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.     

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.     

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.     

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/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.     

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.     

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.     

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.     

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.     

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.     

Desensitization of chemical activation by auxiliary subunits: convergence of molecular determinants critical for augmenting KCNQ1 potassium channels

Chemical openers for KCNQ potassium channels are useful probes both for understanding channel gating and for developing therapeutics. The five KCNQ isoforms (KCNQ1 to KCNQ5, or Kv7.1 to Kv7.5) are differentially localized. Therefore, the molecular specificity of chemical openers is an important subject of investigation. Native KCNQ1 normally exists in complex with auxiliary subunits known as KCNE. In cardiac myocytes, the KCNQ1-KCNE1 (IsK or minK) channel is thought to underlie the I(Ks) current, a component critical for membrane repolarization during cardiac action potential. Hence, the molecular and pharmacological differences between KCNQ1 and KCNQ1-KCNE1 channels have been important topics. Zinc pyrithione (ZnPy) is a newly identified KCNQ channel opener, which potently activates KCNQ2, KCNQ4, and KCNQ5. However, the ZnPy effects on cardiac KCNQ1 potassium channels remain largely unknown. Here we show that ZnPy effectively augments the KCNQ1 current, exhibiting an increase in current amplitude, reduction of inactivation, and slowing of both activation and deactivation. Some of these are reminiscent of effects by KCNE1. In addition, neither the heteromultimeric KCNQ1-KCNE1 channels nor native I(Ks) current displayed any sensitivity to ZnPy, indicating that the static occupancy by a KCNE subunit desensitizes the reversible effects by a chemical opener. Site-directed mutagenesis of KCNQ1 reveals that residues critical for the potentiation effects by either ZnPy or KCNE are clustered together in the S6 region overlapping with the critical gating determinants. Thus, the convergence of potentiation effects and molecular determinants critical for both an auxiliary subunit and a chemical opener argue for a mechanistic overlap in causing potentiation.     

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.     

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.     

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.     

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.     

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.     

Ionic permeation and conduction properties of neuronal KCNQ2/KCNQ3 potassium channels

Heteromeric KCNQ2/3 potassium channels are thought to underlie the M-current, a subthreshold potassium current involved in the regulation of neuronal excitability. KCNQ channel subunits are structurally unique, but it is unknown whether these structural differences result in unique conduction properties. Heterologously expressed KCNQ2/3 channels showed a permeation sequence of while showing a conduction sequence of A differential contribution of component subunits to the properties of heteromeric KCNQ2/3 channels was demonstrated by studying homomeric KCNQ2 and KCNQ3 channels, which displayed contrasting ionic selectivities. KCNQ2/3 channels did not exhibit an anomalous mole-fraction effect in mixtures of K(+) and Rb(+). However, extreme voltage-dependence of block by external Cs(+) was indicative of multi-ion pore behavior. Block of KCNQ2/3 channels by external Ba(2+) ions was voltage-independent, demonstrating unusual ionic occupation of the outer pore. Selectivity properties and block of KCNQ2 were altered by mutation of outer pore residues in a manner consistent with the presence of multiple ion-binding sites. KCNQ2/3 channel deactivation kinetics were slowed exclusively by Rb(+), whereas activation of KCNQ2/3 channels was altered by a variety of external permeant ions. These data indicate that KCNQ2/3 channels are multi-ion pores which exhibit distinctive mechanisms of ion conduction and gating.     

TEA(+)-sensitive KCNQ1 constructs reveal pore-independent access to KCNE1 in assembled I(Ks) channels

I(Ks), a slowly activating delayed rectifier K(+) current through channels formed by the assembly of two subunits KCNQ1 (KvLQT1) and KCNE1 (minK), contributes to the control of the cardiac action potential duration. Coassembly of the two subunits is essential in producing the characteristic and physiologically critical kinetics of assembled channels, but it is not yet clear where or how these subunits interact. Previous investigations of external access to the KCNE1 protein in assembled I(Ks) channels relied on occlusion of the pore by extracellular application of TEA(+), despite the very low TEA(+) sensitivity (estimated EC(50) > 100 mM) of channels encoded by coassembly of wild-type KCNQ1 with the wild type (WT) or a series of cysteine-mutated KCNE1 constructs. We have engineered a high affinity TEA(+) binding site into the h-KCNQ1 channel by either a single (V319Y) or double (K318I, V319Y) mutation, and retested it for pore-delimited access to specific sites on coassembled KCNE1 subunits. Coexpression of either KCNQ1 construct with WT KCNE1 in Chinese hamster ovary cells does not alter the TEA(+) sensitivity of the homomeric channels (IC(50) approximately 0.4 mM [TEA(+)](out)), providing evidence that KCNE1 coassembly does not markedly alter the structure of the outer pore of the KCNQ1 channel. Coexpression of a cysteine-substituted KCNE1 (F54C) with V319Y significantly increases the sensitivity of channels to external Cd(2+), but neither the extent of nor the kinetics of the onset of (or the recovery from) Cd(2+) block was affected by [TEA(+)](o) at 10x the IC(50) for channel block. These data strongly suggest that access of Cd(2+) to the cysteine-mutated site on KCNE1 is independent of pore occlusion caused by TEA(+) binding to the outer region of the KCNE1/V319Y pore, and that KCNE1 does not reside within the pore region of the assembled channels.     

Characterization of a binding site for anionic phospholipids on KCNQ1

The KCNQ family of potassium channels underlie a repolarizing K(+) current in the heart and the M-current in neurones. The assembly of KCNQ1 with KCNE1 generates the delayed rectifier current I(Ks) in the heart. Characteristically these channels are regulated via G(q/11)-coupled receptors and the inhibition seen after phospholipase C activation is now thought to occur from membrane phosphatidylinositol (4,5)-bisphosphate (PIP(2)) depletion. It is not clear how KCNQ1 recognizes PIP(2) and specifically which residues in the channel complex are important. Using biochemical techniques we identify a cluster of basic residues namely, Lys-354, Lys-358, Arg-360, and Lys-362, in the proximal C terminus as being involved in binding anionic phospholipids. The mutation of specific residues in combination, to alanine leads to the loss of binding to phosphoinositides. Functionally, the introduction of these mutations into KCNQ1 leads to shifts in the voltage dependence of channel activation toward depolarized potentials and reductions in current density. Additionally, the biophysical effects of the charge neutralizing mutations, which disrupt phosphoinositide binding, mirror the effects we see on channel function when we deplete cellular PIP(2) levels through activation of a G(q/11)-coupled receptor. Conversely, the addition of diC8-PIP(2) to the wild-type channel, but not a PIP(2) binding-deficient mutant, acts to shift the voltage dependence of channel activation toward hyperpolarized potentials and increase current density. In conclusion, we use a combined biochemical and functional approach to identify a cluster of basic residues important for the binding and action of anionic phospholipids on the KCNQ1/KCNE1 complex.     

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.