KCNE1 Subunits Research Articles

N‐ and O‐linked glycosylation coordinate cell‐surface localization of a cardiac potassium channel

<i>N</i>‐ and <i>O</i>‐linked glycosylation coordinate cell‐surface localization of a cardiac potassium channel

Drosophila KCNQ channel displays evolutionarily conserved electrophysiology and pharmacology with mammalian KCNQ channels.

Of the five human KCNQ (Kv7) channels, KCNQ1 with auxiliary subunit KCNE1 mediates the native cardiac IKs current with mutations causing short and long QT cardiac arrhythmias. KCNQ4 mutations cause deafness. KCNQ2/3 channels form the native M-current controlling excitability of most neurons, with mutations causing benign neonatal febrile convulsions. Drosophila contains a single KCNQ (dKCNQ) that appears to serve alone the functions of all the duplicated mammalian neuronal and cardiac KCNQ channels sharing roughly 50–60% amino acid identity therefore offering a route to investigate these channels. Current information about the functional properties of dKCNQ is lacking therefore we have investigated these properties here. Using whole cell patch clamp electrophysiology we compare the biophysical and pharmacological properties of dKCNQ with the mammalian neuronal and cardiac KCNQ channels expressed in HEK cells. We show that Drosophila KCNQ (dKCNQ) is a slowly activating and slowly-deactivating K+ current open at sub-threshold potentials that has similar properties to neuronal KCNQ2/3 with some features of the cardiac KCNQ1/KCNE1 accompanied by conserved sensitivity to a number of clinically relevant KCNQ blockers (chromanol 293B, XE991, linopirdine) and opener (zinc pyrithione). We also investigate the molecular basis of the differential selectivity of KCNQ channels to the opener retigabine and show a single amino acid substitution (M217W) can confer sensitivity to dKCNQ. We show dKCNQ has similar electrophysiological and pharmacological properties as the mammalian KCNQ channels, allowing future study of physiological and pathological roles of KCNQ in Drosophila and whole organism screening for new modulators of KCNQ channelopathies.

KCNE1 and KCNE2 Provide a Checkpoint Governing Voltage-Gated Potassium Channel α-Subunit Composition

Voltage-gated potassium (Kv) currents generated by N-type α-subunit homotetramers inactivate rapidly because an N-terminal ball domain blocks the channel pore after activation. Hence, the inactivation rate of heterotetrameric channels comprising both N-type and non-N-type (delayed rectifier) α-subunits depends upon the number of N-type α-subunits in the complex. As Kv channel inactivation and inactivation recovery rates regulate cellular excitability, the composition and expression of these heterotetrameric complexes are expected to be tightly regulated. In a companion article, we showed that the single transmembrane segment ancillary (β) subunits KCNE1 and KCNE2 suppress currents generated by homomeric Kv1.4, Kv3.3, and Kv3.4 channels, by trapping them early in the secretory pathway. Here, we show that this trapping is prevented by coassembly of the N-type α-subunits with intra-subfamily delayed rectifier α-subunits. Extra-subfamily delayed rectifier α-subunits, regardless of their capacity to interact with KCNE1 and KCNE2, cannot rescue Kv1.4 or Kv3.4 surface expression unless engineered to interact with them using N-terminal A and B domain swapping. The KCNE1/2-enforced checkpoint ensures N-type α-subunits only reach the cell surface as part of intra-subfamily mixed-α complexes, thereby governing channel composition, inactivation rate, and—by extension—cellular excitability.

KCNE1 and KCNE2 Inhibit Forward Trafficking of Homomeric N-Type Voltage-Gated Potassium Channels

Potassium currents generated by voltage-gated potassium (Kv) channels comprising α-subunits from the Kv1, 2, and 3 subfamilies facilitate high-frequency firing of mammalian neurons. Within these subfamilies, only three α-subunits (Kv1.4, Kv3.3, and Kv3.4) generate currents that decay rapidly in the open state because an N-terminal ball domain blocks the channel pore after activation—a process termed N-type inactivation. Despite its importance to shaping cellular excitability, little is known of the processes regulating surface expression of N-type α-subunits, versus their slowly inactivating (delayed rectifier) counterparts. Here we found that currents generated by homomeric Kv1.4, Kv3.3, and Kv3.4 channels are all strongly suppressed by the single transmembrane domain ancillary (β) subunits KCNE1 and KCNE2. A combination of electrophysiological, biochemical, and immunofluorescence analyses revealed this suppression is due to KCNE1 and KCNE2 retaining Kv1.4 and Kv3.4 intracellularly, early in the secretory pathway. The retention is specific, requires α-β coassembly, and does not involve the dynamin-dependent endocytosis pathway. However, the small fraction of Kv3.4 that escapes KCNE-dependent retention is regulated by dynamin-dependent endocytosis. The findings illustrate two contrasting mechanisms controlling surface expression of N-type Kv α-subunits and therefore, potentially, cellular excitability and refractory periods.

