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.     

Klotho Sensitivity of the Neuronal Excitatory Amino Acid Transporters EAAT3 and EAAT4

Klotho, a transmembrane protein, which can be cleaved off as β-glucuronidase and hormone, is released in both, kidney and choroid plexus and encountered in blood and cerebrospinal fluid. Klotho deficiency leads to early appearance of age-related disorders and premature death. Klotho may modify transport by inhibiting 1,25(OH)₂D₃ formation or by directly affecting channel and carrier proteins. The present study explored whether Klotho influences the activity of the Na⁺-coupled excitatory amino acid transporters EAAT3 and EAAT4, which are expressed in kidney (EAAT3), intestine (EAAT3) and brain (EAAT3 and EAAT4). To this end, cRNA encoding EAAT3 or EAAT4 was injected into Xenopus oocytes with and without additional injection of cRNA encoding Klotho. EAAT expressing Xenopus oocytes were further treated with recombinant human β-Klotho protein with or without β-glucuronidase inhibitor D-saccharic acid 1,4-lactone monohydrate (DSAL). Electrogenic excitatory amino acid transport was determined as L-glutamate-induced current (I_glu) in two electrode voltage clamp experiments. EAAT3 and EAAT4 protein abundance in the Xenopus oocyte cell membrane was visualized by confocal microscopy and quantified utilizing chemiluminescence. As a result, coexpression of Klotho cRNA significantly increased I_glu in both, EAAT3 or EAAT4-expressing Xenopus oocytes. Klotho cRNA coexpression significantly increased the maximal current and cell membrane protein abundance of both EAAT3 and EAAT4. The effect of Klotho coexpression on EAAT3 and EAAT4 activity was mimicked by treating EAAT3 or EAAT4-expressing Xenopus oocytes with recombinant human β-Klotho protein. The effects of Klotho coexpression and of treatment with recombinant human β-Klotho protein were both abrogated in the presence of DSAL (10 µM). In conclusion, Klotho is a novel, powerful regulator of the excitatory amino acid transporters EAAT3 and EAAT4.     

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.     

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.     

Discovery of a Novel Activator of KCNQ1-KCNE1 K+ Channel Complexes

KCNQ1 voltage-gated K⁺ channels (Kv7.1) associate with the family of five KCNE peptides to form complexes with diverse gating properties and pharmacological sensitivities. The varied gating properties of the different KCNQ1-KCNE complexes enables the same K⁺ channel to function in both excitable and non excitable tissues. Small molecule activators would be valuable tools for dissecting the gating mechanisms of KCNQ1-KCNE complexes; however, there are very few known activators of KCNQ1 channels and most are ineffective on the physiologically relevant KCNQ1-KCNE complexes. Here we show that a simple boronic acid, phenylboronic acid (PBA), activates KCNQ1/KCNE1 complexes co-expressed in Xenopus oocytes at millimolar concentrations. PBA shifts the voltage sensitivity of KCNQ1 channel complexes to favor the open state at negative potentials. Analysis of different-sized charge carriers revealed that PBA also targets the permeation pathway of KCNQ1 channels. Activation by the boronic acid moiety has some specificity for the Kv7 family members (KCNQ1, KCNQ2/3, and KCNQ4) since PBA does not activate Shaker or hERG channels. Furthermore, the commercial availability of numerous PBA derivatives provides a large class of compounds to investigate the gating mechanisms of KCNQ1-KCNE complexes.     

A carboxy-terminal domain determines the subunit specificity of KCNQ K+ channel assembly

Mutations in KCNQ K⁺ channel genes underlie several human pathologies. KCNQ α-subunits form either homotetramers or hetero-oligomers with a restricted subset of other KCNQ α-subunits or with KCNE β-subunits. KCNQ1 assembles with KCNE β-subunits but not with other KCNQ α-subunits. By contrast, KCNQ3 interacts with KCNQ2, KCNQ4 and KCNQ5. Using a chimaeric strategy, we show that a cytoplasmic carboxy-terminal subunit interaction domain (sid) suffices to transfer assembly properties between KCNQ3 and KCNQ1. A chimaera (KCNQ1-sid_Q3) carrying the si domain of KCNQ3 within the KCNQ1 backbone interacted with KCNQ2, KCNQ3 and KCNQ4 but not with KCNQ1. This interaction was shown by enhancement of KCNQ2 currents, testing for dominant-negative effects of pore mutants, determining its effects on surface expression and co-immunoprecipitation experiments. Conversely, a KCNQ3-sid_Q1 chimaera no longer affects KCNQ2 but interacts with KCNQ1. We conclude that the si domain suffices to determine the subunit specificity of KCNQ channel assembly.     

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.     

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.     

Identification of a protein–protein interaction between KCNE1 and the activation gate machinery of KCNQ1

KCNQ1 channels assemble with KCNE1 transmembrane (TM) peptides to form voltage-gated K⁺ channel complexes with slow activation gate opening. The cytoplasmic C-terminal domain that abuts the KCNE1 TM segment has been implicated in regulating KCNQ1 gating, yet its interaction with KCNQ1 has not been described. Here, we identified a protein–protein interaction between the KCNE1 C-terminal domain and the KCNQ1 S6 activation gate and S4–S5 linker. Using cysteine cross-linking, we biochemically screened over 300 cysteine pairs in the KCNQ1–KCNE1 complex and identified three residues in KCNQ1 (H363C, P369C, and I257C) that formed disulfide bonds with cysteine residues in the KCNE1 C-terminal domain. Statistical analysis of cross-link efficiency showed that H363C preferentially reacted with KCNE1 residues H73C, S74C, and D76C, whereas P369C showed preference for only D76C. Electrophysiological investigation of the mutant K⁺ channel complexes revealed that the KCNQ1 residue, H363C, formed cross-links not only with KCNE1 subunits, but also with neighboring KCNQ1 subunits in the complex. Cross-link formation involving the H363C residue was state dependent, primarily occurring when the KCNQ1–KCNE1 complex was closed. Based on these biochemical and electrophysiological data, we generated a closed-state model of the KCNQ1–KCNE1 cytoplasmic region where these protein–protein interactions are poised to slow activation gate opening.     

