Nano-environmental changes by KCNE proteins modify KCNQ channel function

The KCNQ1 channel is a voltage-dependent potassium channel, which is widely expressed in various tissues of the human body including heart, inner ear, intestine, kidney and pancreas. The ion channel properties of KCNQ1 change remarkably when auxiliary subunit KCNE proteins co-exist. The mechanisms of KCNQ1 channel regulation by KCNE proteins are of longstanding interest but are still far from being fully understood. The pore region (S5-S6 segments) of KCNQ1 is thought to be the main interaction site for KCNE proteins. However, some recent reports showed that the voltage-sensing domain (S1-S4 segments) is critically involved in the regulation of KCNQ1 by KCNE proteins. In addition, we recently re-examined the stoichiometry of the KCNQ1-KCNE1 complex and found that the stoichiometry is not fixed but rather flexible and the KCNQ1 channel can have up to four associated KCNE1 proteins. We will review these recent findings concerning the mechanisms of KCNQ1 regulation by KCNE proteins.     

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.     

Probing Binding Sites and Mechanisms of Action of an IKs Activator by Computations and Experiments

The slow delayed rectifier (I_Ks) channel is composed of the KCNQ1 channel and KCNE1 auxiliary subunit, and functions to repolarize action potentials in the human heart. I_Ks activators may provide therapeutic efficacy for treating long QT syndromes. Here, we show that a new KCNQ1 activator, ML277, can enhance I_Ks amplitude in adult guinea pig and canine ventricular myocytes. We probe its binding site and mechanism of action by computational analysis based on our recently reported KCNQ1 and KCNQ1/KCNE1 3D models, followed by experimental validation. Results from a pocket analysis and docking exercise suggest that ML277 binds to a side pocket in KCNQ1 and the KCNE1-free side pocket of KCNQ1/KCNE1. Molecular-dynamics (MD) simulations based on the most favorable channel/ML277 docking configurations reveal a well-defined ML277 binding space surrounded by the S2-S3 loop and S4-S5 helix on the intracellular side, and by S4–S6 transmembrane helices on the lateral sides. A detailed analysis of MD trajectories suggests two mechanisms of ML277 action. First, ML277 restricts the conformational dynamics of the KCNQ1 pore, optimizing K⁺ ion coordination in the selectivity filter and increasing current amplitudes. Second, ML277 binding induces global motions in the channel, including regions critical for KCNQ1 gating transitions. We conclude that ML277 activates I_Ks by binding to an intersubunit space and allosterically influencing pore conductance and gating transitions. KCNE1 association protects KCNQ1 from an arrhythmogenic (constitutive current-inducing) effect of ML277, but does not preclude its current-enhancing effect.     

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.     

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.     

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.     

Characterization of KCNQ1 atrial fibrillation mutations reveals distinct dependence on KCNE1

The I(Ks) potassium channel, critical to control of heart electrical activity, requires assembly of α (KCNQ1) and β (KCNE1) subunits. Inherited mutations in either I(Ks) channel subunit are associated with cardiac arrhythmia syndromes. Two mutations (S140G and V141M) that cause familial atrial fibrillation (AF) are located on adjacent residues in the first membrane-spanning domain of KCNQ1, S1. These mutations impair the deactivation process, causing channels to appear constitutively open. Previous studies suggest that both mutant phenotypes require the presence of KCNE1. Here we found that despite the proximity of these two mutations in the primary protein structure, they display different functional dependence in the presence of KCNE1. In the absence of KCNE1, the S140G mutation, but not V141M, confers a pronounced slowing of channel deactivation and a hyperpolarizing shift in voltage-dependent activation. When coexpressed with KCNE1, both mutants deactivate significantly slower than wild-type KCNQ1/KCNE1 channels. The differential dependence on KCNE1 can be correlated with the physical proximity between these positions and KCNE1 as shown by disulfide cross-linking studies: V141C forms disulfide bonds with cysteine-substituted KCNE1 residues, whereas S140C does not. These results further our understanding of the structural relationship between KCNE1 and KCNQ1 subunits in the I(Ks) channel, and provide mechanisms for understanding the effects on channel deactivation underlying these two atrial fibrillation mutations.     

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.     

Gating-Related Molecular Motions in the Extracellular Domain of the I<Subscript>Ks</Subscript> Channel: Implications for I<Subscript>Ks</Subscript> Channelopathy

