. These results demonstrate that the KCNE3 transmembrane domain is sufficient for assembly with and modulation of KCNQ1 channels and suggests a bipartite model for KCNQ1 modulation by KCNE1 and KCNE3 subunits. In this model, the KCNE3 transmembrane domain is active in modulation and overrides the COOH terminus' contribution, whereas the KCNE1 transmembrane domain is passive and reveals COOH-terminal modulation of KCNQ1 channels. We furthermore test the validity of this model by using the active KCNE3 transmembrane domain to functionally rescue a nonconducting, yet assembly and trafficking competent, long QT mutation located in the conserved COOH-terminal region of KCNE1.     

Differential roles of S6 domain hinges in the gating of KCNQ potassium channels

Voltage-gated K+ channel activation is proposed to result from simultaneous bending of all S6 segments away from the central axis, enlarging the aperture of the pore sufficiently to permit diffusion of K+ into the water-filled central cavity. The hinge position for the bending motion of each S6 segment is proposed to be a Gly residue and/or a Pro-Val-Pro motif in Kv1-Kv4 channels. The KCNQ1 (Kv7.1) channel has Ala-336 in the Gly-hinge position and Pro-Ala-Gly. Here we show that mutation of Ala-336 to Gly in KCNQ1 increased current amplitude and shifted the voltage dependence of activation to more negative potentials, consistent with facilitation of hinge activity that favors the open state. In contrast, mutation of Ala-336 to Cys or Thr shifted the voltage dependence of activation to more positive potentials and reduced current amplitude. Mutation of the putative Gly hinge to Ala in KCNQ2 (Kv7.2) abolished channel function. Mutation-dependent changes in current amplitude, but not kinetics, were found in heteromeric KCNQ1/KCNE1 channels. Mutation of the Pro or Gly of the Pro-Ala-Gly motif to Ala abolished KCNQ1 function and introduction of Gly in front of the Ala-mutations partially recovered channel function, suggesting that flexibility at the PAG is important for channel activation.     

The small conductance K+ channel, KCNQ1: expression, function, and subunit composition in murine trachea

The gene KCNQ1 encodes a K(+) channel alpha-subunit important for cardiac repolarization, formerly known as K(v)LQT1. In large and small intestine a channel complex consisting of KCNQ1 and the beta-subunit KCNE3 (MiRP2) is known to mediate the cAMP-activated basolateral K(+) current, which is essential for luminal Cl(-) secretion. Northern blot experiments revealed an expression of both subunits in lung tissue. However, previous reports suggested a role of KCNE1 (minK, Isk) but not KCNE3 in airway epithelial cells. Here we give evidence that KCNE1 is not detected in murine tracheal epithelial cells and that Cl(-) secretion by these cells is not reduced by the knock-out of the KCNE1 gene. In contrast we show that a complex consisting of KCNQ1 and KCNE3 probably forms a basolateral K(+) channel in murine tracheal epithelial cells. As described for colonic epithelium, the current through KCNQ1 complexes in murine trachea is specifically inhibited by the chromanol 293B. A 293B-sensitive current was present after stimulation with forskolin and agonists that increase Ca(2+) as well as after administration of the pharmacological K(+) channel activator, 1-EBIO. A 293B-inhibitable current was already present under control conditions and reduced after administration of amiloride indicating a role of this K(+) channel not only for Cl(-) secretion but also for Na(+) reabsorption. We conclude that at least in mice a KCNQ1 channel complex seems to be the dominant basolateral K(+) conductance in tracheal epithelial cells.     