Identification and characterisation of a novel KCNQ1 mutation in a family with Romano-Ward syndrome

Romano-Ward syndrome (RWS), the autosomal dominant form of the congenital long QT syndrome, is characterised by prolongation of the cardiac repolarisation process associated with ventricular tachyarrhythmias of the torsades de pointes type. Genetic studies have identified mutations in six ion channel genes, KCNQ1, KCNH2, SCN5A, KCNE1 and KCNE2 and the accessory protein Ankyrin-B gene, to be responsible for this disorder. Single-strand conformation polymorphism (SSCP) analysis and subsequent DNA sequence analysis have identified a KCNQ1 mutation in a family that were clinically conspicuous due to several syncopes and prolonged QTc intervals in the ECG. The mutant subunit was expressed and functionally characterised in the Xenopus oocyte expression system. A novel heterozygous missense mutation with a C to T transition at the first position of codon 343 (CCA) of the KCNQ1 gene was identified in three concerned family members (QTc intervals: 500, 510 and 530 ms, respectively). As a result, proline 343 localised within the highly conserved transmembrane segment S6 of the KCNQ1 channel is replaced by a serine. Co-expression of mutant (KCNQ1-P343S) and wild-type (KCNQ1) cRNA in Xenopus oocytes produced potassium currents reduced by approximately 92%, while IKs reconstitution experiments with a combination of KCNQ1 mutant, wild-type and KCNE1 subunits yielded currents reduced by approximately 60%. A novel mutation (P343S) identified in the KCNQ1 subunit gene of three members of a RWS family showed a dominant-negative effect on native IKs currents leading to prolongation of the heart repolarisation and possibly increases the risk of malign arrhythmias with sudden cardiac death.     

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.     

Building KCNQ1/KCNE1 Channel Models and Probing their Interactions by Molecular-Dynamics Simulations

The slow delayed rectifier (I_Ks) channel is composed of KCNQ1 (pore-forming) and KCNE1 (auxiliary) subunits, and functions as a repolarization reserve in the human heart. Design of I_Ks-targeting anti-arrhythmic drugs requires detailed three-dimensional structures of the KCNQ1/KCNE1 complex, a task made possible by Kv channel crystal structures (templates for KCNQ1 homology-modeling) and KCNE1 NMR structures. Our goal was to build KCNQ1/KCNE1 models and extract mechanistic information about their interactions by molecular-dynamics simulations in an explicit lipid/solvent environment. We validated our models by confirming two sets of model-generated predictions that were independent from the spatial restraints used in model-building. Detailed analysis of the molecular-dynamics trajectories revealed previously unrecognized KCNQ1/KCNE1 interactions, whose relevance in I_Ks channel function was confirmed by voltage-clamp experiments. Our models and analyses suggest three mechanisms by which KCNE1 slows KCNQ1 activation: by promoting S6 bending at the Pro hinge that closes the activation gate; by promoting a downward movement of gating charge on S4; and by establishing a network of electrostatic interactions with KCNQ1 on the extracellular surface that stabilizes the channel in a pre-open activated state. Our data also suggest how KCNE1 may affect the KCNQ1 pore conductance.     

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.     

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.     

Preparation, functional characterization, and NMR studies of human KCNE1, a voltage-gated potassium channel accessory subunit associated with deafness and long QT syndrome

KCNE1, also known as minK, is a member of the KCNE family of membrane proteins that modulate the function of KCNQ1 and certain other voltage-gated potassium channels (KV). Mutations in human KCNE1 cause congenital deafness and congenital long QT syndrome, an inherited predisposition to potentially life-threatening cardiac arrhythmias. Although its modulation of KCNQ1 function has been extensively characterized, many questions remain regarding KCNE1's structure and location within the channel complex. In this study, KCNE1 was overexpressed in Escherichia coli and purified. Micellar solutions of the protein were then microinjected into Xenopus oocytes expressing KCNQ1 channels, followed by electrophysiological recordings aimed at testing whether recombinant KCNE1 can co-assemble with the channel. Nativelike modulation of channel properties was observed following injection of KCNE1 in lyso-myristoylphosphatidylglycerol (LMPG) micelles, indicating that KCNE1 is not irreversibly misfolded and that LMPG is able to act as a vehicle for delivering membrane proteins into the membranes of viable cells. 1H-15N TROSY NMR experiments indicated that LMPG micelles are well-suited for structural studies of KCNE1, leading to assignment of its backbone resonances and to relaxation studies. The chemical shift data confirmed that KCNE1's secondary structure includes several alpha-helices and demonstrated that its distal C-terminus is disordered. Surprisingly, for KCNE1 in LMPG micelles, there appears to be a break in alpha-helicity at sites 59-61, near the middle of the transmembrane segment, a feature that is accompanied by increased local backbone mobility. Given that this segment overlaps with sites 57-59, which are known to play a critical role in modulating KCNQ1 channel activation kinetics, this unusual structural feature likely has considerable functional relevance.     

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.