KCNE1 Subunits Research Articles

KCNE gene expression is dependent on the proliferation and mode of activation of leukocytes

Voltage-dependent K+ (Kv) channels are tightly regulated during the immune system response. Leukocytes have a limited repertoire of Kv channels, whose physiological role is under intense investigation. A functional Kv channel is an oligomeric complex composed of pore-forming and ancillary subunits. The KCNE gene family is a novel group of modulatory Kv channel elements in leukocytes. Here, we characterized the gene expression of KCNEs (1–5) in leukocytes and investigated their regulation during leukocyte proliferation and mode of activation. Murine bone-marrow-derived macrophages, human Jurkat T-lymphocytes and human Raji B-cells were analyzed. KCNEs (1–5) are expressed in all leukocytes lineages. Most KCNE mRNAs show cell cycle-dependent regulation and are differentially regulated under specific insults. Our results further suggest a new and yet undefined physiological role for KCNE subunits in the immune system. Putative associations of these ancillary proteins with Kv channels would yield a wide variety of biophysically and pharmacologically distinct channels that fine-tune the immunological response.

Probucol and the cholesterol synthesis inhibitors simvastatin and triparanol regulate Iks channel function differently

Channels responsible for slowly activating delayed-rectifier potassium current (I(Ks)) are composed of KCNQ1 and KCNE1 subunits, and these channels play a role in the repolarization of cardiac action potentials. Recently, we showed that the antihyperlipidemic drug probucol, which induces QT prolongation, decreases the I(Ks) after 24-h treatment. In the present study, we investigated the effects of three cholesterol-lowering agents (probucol, an enhancer of cholesterol efflux; simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor; and triparanol, a 3β-hydroxysterol-▵24-reductase inhibitor) on cholesterol synthesis, the KCNQ1 current (I KCNQ1), and the I(Ks) to clarify the differences in the modes of action of these agents on the I(Ks). Probucol did not inhibit cholesterol synthesis and had no effect on I KCNQ1, while I(Ks) decreased after 24-h treatment. Simvastatin inhibited cholesterol synthesis and decreased I KCNQ1 and I(Ks). Additionally, the activation kinetics of I(Ks) became faster, compared with that of control I(Ks). Triparanol inhibited cholesterol synthesis but did not reduce I KCNQ1 and I(Ks). However, the activation kinetics of I(Ks) became faster. Our data indicated that the mechanism by which probucol inhibits I(Ks) was not mediated by the inhibition of cholesterol synthesis but depended on an interaction with the KCNQ1/KCNE1 complex. Meanwhile, the reduction in cholesterol induced by simvastatin and triparanol is one of the mechanisms that affects the kinetics of I(ks).

Trafficking of the IKs‐Complex in MDCK Cells: Site of Subunit Assembly and Determinants of Polarized Localization

The voltage-gated potassium channel KV 7.1 is regulated by non-pore forming regulatory KCNE β-subunits. Together with KCNE1, it forms the slowly activating delayed rectifier potassium current IKs . However, where the subunits assemble and which of the subunits determines localization of the IKs -complex has not been unequivocally resolved yet. We employed trafficking-deficient KV 7.1 and KCNE1 mutants to investigate IKs trafficking using the polarized Madin-Darby Canine Kidney cell line. We find that the assembly happens early in the secretory pathway but provide three lines of evidence that it takes place in a post-endoplasmic reticulum compartment. We demonstrate that KV 7.1 targets the IKs -complex to the basolateral membrane, but that KCNE1 can redirect the complex to the apical membrane upon mutation of critical KV 7.1 basolateral targeting signals. Our data provide a possible explanation to the fact that KV 7.1 can be localized apically or basolaterally in different epithelial tissues and offer a solution to divergent literature results regarding the effect of KCNE subunits on the subcellular localization of KV 7.1/KCNE complexes.