Fenofibrate inhibits intestinal Cl- secretion by blocking basolateral KCNQ1 K+ channels

Fibrates are peroxisome proliferator-activated receptor-alpha (PPARalpha) ligands in widespread clinical use to lower plasma triglyceride levels. We investigated the effect of fenofibrate and clofibrate on ion transport in mouse intestine and in human T84 colonic adenocarcinoma cells through the use of short-circuit current (I(sc)) and ion flux analysis. In mice, oral administration of fenofibrate produced a persistent inhibition of cAMP-stimulated electrogenic Cl(-) secretion by isolated jejunum and colon without affecting electroneutral fluxes of (22)Na(+) or (86)Rb(+) (K(+)) across unstimulated colonic mucosa. When applied acutely to isolated mouse intestinal mucosa, 100 microM fenofibrate inhibited cAMP-stimulated I(sc) within 5 min. In T84 cells, fenofibrate rapidly inhibited approximately 80% the Cl(-) secretory responses to forskolin (cAMP) and to heat stable enterotoxin STa (cGMP) without affecting the response to carbachol (Ca(2+)). Both fenofibrate and clofibrate inhibited cAMP-stimulated I(sc) with an IC(50) approximately 1 muM, whereas other PPARalpha activators (gemfibrozil and Wy-14,643) were without effect. Membrane permeabilization experiments on T84 cells indicated that fenofibrate inhibits basolateral cAMP-stimulated K(+) channels (putatively KCNQ1/KCNE3) without affecting Ca(2+)-stimulated K(+) channel activity, whereas clofibrate inhibits both K(+) pathways. Fenofibrate had no effect on apical cAMP-stimulated Cl(-) channel activity. Patch-clamp analysis of HEK-293T cells confirmed that 100 microM fenofibrate rapidly inhibits K(+) currents associated with ectopic expression of human KCNQ1 with or without the KCNE3 beta-subunit. We conclude that fenofibrate inhibits intestinal cAMP-stimulated Cl(-) secretion through a nongenomic mechanism that involves a selective inhibition of basolateral KCNQ1/KCNE3 channel complexes. Our findings raise the prospect of fenofibrate as a safe and effective antidiarrheal agent.     

KCNE peptides differently affect voltage sensor equilibrium and equilibration rates in KCNQ1 K+ channels

KCNQ1 voltage-gated K(+) channels assemble with the family of KCNE type I transmembrane peptides to afford membrane-embedded complexes with diverse channel gating properties. KCNQ1/KCNE1 complexes generate the very slowly activating cardiac I(Ks) current, whereas assembly with KCNE3 produces a constitutively conducting complex involved in K(+) recycling in epithelia. To determine whether these two KCNE peptides influence voltage sensing in KCNQ1 channels, we monitored the position of the S4 voltage sensor in KCNQ1/KCNE complexes using cysteine accessibility experiments. A panel of KCNQ1 S4 cysteine mutants was expressed in Xenopus oocytes, treated with the membrane-impermeant cysteine-specific reagent 2-(trimethylammonium) ethyl methanethiosulfonate (MTSET), and the voltage-dependent accessibility of each mutant was determined. Of these S4 cysteine mutants, three (R228C, G229C, I230C) were modified by MTSET only when KCNQ1 was depolarized. We then employed these state-dependent residues to determine how assembly with KCNE1 and KCNE3 affects KCNQ1 voltage sensor equilibrium and equilibration rates. In the presence of KCNE1, MTSET modification rates for the majority of the cysteine mutants were approximately 10-fold slower, as was recently reported to indicate that the kinetics of the KCNQ1 voltage sensor are slowed by KCNE1 (Nakajo, K., and Y. Kubo. 2007 J. Gen. Physiol. 130:269-281). Since MTS modification rates reflect an amalgam of reagent accessibility, chemical reactivity, and protein conformational changes, we varied the depolarization pulse duration to determine whether KCNE1 slows the equilibration rate of the voltage sensors. Using the state-dependent cysteine mutants, we determined that MTSET modification rates were essentially independent of depolarization pulse duration. These results demonstrate that upon depolarization the voltage sensors reach equilibrium quickly in the presence of KCNE1 and the slow gating of the channel complex is not due to slowly moving voltage sensors. In contrast, all cysteine substitutions in the S4 of KCNQ1/KCNE3 complexes were freely accessible to MTSET independent of voltage, which is consistent with KCNE3 shifting the voltage sensor equilibrium to favor the active state at hyperpolarizing potentials. In total, these results suggest that KCNE peptides differently modulate the voltage sensor in KCNQ1 K(+) channels.