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.     

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.     

Regulation of human cardiac KCNQ1/KCNE1 channel by epidermal growth factor receptor kinase

The aim of the present study was to investigate whether/how the recombinant human cardiac I_Ks could be regulated by epidermal growth factor receptor kinase in HEK 293 cells stably expressing hKCNQ1/hKCNE1 genes using the approaches of perforated patch clamp technique, immunoprecipitation and Western blot analysis. It was found that the broad spectrum isoflavone tyrosine kinase inhibitor genistein and the selective epidermal growth factor receptor kinase inhibitor tyrphostin AG556 suppressed the recombinant I_Ks, and their inhibition was countered by the protein tyrosine phosphatase inhibitor orthovanadate. The Src-family kinase inhibitor PP2 reduced the current, but the effect was not antagonized by orthovanadate. Immunoprecipitation and Western blot analysis revealed that tyrosine phosphorylation level of hKCNQ1 protein was decreased by genistein or AG556, but not by PP2. These results provide the novel information that epidermal growth factor receptor kinase, but not Src-family kinases, regulates the recombinant cardiac I_Ks stably expressed in HEK 293 cells via phosphorylating KCNQ1 protein of the channel.     

Inhibition of the heterotetrameric K+ channel KCNQ1/KCNE1 by the AMP-activated protein kinase

Abstract

The heterotetrameric K⁺-channel KCNQ1/KCNE1 is expressed in heart, skeletal muscle, liver and several epithelia including the renal proximal tubule. In the heart, it contributes to the repolarization of cardiomyocytes. The repolarization is impaired in ischemia. Ischemia stimulates the AMP-activated protein kinase (AMPK), a serine/threonine kinase, sensing energy depletion and stimulating several cellular mechanisms to enhance energy production and to limit energy utilization. AMPK has previously been shown to downregulate the epithelial Na⁺ channel ENaC, an effect mediated by the ubiquitin ligase Nedd4-2. The present study explored whether AMPK regulates KCNQ1/KCNE1. To this end, cRNA encoding KCNQ1/KCNE1 was injected into Xenopus oocytes with and without additional injection of wild type AMPK (AMPKα1 + AMPKβ1 + AMPKγ1), of the constitutively active ^γR70QAMPK (α1β1γ1(R70Q)), of the kinase dead mutant ^αK45RAMPK (α1(K45R)β1γ1), or of the ubiquitin ligase Nedd4-2. KCNQ1/KCNE1 activity was determined in two electrode voltage clamp experiments. Moreover, KCNQ1 abundance in the cell membrane was determined by immunostaining and subsequent confocal imaging. As a result, wild type and constitutively active AMPK significantly reduced KCNQ1/KCNE1-mediated currents and reduced KCNQ1 abundance in the cell membrane. Similarly, Nedd4-2 decreased KCNQ1/KCNE1-mediated currents and KCNQ1 protein abundance in the cell membrane. Activation of AMPK in isolated perfused proximal renal tubules by AICAR (10 mM) was followed by significant depolarization. In conclusion, AMPK is a potent regulator of KCNQ1/KCNE1.     

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.     

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.     

Inhibition of the heterotetrameric K+ channel KCNQ1/KCNE1 by the AMP-activated protein kinase

The heterotetrameric K(+)-channel KCNQ1/KCNE1 is expressed in heart, skeletal muscle, liver and several epithelia including the renal proximal tubule. In the heart, it contributes to the repolarization of cardiomyocytes. The repolarization is impaired in ischemia. Ischemia stimulates the AMP-activated protein kinase (AMPK), a serine/threonine kinase, sensing energy depletion and stimulating several cellular mechanisms to enhance energy production and to limit energy utilization. AMPK has previously been shown to downregulate the epithelial Na(+) channel ENaC, an effect mediated by the ubiquitin ligase Nedd4-2. The present study explored whether AMPK regulates KCNQ1/KCNE1. To this end, cRNA encoding KCNQ1/KCNE1 was injected into Xenopus oocytes with and without additional injection of wild type AMPK (AMPKα1 + AMPKβ1 + AMPKγ1), of the constitutively active (γR70Q)AMPK (α1β1γ1(R70Q)), of the kinase dead mutant (αK45R)AMPK (α1(K45R)β1γ1), or of the ubiquitin ligase Nedd4-2. KCNQ1/KCNE1 activity was determined in two electrode voltage clamp experiments. Moreover, KCNQ1 abundance in the cell membrane was determined by immunostaining and subsequent confocal imaging. As a result, wild type and constitutively active AMPK significantly reduced KCNQ1/KCNE1-mediated currents and reduced KCNQ1 abundance in the cell membrane. Similarly, Nedd4-2 decreased KCNQ1/KCNE1-mediated currents and KCNQ1 protein abundance in the cell membrane. Activation of AMPK in isolated perfused proximal renal tubules by AICAR (10 mM) was followed by significant depolarization. In conclusion, AMPK is a potent regulator of KCNQ1/KCNE1.     