Long QT Mutations and KCNE1 β-Subunit Modulate S4 Movement in KCNQ1 Channel

The KCNQ1 potassium channel is expressed in many different cell types and plays widely different physiological roles. For example, in the heart KCNQ1 forms part of the voltage-gated IKs channels that contributes to limiting the duration of the cardiac action potential. The different functions of the KCNQ1 channel are mainly due to the co-assembly of the KCNQ1 channel with different beta subunits from the KCNE family. The IKS channel consists of 4 α-subunits (KCNQ1) which assemble with 2 to 4 β subunits (KCNE1). Mutations in either KCNQ1 or KCNE1 subunit cause multiple cardiac arrhythmia syndromes such as long QT syndrome, short QT syndrome, and familial atrial fibrillation. Here we use Voltage clamp fluorometry (VCF) to directly study the effects of wild type, mutant KCNQ1 and KCNE1 subunits on the voltage sensor movement in KCNQ1 channel. We assess the voltage sensor movement (fluorescence) and channel opening (current), in order to understand the coupling between the KCNQ1 voltage sensor and channel gate in the presence of KCNE1. Our data show that KCNE1 splits the voltage sensor movement in two separates phases. An early phase (1) involving voltage sensor movements to its active state upon changes in the membrane potential occurs at hyperpolarized potentials. A second phase (2) that happens at much more depolarized potentials involves an additional voltage sensor movement which is tightly coupled to channel opening. Mutations in KCNE1 that cause arrhythmia shift the voltage dependence of the voltage sensor movements of either phases 1 or 2 (or both), revealing some of the molecular mechanisms underlying the pathophysiology of these arrhythmia-causing mutations. Our data suggests a putative mechanism for how KCNE1 exerts its effects on the voltage sensor movement during IKs channel activation.

Regulation of Voltage Sensor Movement in KCNQ Channels by KCNE Beta Subunits

The KCNQ1 potassium channel plays widely different physiological roles different cell types. In the heart KCNQ1 forms part of the voltage-gated IKs channels that limit the duration of the cardiac action potential, whereas in epithelial cells KCNQ1 forms part of a voltage-independent K+ channel that is important for ion secretion. The different functions of KCNQ1 are mainly due to the co-assembly of KCNQ1 with different KCNE beta subunits. For example, the IKS channel consists of 4 α-subunits (KCNQ1) which assemble with 2 to 4 β subunits (KCNE1), whereas in epithelial cells KCNQ1 co-assembles with KCNE3 to form voltage-independent K+ channels. Mutations in either KCNQ1 or KCNE1 cause cardiac arrhythmia syndromes. Here we use Voltage clamp fluorometry (VCF) to directly study the effects of wild type and mutant KCNE subunits on the voltage sensor movement in KCNQ1 channel. We assess the voltage sensor movement (fluorescence) and channel opening (current), in order to understand the coupling between the KCNQ1 voltage sensor and channel gate in the presence of KCNE subunits. Our data shows that KCNE1 splits the voltage sensor movement in two separates phases: one at hyperpolarized potentials that moves the voltage sensor to an active state and a second at more depolarized potentials, which is tightly coupled to channel opening. Interestingly, VCF shows that KCNE3 locks the voltage sensor of KCNQ1, presumably in its activated state, thereby generating a voltage-independent K+ channel. Furthermore, arrhythmia-causing mutations in KCNE1 shift the voltage dependence of the two different voltage sensor movements, revealing some of the molecular mechanisms underlying these arrhythmia-causing mutations. Our data suggests a putative mechanism for how KCNE1 subunit exerts its effects on the voltage sensor movement during IKs channel activation.

