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 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.     

Probing the Interaction Between KCNE2 and KCNQ1 in Their Transmembrane Regions

KCNE1-KCNE5 are single membrane-spanning proteins that associate with voltage-gated potassium channels to diversify their function. Other than the KCNQ1/KCNE1 complex, little is known about how KCNE proteins work. We focus on KCNE2, which associates with KCNQ1 to form K channels critical for gastric acid secretion in parietal cells. We use cysteine (Cys)-scanning mutagenesis to probe the functional role of residues along the KCNE2 transmembrane domain (TMD) in modulating KCNQ1 function. There is an α-helical periodicity in how Cys substitutions along the KCNE2 TMD perturb KCNQ1 pore conductance/ion selectivity. However, positions where Cys substitutions perturb KCNQ1 gating kinetics cluster to the extracellular end and cytoplasmic half of the KCNE2 TMD. This is the first systematic perturbation analysis of a KCNE TMD. We propose that the KCNE2 TMD adopts an α-helical secondary structure with one face making intimate contact with the KCNQ1 pore domain, while the contacts with the KCNQ1 voltage-sensing domain appear more dynamic.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.