Structure of KCNE1 and implications for how it modulates the KCNQ1 potassium channel

KCNE1 is a single-span membrane protein that modulates the voltage-gated potassium channel KCNQ1 (K V7.1) by slowing activation and enhancing channel conductance to generate the slow delayed rectifier current ( I Ks) that is critical for the repolarization phase of the cardiac action potential. Perturbation of channel function by inherited mutations in KCNE1 or KCNQ1 results in increased susceptibility to cardiac arrhythmias and sudden death with or without accompanying deafness. Here, we present the three-dimensional structure of KCNE1. The transmembrane domain (TMD) of KCNE1 is a curved alpha-helix and is flanked by intra- and extracellular domains comprised of alpha-helices joined by flexible linkers. Experimentally restrained docking of the KCNE1 TMD to a closed state model of KCNQ1 suggests that KCNE1 slows channel activation by sitting on and restricting the movement of the S4-S5 linker that connects the voltage sensor to the pore domain. We postulate that this is an adhesive interaction that must be disrupted before the channel can be opened in response to membrane depolarization. Docking to open KCNQ1 indicates that the extracellular end of the KCNE1 TMD forms an interface with an intersubunit cleft in the channel that is associated with most known gain-of-function disease mutations. Binding of KCNE1 to this "gain-of-function cleft" may explain how it increases conductance and stabilizes the open state. These working models for the KCNE1-KCNQ1 complexes may be used to formulate testable hypotheses for the molecular bases of disease phenotypes associated with the dozens of known inherited mutations in KCNE1 and KCNQ1.     

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.     

KCNE2 confers background current characteristics to the cardiac KCNQ1 potassium channel

Mutations in HERG and KCNQ1 (or KVLQT1) genes cause the life-threatening Long QT syndrome. These genes encode K(+) channel pore-forming subunits that associate with ancillary subunits from the KCNE family to underlie the two components, I(Kr) and I(Ks), of the human cardiac delayed rectifier current I(K). The KCNE family comprises at least three members. KCNE1 (IsK or MinK) recapitulates I(Ks) when associated with KCNQ1, whereas it augments the amplitude of an I(Kr)-like current when co-expressed with HERG. KCNE3 markedly changes KCNQ1 as well as HERG current properties. So far, KCNE2 (MirP1) has only been shown to modulate HERG current. Here we demonstrate the interaction of KCNE2 with the KCNQ1 subunit, which results in a drastic change of KCNQ1 current amplitude and gating properties. Furthermore, KCNE2 mutations also reveal their specific functional consequences on KCNQ1 currents. KCNQ1 and HERG appear to share unique interactions with KCNE1, 2 and 3 subunits. With the exception of KCNE3, mutations in all these partner subunits have been found to lead to an increased propensity for cardiac arrhythmias.     