Polyunsaturated Fatty acids as Kv7 Channel Modulators

Lipids in different forms can directly affect the electric activity of excitable tissues, in some cases via direct interactions with ion channels. For instance is the activity of Kv7 (KCNQ) channels dependent on the abundance of the phospholipid PIP2 in the inner leaflet of the plasma membrane. In this work we instead studied the effect of free extracellular polyunsaturated fatty acids (PUFAs) on Kv7 channels expressed in Xenopus oocytes. We show that ω-3 and ω-6 PUFAs affected the voltage dependence of Kv7.1 and Kv7.2/3 by shifting the conductance versus voltage (G(V)) curves in negative direction along the voltage axis. The effect was pH dependent. In contrast, uncharged methyl esters of the PUFAs did not affect the voltage dependences. Fatty acid requirements and PUFA-induced effects were similar to those previously reported for the Shaker K channel suggesting a similar modulatory mechanism of action, i.e. our previously proposed lipoelectric mechanism in which PUFAs electrostatically affect channel opening. The putative PUFA interaction site is close to the positioning of auxiliary KCNE subunits. We therefore also investigated the impact of KCNE subunits and found that PUFA potency was influenced by KCNE association. Kv7.1 and Kv7.2/3 channels are important for cardiac and neuronal excitability, respectively, and mutated channels resulting in loss-of-function cause heart arrhythmias and epilepsy. The PUFA-induced opening of Kv7 channels found in this study may therefore help explain the antiexcitable properties of PUFAs and the fat-rich ketogenic diet. Lipoelectric modification of the channels voltage dependence could be a future new approach for pharmacological treatment.

Structural Basis for Differential KCNQ1 Interactions with KCNE1 and KCNE2 in the Extracellular Juxtamembrane Region

KCNE1 and KCNE2 are both expressed in human heart and can associate with KCNQ1. KCNE1 and KCNE2 share transmembrane topology and sequence homology, yet they differ in KCNQ1 modulation (KCNE1 slows KCNQ1 Closed-to-Open transition, while KCNE2 slows KCNQ1 Open-to-Closed transition) and pharmacology (KCNQ1/KCNE2 is 5 and 10 fold less sensitive than KCNQ1/KCNE1 to niflumic acid, IKs activator, and azimilide, IKs suppressor). Previous work has shown that the extracellular juxtamembrane (EJM) region of KCNE1 interacts with the extracellular surface of KCNQ1 to modulate gating kinetics and to form IKs activator binding sites. This prompts us to compare the EJMs of the two KCNE subunits in terms of KCNQ1 interaction. We apply cysteine (Cys) scanning mutagenesis to EJMs of KCNE1 and KCNE2 and analyze the patterns of functional perturbation when coexpressed with KCNQ1. We use methanethiosulfonate (MTS) reagents to probe the relationship between EJMs of KCNE subunits and KCNQ1. Finally we probe disulfide formation between Cys engineered into the EJMs of KCNE subunits and those engineered into extracellular surface of KCNQ1. The EJM of KCNE1 makes frequent contacts with KCNQ1, so that MTS modification of exposed Cys side chains can affect channel gating. KCNQ1/KCNE2 is largely indifferent to MTS modification of exposed Cys side chains engineered to EJM of KCNE2. However, MTS can slowly access 7 consecutive hydrophobic positions in the beginning of KCNE2 transmembrane domain, as if there is a crevice between KCNQ1 and KCNE2. Disulfide trapping experiments suggest that KCNE2 is leaning more toward KCNQ1 S2 than KCNE1. We propose that KCNE2 interferes with S2-S4 interactions during KCNQ1 Open-to-Closed transition and thus slows deactivation. This weakens sensitivity to niflumic acid and azimilide by affecting binding site directly or by an allosteric mechanism.

Dynamic Control of IKs Current Amplitude by the ‘Late-Assembly’ Strategy of Channel Subunit Association

Background: Ion channels composed of α (pore-forming) and β (auxiliary) subunits use an ‘early-assembly' strategy (Kir6.x/SUR assembly in ER) to control cell surface expression, or a ‘late-assembly' strategy (α/β2 of rat brain Nav channels, both subunits independently traffic to cell surface) to allow dynamic control of current amplitude/gating kinetics. Slow delayed rectifier (IKs) channel is composed of KCNQ1 (Q1, α) and KCNE1 (E1, β) subunits, and functions as ‘repolarization-reserve' in human heart. It is not clear which of the 2 assembly strategies Q1 and E1 use in forming IKs. Methods: We express Q1 and E1 tagged with fluorescent-protein (Q1-GFP and E1-dsR) or extracellular epitope (AU5-Q1 and HA-E1) in COS-7 and neonatal rat ventricular myocytes (NRVM), and use confocal imaging to track Q1 & E1 movements in cells. Results: In COS-7 cells 3 hr after transfection, Q1 and E1 travel in separate transport intermediates without mixing/fusion. E1 reaches the cell surface before Q1. By 24 hr, Q1 and E1 are colocalized on cell surface. Brefeldin-A (blocking protein export from ER) prevents surface expression of Q1-147C/E1-40C and reduces disulfide formation between the two (as a measure of functional Q1/E1 assembly). In NRVM during in vitro development, Q1-GFP and E1-dsR travel in distinctly different transport intermediates. As NRVM develops into mature-like phenotype, Q1-GFP and E1-dsR are colocalized on the cell surface, with a separate cytosolic Q1-GFP pool colocalized with α-actinin and calnexin (z-line and ER/SR markers, respectively). Conclusions: Q1 and E1 use the ‘late-assembly' strategy to afford dynamic control of IKs current amplitude. This explains why native Q1 and E1 in adult ventricular myocytes are often not well-colocalized. We propose that cardiac myocytes regulate IKs amplitude dynamically by adjusting the degree of Q1/E1 colocalization on cell surface.