Post-translational N-Glycosylation of Type I Transmembrane KCNE1 Peptides: IMPLICATIONS FOR MEMBRANE PROTEIN BIOGENESIS AND DISEASE

N-Glycosylation of membrane proteins is critical for their proper folding, co-assembly and subsequent matriculation through the secretory pathway. Here, we examine the kinetics of N-glycan addition to type I transmembrane KCNE1 K(+) channel β-subunits, where point mutations that prevent N-glycosylation at one consensus site give rise to disorders of the cardiac rhythm and congenital deafness. We show that KCNE1 has two distinct N-glycosylation sites: a typical co-translational site and a consensus site ∼20 residues away that unexpectedly acquires N-glycans after protein synthesis (post-translational). Mutations that ablate the co-translational site concomitantly reduce glycosylation at the post-translational site, resulting in unglycosylated KCNE1 subunits that cannot reach the cell surface with their cognate K(+) channel. This long range inhibition is highly specific for post-translational N-glycosylation because mutagenic conversion of the KCNE1 post-translational site into a co-translational site restored both monoglycosylation and anterograde trafficking. These results directly explain how a single point mutation can prevent N-glycan attachment at multiple sites, providing a new biogenic mechanism for human disease.

Understanding arrhythmogenesis by looking inside the cell

c a k l i a In human ventricular myocardium, the cardiac delayedrectifier potassium current (IK) has two biophysically distinct components (IKr and IKs) and terminates repolarization together with the inward-rectifying current (IK1). The slow delayed-rectifier current (IKs) is an important ionic current roviding a component of the ventricular repolarization eserve. The native channel’s constituents (pore-forming -subunit KCNQ1 and modulatory -subunit KCNE1) asemble with presumably variable stoichiometry to generate unctional complexes with varying biophysical properties. Both delayed-rectifier components have been scrutinized with respect to modulation of synthesis, post-translational processing, trafficking, expression at the membrane, and channel degradation. Each of these processes needs to be functional to maintain a physiological steady state. Changes in the amount of IKs that is available for cardiomyocyte repolarization influence the propensity to arrhythmias in various cardiac conditions (genetically determined arrhythmia syndromes or acquired conditions). Membranes of cardiomyocytes are not immobile and static barriers—rather they represent highly regulated flexible systems with many important functions. After membrane proteins (like ion channels) have been synthesized in the endoplasmic reticulum, they often undergo post-translational modifications and are subsequently trafficked to the cell membrane where they are exposed to fulfill their functional role. In the case of ion channels, these functions directly relate to cellular excitability and repolarization. Malprocessed or malfunctioning proteins are degraded through internal quality control mechanisms before reaching the membrane. Once exposed to the cell exterior, proteins are again removed via endocytosis. Endocytosis is a tightly regulated, energy-consuming process that is crucial for the survival of eukaryotic cells. Clathrin-mediated enocytosis accounts for the greatest contribution to houseeeping or other specialized cellular functions and has so far een studied in the most detail. Adaptation of cells to nvironmental changes (e.g., electrical remodeling) dictates ubsequent alterations in membrane representations of ion hannel proteins. Classically, transcriptional, translational,

KCNE4 Juxtamembrane Region Is Required for Interaction with Calmodulin and for Functional Suppression of KCNQ1

Voltage-gated potassium (K(V)) channels, such as KCNQ1 (K(V)7.1), are modulated by accessory subunits and regulated by intracellular second messengers. Accessory subunits belonging to the KCNE family exert diverse functional effects on KCNQ1, have been implicated in the pathogenesis of various genetic disorders of heart rhythm, and contribute to transducing intracellular signaling events into changes in K(V) channel activity. We investigated the interactions between calmodulin (CaM), the ubiquitous Ca(2+)-transducing protein that binds and confers Ca(2+) sensitivity to the biophysical properties of KCNQ1, and KCNE4. These studies were motivated by the observed similarities between the suppression of KCNQ1 function by pharmacological disruption of KCNQ1-CaM interactions and the effects of KCNE4 co-expression on the channel. We determined that KCNE4, but not KCNE1, can biochemically interact with CaM and that this interaction is Ca(2+)-dependent and requires a tetraleucine motif in the juxtamembrane region of the KCNE4 C terminus. Furthermore, disruption of the KCNE4-CaM interaction either by mutagenesis of the tetraleucine motif or by acute Ca(2+) chelation impairs the ability of KCNE4 to inhibit KCNQ1. Our findings have potential relevance to KCNQ1 regulation both by KCNE accessory subunits and by an important intracellular signaling molecule.