Upregulation of KCNQ1/KCNE1 K+ channels by Klotho

Klotho is a transmembrane protein expressed primarily in kidney, parathyroid gland, and choroid plexus. The extracellular domain could be cleaved off and released into the systemic circulation. Klotho is in part effective as β-glucuronidase regulating protein stability in the cell membrane. Klotho is a major determinant of aging and life span. Overexpression of Klotho increases and Klotho deficiency decreases life span. Klotho deficiency may further result in hearing loss and cardiac arrhythmia. The present study explored whether Klotho modifies activity and protein abundance of KCNQ1/KCNE1, a K⁺ channel required for proper hearing and cardiac repolarization. To this end, cRNA encoding KCNQ1/KCNE1 was injected in Xenopus oocytes with or without additional injection of cRNA encoding Klotho. KCNQ1/KCNE1 expressing oocytes were treated with human recombinant Klotho protein (30 ng/ml) for 24 h. Moreover, oocytes which express both KCNQ1/KCNE1 and Klotho were treated with 10 µM DSAL (D-saccharic acid-1,4-lactone), a β-glucuronidase inhibitor. The KCNQ1/KCNE1 depolarization-induced current (I_Ks) was determined utilizing dual electrode voltage clamp, while KCNQ1/KCNE1 protein abundance in the cell membrane was visualized utilizing specific antibody binding and quantified by chemiluminescence. KCNQ1/KCNE1 channel activity and KCNQ1/KCNE1 protein abundance were upregulated by coexpression of Klotho. The effect was mimicked by treatment with human recombinant Klotho protein (30 ng/ml) and inhibited by DSAL (10 µM). In conclusion, Klotho upregulates KCNQ1/KCNE1 channel activity by 'mainly' enhancing channel protein abundance in the plasma cell membrane, an effect at least partially mediated through the β-glucuronidase activity of Klotho protein.     

Upregulation of KCNQ1/KCNE1 K+ channels by Klotho

Klotho is a transmembrane protein expressed primarily in kidney, parathyroid gland, and choroid plexus. The extracellular domain could be cleaved off and released into the systemic circulation. Klotho is in part effective as β-glucuronidase regulating protein stability in the cell membrane. Klotho is a major determinant of aging and life span. Overexpression of Klotho increases and Klotho deficiency decreases life span. Klotho deficiency may further result in hearing loss and cardiac arrhythmia. The present study explored whether Klotho modifies activity and protein abundance of KCNQ1/KCNE1, a K⁺ channel required for proper hearing and cardiac repolarization. To this end, cRNA encoding KCNQ1/KCNE1 was injected in Xenopus oocytes with or without additional injection of cRNA encoding Klotho. KCNQ1/KCNE1 expressing oocytes were treated with human recombinant Klotho protein (30 ng/ml) for 24 h. Moreover, oocytes which express both KCNQ1/KCNE1 and Klotho were treated with 10 µM DSAL (D-saccharic acid-1,4-lactone), a β-glucuronidase inhibitor. The KCNQ1/KCNE1 depolarization-induced current (I_Ks) was determined utilizing dual electrode voltage clamp, while KCNQ1/KCNE1 protein abundance in the cell membrane was visualized utilizing specific antibody binding and quantified by chemiluminescence. KCNQ1/KCNE1 channel activity and KCNQ1/KCNE1 protein abundance were upregulated by coexpression of Klotho. The effect was mimicked by treatment with human recombinant Klotho protein (30 ng/ml) and inhibited by DSAL (10 µM). In conclusion, Klotho upregulates KCNQ1/KCNE1 channel activity by 'mainly' enhancing channel protein abundance in the plasma cell membrane, an effect at least partially mediated through the β-glucuronidase activity of Klotho protein.     

Probing the structural basis for differential KCNQ1 modulation by KCNE1 and KCNE2

KCNE1 associates with KCNQ1 to increase its current amplitude and slow the activation gating process, creating the slow delayed rectifier channel that functions as a “repolarization reserve” in human heart. The transmembrane domain (TMD) of KCNE1 plays a key role in modulating KCNQ1 pore conductance and gating kinetics, and the extracellular juxtamembrane (EJM) region plays a modulatory role by interacting with the extracellular surface of KCNQ1. KCNE2 is also expressed in human heart and can associate with KCNQ1 to suppress its current amplitude and slow the deactivation gating process. KCNE1 and KCNE2 share the transmembrane topology and a high degree of sequence homology in TMD and surrounding regions. The structural basis for their distinctly different effects on KCNQ1 is not clear. To address this question, we apply cysteine (Cys) scanning mutagenesis to TMDs and EJMs of KCNE1 and KCNE2. We analyze the patterns of functional perturbation to identify high impact positions, and probe disulfide formation between engineered Cys side chains on KCNE subunits and native Cys on KCNQ1. We also use methanethiosulfonate reagents to probe the relationship between EJMs of KCNE subunits and KCNQ1. Our data suggest that the TMDs of both KCNE subunits are at about the same location but interact differently with KCNQ1. In particular, the much closer contact of KCNE2 TMD with KCNQ1, relative to that of KCNE1, is expected to impact the allosteric modulation of KCNQ1 pore conductance and may explain their differential effects on the KCNQ1 current amplitude. KCNE1 and KCNE2 also differ in the relationship between their EJMs and KCNQ1. Although the EJM of KCNE1 makes intimate contacts with KCNQ1, there appears to be a crevice between KCNQ1 and KCNE2. This putative crevice may perturb the electrical field around the voltage-sensing domain of KCNQ1, contributing to the differential effects of KCNE2 versus KCNE1 on KCNQ1 gating kinetics.     