Global Ischemia Upregulates KCNQ1 Potassium Channel Activity in Neurons

Transient forebrain or global ischemia induces delayed cell death of hippocampal CA1 pyramidal neurons and impaired cognition. Potassium (K+) channels are implicated as key players in ischemia-induced death. The KCNQ1 K+ channel assembles with the auxiliary subunit KCNE1 to produce the slow component of Ik in the heart. Emerging evidence indicates that KCNQ1 mRNA and protein are expressed not only in heart, but also brain, although its function in neurons is, as yet, unclear.The transcription factor REST (repressor element-1 silencing transcription factor)/NRSF (neuron-specific silencing factor) is upregulated in response to global ischemia and contributes to neuronal death. We recently showed that REST is enriched at the promoter of the KCNQ1 K+ channel in postischemic CA1, assessed by targeted ChIP-chip. Here we set out to assess a possible role for KCNQ1 in ischemia-induced neuronal death. Global ischemia in vivo elicited an increase in KCNQ1 mRNA at 24 (∼1.4 fold) and 48 h (∼3.5-fold; p < 0.04), as assessed by qPCR. Moreover, ischemia induced an increase in KCNQ1 protein at 24 (∼1.4 fold) and 48 h (∼1.6 fold; p < 0.004), as assessed by Western analysis. To examine KCNQ1 function in brain, we identified and recorded K+ currents from cultured hippocampal neurons with the specific KCNQ1 inhibitor Chromanol 293B (IC50=62μM) and confirmed them with a dominant-negative KCNQ1 mutant S277L which reduced the endogenous current by ∼50% (p < 0.005). Cultures exposed to oxygen-glucose deprivation (an in vitro model of ischemia) showed a marked increase in KCNQ1 currents at 48 h post ischemia (∼28%, p < 0.03). These results document the presence of functional KCNQ1 channels in hippocampal neurons, and their upregulation in response to ischemia, consistent with a possible contribution to delayed neuronal death of the CA1.

Hexachlorophene Is a Potent KCNQ1/KCNE1 Potassium Channel Activator Which Rescues LQTs Mutants

The voltage-gated KCNQ1 potassium channel is expressed in cardiac tissues, and coassembly of KCNQ1 with an auxiliary KCNE1 subunit mediates a slowly activating current that accelerates the repolarization of action potential in cardiomyocytes. Mutations of KCNQ1 genes that result in reduction or loss of channel activity cause prolongation of repolarization during action potential, thereby causing long QT syndrome (LQTs). Small molecule activators of KCNQ1/KCNE1 are useful both for understanding the mechanism of the complex activity and for developing therapeutics for LQTs. In this study we report that hexachlorophene (HCP), the active component of the topical anti-infective prescription drug pHisoHex, is a KCNQ1/KCNE1 activator. HCP potently increases the current amplitude of KCNQ1/KCNE1 expressed by stabilizing the channel in an open state with an EC50 of 4.61±1.29 μM. Further studies in cardiomyocytes showed that HCP significantly shortens the action potential duration at 1 μM. In addition, HCP is capable of rescuing the loss of function of the LQTs mutants caused by either impaired activation gating or phosphatidylinositol-4,5-bisphosphate (PIP2) binding affinity. Our results indicate HCP is a novel KCNQ1/KCNE1 activator and may be a useful tool compound for the development of LQTs therapeutics.