Cardiac slow delayed rectifier (I_Ks) channel complex consists of KCNQ1 channel and KCNE1 auxiliary subunits. The extracellular juxtamembranous region of KCNE1 is an unstructured loop that contacts multiple KCNQ1 positions in a gating-state-dependent manner. Congenital arrhythmia-related mutations have been identified in the extracellular S1–S2 linker of KCNQ1. These mutations manifest abnormal phenotypes only when coexpressed with KCNE1, pointing to the importance of proper KCNQ1/KCNE1 interactions here in I_Ks channel function. We investigate the interactions between the KCNE1 loop (positions 36–47) and KCNQ1 S1–S2 linker (positions 140–148) by means of disulfide trapping and voltage clamp techniques. During transitions among the resting-state conformations, KCNE1 positions 36–43 make contacts with KCNQ1 positions 144, 145, and 147 in a parallel fashion. During conformational changes in the activated state, KCNE1 position 40 can make contacts with all three KCNQ1 positions, while the neighboring KCNE1 positions (36, 38, 39, and 41) can make contact with KCNQ1 position 147. Furthermore, KCNQ1 positions 143 and 146 are high-impact positions that cannot tolerate cysteine substitution. To maintain the proper I_Ks channel function, position 143 requires a small side chain with a hydroxyl group, and position 146 requires a negatively charged side chain. These data and the proposed molecular motions provide insights into the mechanisms by which mutations in the extracellular juxtamembranous region of the I_Ks channel impair its function.     

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.     

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.     

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     

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.     

Biophysical characteristics of a new mutation on the KCNQ1 potassium channel (L251P) causing long QT syndrome

The congenital long QT syndrome (LQTS) is a hereditary cardiac disease characterized by prolonged ventricular repolarization, syncope, and sudden death. Mutations causing LQTS have been identified in various genes that encode for ionic channels or their regulatory subunits. Several of these mutations have been reported on the KCNQ1 gene encoding for a potassium channel or its regulatory subunit (KCNE1). In this study, we report the biophysical characteristics of a new mutation (L251P) in the transmembrane segment 5 (S5) of the KCNQ1 potassium channel. Potassium currents were recorded from CHO cells transfected with either wild type or mutant KCNQ1 in the presence or in the absence of its regulatory subunit (KCNE1), using the whole-cell configuration of the patch clamp technique. Wild-type KCNQ1 current amplitudes are increased particularly by KCNE1 co-expression but no current is observed with the KCNQ1 (L251P) mutant either in the presence or in the absence of KCNE1. Coexpressing KCNE1 with equal amount of cDNAs encoding wild type and mutant KCNQ1 results in an 11-fold reduction in the amplitude of potassium currents. The kinetics of activation and inactivation and the activation curve are minimally affected by this mutation. Our results suggest that the dominant negative effect of the P251L mutation on KCNQ1 channel explains the prolonged repolarization in patients carrying this mutation.     

KCNE1 and KCNE3 stabilize and/or slow voltage sensing S4 segment of KCNQ1 channel

KCNQ1 is a voltage-dependent K(+) channel whose gating properties are dramatically altered by association with auxiliary KCNE proteins. For example, KCNE1, which is mainly expressed in heart and inner ear, markedly slows the activation kinetics of KCNQ1. Whether the voltage-sensing S4 segment moves differently in the presence of KCNE1 is not yet known, however. To address that question, we systematically introduced cysteine mutations, one at a time, into the first half of the S4 segment of human KCNQ1. A226C was found out as the most suited mutant for a methanethiosulfonate (MTS) accessibility analysis because it is located at the N-terminal end of S4 segment and its current was stable with repetitive stimuli in the absence of MTS reagent. MTS accessibility analysis revealed that the apparent second order rate constant for modification of the A226C mutant was state dependent, with faster modification during depolarization, and was 13 times slower in the presence of KCNE1 than in its absence. In the presence of KCNE3, on the other hand, the second order rate constant for modification was not state dependent, indicating that the C226 residue was always exposed to the extracellular milieu, even at the resting membrane potential. Taken together, these results suggest that KCNE1 stabilizes the S4 segment in the resting state and slows the rate of transition to the active state, while KCNE3 stabilizes the S4 segment in the active state. These results offer new insight into the mechanism of KCNQ1 channel modulation by KCNE1 and KCNE3.     

Identification of a KCNE2 gain-of-function mutation in patients with familial atrial fibrillation

Atrial fibrillation (AF) is the most common cardiac arrhythmia encountered in clinical practice. We first reported an S140G mutation of KCNQ1, an alpha subunit of potassium channels, in one Chinese kindred with AF. However, the molecular defects and cellular mechanisms in most patients with AF remain to be identified. We evaluated 28 unrelated Chinese kindreds with AF and sequenced eight genes of potassium channels (KCNQ1, HERG, KCNE1, KCNE2, KCNE3, KCNE4, KCNE5, and KCNJ2). An arginine-to-cysteine mutation at position 27 (R27C) of KCNE2, the beta subunit of the KCNQ1-KCNE2 channel responsible for a background potassium current, was found in 2 of the 28 probands. The mutation was present in all affected members in the two kindreds and was absent in 462 healthy unrelated Chinese subjects. Similar to KCNQ1 S140G, the mutation had a gain-of-function effect on the KCNQ1-KCNE2 channel; unlike long QT syndrome-associated KCNE2 mutations, it did not alter HERG-KCNE2 current. The mutation did not alter the functions of the HCN channel family either. Thus, KCNE2 R27C is a gain-of-function mutation associated with the initiation and/or maintenance of AF.     

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.     

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.