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.     

Working model for the structural basis for KCNE1 modulation of the KCNQ1 potassium channel

The voltage-gated potassium channel KCNQ1 (Kv7.1) is modulated by KCNE1 (minK) to generate the I(Ks) current crucial to heartbeat. Defects in either protein result in serious cardiac arrhythmias. Recently developed structural models of the open and closed state KCNQ1/KCNE1 complexes offer a compelling explanation for how KCNE1 slows channel opening and provides a platform from which to refine and test hypotheses for other aspects of KCNE1 modulation. These working models were developed using an integrative approach based on results from nuclear magnetic resonance spectroscopy, electrophysiology, biochemistry, and computational methods-an approach that can be applied iteratively for model testing and revision. We present a critical review of these structural models, illustrating the strengths and challenges of the integrative approach.     

KCNQ1 and KCNE1 in the IKs channel complex make state-dependent contacts in their extracellular domains

KCNQ1 and KCNE1 (Q1 and E1) associate to form the slow delayed rectifier I(Ks) channels in the heart. A short stretch of eight amino acids at the extracellular end of S1 in Q1 (positions 140-147) harbors six arrhythmia-associated mutations. Some of these mutations affect the Q1 channel function only when coexpressed with E1, suggesting that this Q1 region may engage in the interaction with E1 critical for the I(Ks) channel function. Identifying the Q1/E1 contact points here may provide new insights into how the I(Ks) channel operates. We focus on Q1 position 145 and E1 positions 40-43. Replacing all native cysteine (Cys) in Q1 and introducing Cys into the above Q1 and E1 positions do not significantly perturb the Q1 channel function or Q1/E1 interactions. Immunoblot experiments on COS-7 cells reveal that Q1 145C can form disulfide bonds with E1 40C and 41C, but not E1 42C or 43C. Correspondingly, voltage clamp experiments in oocytes reveal that Q1 145C coexpressed with E1 40C or E1 41C manifests unique gating behavior and DTT sensitivity. Our data suggest that E1 40C and 41C come close to Q1 145C in the activated and resting states, respectively, to allow disulfide bond formation. These data and those in the literature lead us to propose a structural model for the Q1/E1 channel complex, in which E1 is located between S1, S4, and S6 of three separate Q1 subunits. We propose that E1 is not a passive partner of the Q1 channel, but instead can engage in molecular motions during I(Ks) gating.     

Expression and coassociation of ERG1, KCNQ1, and KCNE1 potassium channel proteins in horse heart

In dogs and in humans, potassium channels formed by ether-a-go-go-related gene 1 protein ERG1 (KCNH2) and KCNQ1 alpha-subunits, in association with KCNE beta-subunits, play a role in normal repolarization and may contribute to abnormal repolarization associated with long QT syndrome (LQTS). The molecular basis of repolarization in horse heart is unknown, although horses exhibit common cardiac arrhythmias and may receive drugs that induce LQTS. In horse heart, we have used immunoblotting and immunostaining to demonstrate the expression of ERG1, KCNQ1, KCNE1, and KCNE3 proteins and RT-PCR to detect KCNE2 message. Peptide N-glycosidase F-sensitive forms of horse ERG1 (145 kDa) and KCNQ1 (75 kDa) were detected. Both ERG1 and KCNQ1 coimmunoprecipitated with KCNE1. Cardiac action potential duration was prolonged by antagonists of either ERG1 (MK-499, cisapride) or KCNQ1/KCNE1 (chromanol 293B). Patch-clamp analysis confirmed the presence of a slow delayed rectifier current. These data suggest that repolarizing currents in horses are similar to those of other species, and that horses are therefore at risk for acquired LQTS. The data also provide unique evidence for coassociation between ERG1 and KCNE1 in cardiac tissue.     