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.

Abstract 19413: Regulation of I Ks Current Amplitude by Differential Trafficking Patterns and Interactions Of KCNQ1 and KCNE Subunits

KCNQ1 (Q1, pore-forming subunit) and KCNE1 (E1, modulatory subunit) associate to form the slow delayed rectifier (I Ks ) channel that functions as ‘repolarization reserve’ in human heart. We have shown that in adult guinea pig (GP) cardiomyocytes Q1 &amp; E1 cluster to different subcellular compartments, and the degree of Q1/E1 colocalization affects the I Ks amplitude. We have also shown that KCNE2 (E2) is expressed in human heart, and can associate with Q1 or Q1/E1 to suppress the I Ks amplitude. Objectives: To study the patterns of trafficking and interactions of Q1, E1 and E2. Methods: Q1, E1 and E2 are labeled with eGFP (Q1-G) or mono-dsR (Ex-R, x = 1 or 2) and expressed in COS-7 cells, neonatal rat (NRVM) and adult GP (AGPV) ventricular myocytes; the latter 2 by adenoviral (Adv) gene transfer. Protein trafficking/interactions are monitored by live cell imaging, in conjunction with patch clamping, immunocytochemistry &amp; immunoblotting. Results: Time courses of Q1-G &amp; Ex-R distribution in COS-7 cells suggest that they are translated, packaged and shipped to the cell surface independent of each other. They meet on the cell surface and form Q1-G/Ex-R complexes. Q1-G/Ex-R complexes are endocytosed and colocalized in a cytosolic tubulo-vesicular system. We identify 2 main effects of Ex-R on Q1-G trafficking: (1) Ex-R accelerates Q1-G recovery after photobleaching, (2) 24-48 hr after transfection, E2-R brings Q1-G with it to lysosomes for increased degradation. The distribution patterns of Q1-G and Ex-R in NRVM are similar to those in COS-7 cells. However, under the same Adv incubation conditions, Q1-G and Ex-R are expressed at a much lower level in AGPV than NRVM. In AGPV, Q1-G is distributed mainly as cytosolic striations while Ex-R mainly on cell surface, similar to the native Q1 and Ex immunofluorescence patterns seen in control myocytes. Conclusions: Assembly of Q1 &amp; E1 into I Ks does not occur early during biogenesis. Q1 &amp; E1 meet after they reach the plasma membrane and travel together thereafter. E1 increases the mobility of Q1, while E2 increases Q1 degradation in lysosomes. In adult cardiomyocytes the expression levels and subcellular distribution patterns of Q1 &amp; Ex are tightly regulated, with the sarcomere structure playing a key role in Q1 localization.

How Does KCNE1 Regulate the Kv7.1 Potassium Channel? Model-Structure, Mutations, and Dynamics of the Kv7.1-KCNE1 Complex

The voltage-gated potassium channel Kv7.1 and its auxiliary subunit KCNE1 are expressed in the heart and give rise to the major repolarization current. The interaction of Kv7.1 with the single transmembrane helix of KCNE1 considerably slows channel activation and deactivation, raises single-channel conductance, and prevents slow voltage-dependent inactivation. We built a Kv7.1-KCNE1 model-structure. The model-structure agrees with previous disulfide mapping studies and enables us to derive molecular interpretations of electrophysiological recordings that we obtained for two KCNE1 mutations. An elastic network analysis of Kv7.1 fluctuations in the presence and absence of KCNE1 suggests a mechanistic perspective on the known effects of KCNE1 on Kv7.1 function: slow deactivation is attributed to the low mobility of the voltage-sensor domains upon KCNE1 binding, abolishment of voltage-dependent inactivation could result from decreased fluctuations in the external vestibule, and amalgamation of the fluctuations in the pore region is associated with enhanced ion conductivity.

Mutations in the potassium channel subunit KCNE1 are associated with early-onset familial atrial fibrillation

BackgroundAtrial fibrillation (AF) is the most common arrhythmia. The potassium current IKs is essential for cardiac repolarization. Gain-of-function mutations in KV7.1, the pore-forming α-subunit of the IKs channel, have been associated with AF. We hypothesized that early-onset lone AF is associated with mutations in the IKs channel regulatory subunit KCNE1.MethodsIn 209 unrelated early-onset lone AF patients (< 40 years) the entire coding sequence of KCNE1 was bidirectionally sequenced. We analyzed the identified KCNE1 mutants electrophysiologically in heterologous expression systems.ResultsTwo non-synonymous mutations G25V and G60D were found in KCNE1 that were not present in the control group (n = 432 alleles) and that have not previously been reported in any publicly available databases or in the exom variant server holding exom data from more than 10.000 alleles. Proband 1 (female, age 45, G25V) had onset of paroxysmal AF at the age of 39 years. Proband 2 (G60D) was diagnosed with lone AF at the age of 33 years. The patient has inherited the mutation from his mother, who also has AF. Both probands had no mutations in genes previously associated with AF. In heterologous expression systems, both mutants showed significant gain-of-function for IKs both with respect to steady-state current levels, kinetic parameters, and heart rate-dependent modulation.ConclusionsMutations in KV7.1 leading to gain-of-function of IKs current have previously been described in lone AF, yet this is the first time a mutation in the beta-subunit KCNE1 is associated with the disease. This finding further supports the hypothesis that increased potassium current enhances AF susceptibility.

Stoichiometry of the Slow Iks Potassium Channel in Human Embryonic Stem Cell-Derived Myocytes

The delayed rectifier I(ks) potassium channel is composed of α-(KCNQ1) and β-(KCNE1) subunits. The stoichiometry of I(ks) channels is a matter of some debate. Recently some investigators proposed that the number of KCNE1 subunits per KCNQ1 tetramer could be vary from one to four depending on the relative expression of these two genes. Here we review our previous study of biophysical properties of I(ks) in human embryonic stem cell-derived cardiomyocytes (hESC-CMs) showed that I(ks) in hESC-CMs is a coassembly channel with a stoichiometry other than 1:1, which could be further modulated by additional KCNE1.

Characterization of KCNQ1 atrial fibrillation mutations reveals distinct dependence on KCNE1

The IKs potassium channel, critical to control of heart electrical activity, requires assembly of α (KCNQ1) and β (KCNE1) subunits. Inherited mutations in either IKs 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 IKs channel, and provide mechanisms for understanding the effects on channel deactivation underlying these two atrial fibrillation mutations.

Selective Formation of Oligomeric Kv7.5 (KCNQ5)/KCNE1 and Kv7.5 (KCNQ5)/KCNE3 Channels. Differential Targeting to Membrane Surface Microdomains

Kv7 (KCNQ) proteins form a family of voltage-gated potassium channels that is comprised of five members, Kv7.1-Kv7.5. While Kv7.1 is crucial in the heart, the Kv7.2-Kv7.5 channels contribute to the M-current in the nervous system. In addition, Kv7.5 is expressed in muscles, where its physiological role is currently under evaluation. Kv7 associations with KCNE accessory subunits (KCNE1-5) enhance channel diversity. KCNE peptides control the surface expression, voltage-dependence, kinetics of gating, unitary conductance, ion selectivity and pharmacology of several channels. KCNE subunits have been primarily studied in the heart; however, their activity in the brain and in many other tissues is being increasingly recognized. Here, we found that Kv7.5 and KCNE subunits are present in myoblasts. Therefore, oligomeric associations may underlie some Kv7.5 functional diversity in skeletal muscle. Expression in Xenopus oocytes and HEK-293 cells demonstrates that KCNE1 and KCNE3, but none of the other KCNE subunits, associate to Kv7.5. While KCNE1 slows activation and suppresses inward rectification, KCNE3 inhibits Kv7.5 currents. Furthermore, KCNE1 increases Kv7.5 currents in HEK cells. The membrane targeting is also affected. Biochemical isolation of lipid rafts demonstrates that Kv7.5 barely locates in rafts. KCNE1 and KCNE3 show differential targeting. While KCNE3 targets to rafts, KCNE1 does not. Association of Kv7.5 with KCNE3 impairs KCNE3 targeting to rafts and this is further supported by FRAP analysis. Our results have physiological relevance since Kv7.5 is abundant in skeletal and smooth muscle and its association with KCNE peptides may fine-tune cellular responses.Supported by the Ministerio de Ciencia e Innovacion (MICINN), Spain (BFU2005-00695, BFU2008-00431 and CSD2008-00005).