Impact of KCNE subunits on KCNQ1 (Kv7.1) channel membrane surface targeting

The KCNQ1 (Kv7.1) channel plays an important role in cardiovascular physiology. Cardiomyocytes co‐express KCNQ1 with KCNE1‐5 proteins. KCNQ1 may co‐associate with multiple KCNE regulatory subunits to generate different biophysically and pharmacologically distinct channels. Increasing evidence indicates that the location and targeting of channels are important determinants of their function. In this context, the presence of K⁺ channels in sphingolipid–cholesterol‐enriched membrane microdomains (lipid rafts) is under investigation. Lipid rafts are important for cardiovascular functioning. We aimed to determine whether KCNE subunits modify the localization and targeting of KCNQ1 channels in lipid rafts microdomains. HEK‐293 cells were transiently transfected with KCNQ1 and KCNE1–5, and their traffic and presence in lipid rafts were analyzed. Only KCNQ1 and KCNE3, when expressed alone, co‐localized in raft fractions. In addition, while KCNE2 and KCNE5 notably stained the cell surface, KCNQ1 and the rest of the KCNEs showed strong intracellular retention. KCNQ1 targets multiple membrane surface microdomains upon association with KCNE peptides. Thus, while KCNQ1/KCNE1 and KCNQ1/KCNE2 channels target lipid rafts, KCNQ1 associated with KCNE3–5 did not. Channel membrane dynamics, analyzed by fluorescence recovery after photobleaching (FRAP) experiments, further supported these results. In conclusion, the trafficking and targeting pattern of KCNQ1 can be influenced by its association with KCNEs. Since KCNQ1 is crucial for cardiovascular physiology, the temporal and spatial regulations that different KCNE subunits may confer to the channels could have a dramatic impact on membrane electrical activity and putative endocrine regulation. J. Cell. Physiol. 225: 692–700, 2010. © 2010 Wiley‐Liss, Inc.     

Gating and flickery block differentially affected by rubidium in homomeric KCNQ1 and heteromeric KCNQ1/KCNE1 potassium channels

The voltage-gated potassium channel KCNQ1 associates with the small KCNE1 subunit to form the cardiac IKs delayed rectifier potassium current and mutations in both genes can lead to the long QT syndrome. KCNQ1 can form functional homotetrameric channels, however with drastically different biophysical properties compared to heteromeric KCNQ1/KCNE1 channels. We analyzed gating and conductance of these channels expressed in Xenopus oocytes using the two-electrode voltage-clamp and the patch-clamp technique and high extracellular potassium (K) and rubidium (Rb) solutions. Inward tail currents of homomeric KCNQ1 channels are increased about threefold upon substitution of 100 mM potassium with 100 mM rubidium despite a smaller rubidium permeability, suggesting an effect of rubidium on gating. However, the kinetics of tail currents and the steady-state activation curve are only slightly changed in rubidium. Single-channel amplitude at negative voltages was estimated by nonstationary noise analysis, and it was found that rubidium has only a small effect on homomeric channels (1.2-fold increase) when measured at a 5-kHz bandwidth. The apparent single-channel conductance was decreased after filtering the data at lower cutoff frequencies indicative of a relatively fast "flickery/block" process. The relative conductance in rubidium compared to potassium increased at lower cutoff frequencies (about twofold at 10 Hz), suggesting that the main effect of rubidium is to decrease the probability of channel blockage leading to an increase of inward currents without large changes in gating properties. Macroscopic inward tail currents of heteromeric KCNQ1/KCNE1 channels in rubidium are reduced by about twofold and show a pronounced sigmoidal time course that develops with a delay similar to the inactivation process of homomeric KCNQ1, and is indicative of the presence of several open states. The single channel amplitude of heteromers is about twofold smaller in rubidium than in potassium at a bandwidth of 5 kHz. Filtering at lower cutoff frequencies reduces the apparent single-channel conductance, the ratio of the conductance in rubidium versus potassium is, however, independent of the cutoff frequency. Our results suggest the presence of a relatively rapid process (flicker) that can occur almost independently of the gating state. Occupancy by rubidium at negative voltages favors the flicker-open state and slows the flickering rate in homomeric channels, whereas rubidium does not affect the flickering in heteromeric channels. The effects of KCNE1 on the conduction properties are consistent with an interaction of KCNE1 in the outer vestibule of the channel.