Illuminating Trafficking of KCNQ1/KCNE1 Channels in Heart

In human heart, pore-forming KCNQ1 subunits associate with auxiliary KCNE1 subunits to generate the slowly activating, delayed rectifier potassium current, IKs. Decreases in IKs, due to either congenital mutations in KCNQ1/KCNE1 subunits or the adverse neuro-hormonal milieu accompanying heart failure, delay cardiac repolarization, leading to long QT syndrome (LQTS). LQTS predisposes to lethal ventricular arrhythmias and sudden cardiac death. How KCNQ1/KCNE1 channel trafficking in the heart proceeds and is regulated is unknown. However, understanding this process is critical both for fundamental mechanistic insights into LQTS, and for rational development of potential new therapies for this condition. We sought to develop optical tools that permit direct visualization of KCNQ1/KCNE1 subunit assembly and trafficking in heart cells. KCNQ1 and KCNE1 were tagged intra-cellularly with YFP and CFP, respectively, and extra-cellularly with a 13-residue high affinity α-bungarotoxin (BTX) binding site (BBS). The tagged proteins were viable as determined by functional electrophysiological assays in Chinese hamster ovary (CHO) cells– BBS-KCNQ1-YFP expressed alone gave rise to rapidly-activating outward currents, and co-expression with KCNE1-CFP resulted in the slowly activating kinetic signature of IKs. Cell surface BBS-tagged channel subunits were selectively detected with quantum dot. Optical pulse chase assays in human embryonic kidney (HEK 293) cells revealed that surface KCNQ1 channels undergo rapid endocytosis and recycling, and this process is largely unaffected by KCNE1. Tagged KCNQ1/KCNE1 subunits expressed in guinea pig heart cells using adenovirus were targeted to the surface sarcolemma, t-tubules and intercalated disks, similar to endogenous channels. Surprisingly, IKs amplitude remained unchanged with over-expression of either KCNQ1 or KCNE1, suggesting a tightly regulated system for maintaining KCNQ1/KCNE1 surface density.

Biophysical properties of slow potassium channels in human embryonic stem cell derived cardiomyocytes implicate subunit stoichiometry

Human embryonic stem cells (hESCs) are an important cellular model for studying ion channel function in the context of a human cardiac cell and will provide a wealth of information about both heritable arrhythmias and acquired electrophysiological disorders. However, detailed electrophysiological characterization of the important cardiac ion channels has been so far overlooked. Because mutations in the gene for the I(Ks) α subunit, KCNQ1, constitute the majority of long QT syndrome (LQT-1) cases, we have carried out a detailed biophysical analysis of this channel expressed in hESCs to establish baseline I(Ks) channel biophysical properties in cardiac myocytes derived from hESCs (hESC-CMs). I(Ks) channels are heteromultimeric proteins consisting of four identical α-subunits (KCNQ1) assembled with auxiliary β-subunits (KCNE1). We found that the half-maximal I(Ks) activation voltage in hESC-CMs and in myocytes derived from human induced pluripotent stems cells (hiPSC-CMs) falls between that of KCNQ1 channels expressed alone and with full complement of KCNE1, the major KCNE subunit expressed in hESC-CMs as shown by qPCR analysis. Overexpression of KCNE1 by transfection of hESC-CMs markedly shifted and slowed native I(Ks) activation implying assembly of additional KCNE1 subunits with endogenous channels. Our results in hESC-CMs, which indicate an I(Ks) subunit stoichiometry that can be altered by variable KCNE1 expression, suggest the possibility for variable I(Ks) function in the developing heart, in different tissues in the heart, and in disease. This establishes a new baseline for I(Ks) channel properties in myocytes derived from pluripotent stem cells and will guide future studies in patient-specific hiPSCs.

Sex specific association of potassium channel subunits

Sex specific association of potassium channel subunits