Plasticity in KCNQ1 Subcellular Distribution and Partnership with Different KCNE Subunits Contribute to Variations in IKs Channel Function in the Heart

Background: KCNQ1 can associate with KCNE1 - KCNE5 subunits, singly or in combination, and assume different channel phenotypes in current amplitude and gating kinetics. In the heart, KCNQ1 is known to partner with KCNE1 to form the slow delayed rectifier (IKs) channel, a major contributor to cardiac electrical heterogeneity and stabilizer of heart rhythm. It is unclear whether KCNQ1 can partner with other KCNE subunits expressed in the heart, and how these potential partnerships impact on IKs channel function. Methods: We study the above issues by patch clamping, immunoblotting, and immunofluorescence/confocal microscopy. Results: (1) IKs current density is higher in guinea pig (GP) atrial (A) than ventricular (V) myocytes. There are 2 contributing factors: KCNQ1 protein level is higher in A than in V, and KCNQ1 is colocalized with KCNE1 in the peripheral cell surface to a higher degree in A than in V. (2) IKs current density in V myocytes is much lower in spontaneously hypertensive rat (SHR) than in GP, despite abundant expression of KCNQ1 and KCNE1 in SHR. This coincides with a higher degree of KCNQ1 colocalization with KCNE2 in SHR than in GP V myocytes, and the current-suppressing effect of KCNE2 on IKs. (3) IKs is upregulated during aging in SHR V myocytes. This correlates with a modest increase in KCNQ1 protein expression and, importantly, a prominent redistribution of KCNQ1 and a higher degree of colocalization with KCNE1 on the peripheral cell surface. Conclusion: We propose that the KCNQ1 channel exhibits plastic subcellular distribution patterns and dynamic partnerships with KCNE1 and KCNE2 in the heart. These factors contribute to IKs channel remodeling in response to variations in work load or changes in the heart rate.

KCNE2 uses More Domains than KCNE1 to Modulate KCNQ1 Channel Function

KCNE1 & KCNE2 are both single membrane-passing peptides with amino- & carboxyl ends in extra- & intra-cellular compartments. They also share high sequence homology in the transmembrane (TM) and juxtamembranous regions. Yet the two have distinctly different effects when associated with the KCNQ1 channel. Understanding the structural basis for differential KCNQ1 modulation by the 2 KCNE subunits is prerequisite to drug design targeting such interactions. We use the techniques of NMR, cysteine (Cys)-scanning mutagenesis and voltage clamping to explore this issue. We learn the following: (1) KCNE2 differs from KCNE1 in the extracellular juxtamembranous region (helix vs unstructured loop). Cys substitution in this region has distinct impact on the gating kinetics and/or pore conductance of the KCNQ1/KCNE2 channel complex, but has little or no effects on the KCNQ1/KCNE1 channel function. (2) KCNE2 & KCNE1 sequences diverge in the carboxyl end. Truncating this region of KCNE1 (93 −129) does not interfere with its ability to modulate KCNQ1, while truncating the corresponding region of KCNE2 (98-123) abolishes its function as a KCNQ1 modulator. Intracellular application of a peptide corresponding to KCNE2 aa 98-123 reduces currents through KCNQ1, increases currents through KCNQ1/KCNE2, but has no effects on currents through KCNQ1/KCNE1. (3) Cys substitution along the TM helices of KCNE1 and KCNE2 affects the gating kinetics and/or pore conductance of the KCNQ1/KCNE channel complexes. Structural alignment suggests a rotation of KCNE2 relative to KCNE1 in terms of helical faces interacting with the pore domain and voltage-sensing domain of the KCNQ1 channel. We propose that while both KCNE subunits utilize their TM helices to interact with KCNQ1, the more rigid helical structure of KCNE2 allows it to allosterically modulate the KCNQ1 gating and ion permeation properties by the extracellular juxtamembranous and cytoplasmic carboxyl domains.

Species-Specific PKC Activation of IK S Channels

Calcium activated potassium channels (IKs) is a slow activating potassium channel that is one of the main channels controlling cardiac repolarization. The IKs channel is formed by a pore subunit, KCNQ1 and an auxiliary subunit, KCNE1. These can be modulated by two different secondary messenger kinase proteins, PKA and PKC. Previous reports have suggested that activation of PKC decreases the IKs current in mice, while in humans, PKC activation increases the IKs current. However the detailed mechanism underlying this species-different regulation of IKs by PKC is not known. Here, we expressed the channel in Xenopus oocytes by injecting them with RNA encoding for the rat and human KCNQ1 and KCNE1 subunits. In addition, the muscarinic receptor (M1), a Gq-protein coupled receptor which downstream signaling activates PKC, was also expressed. We used two-electrode voltage clamp techniques to measure channel current. We measured the effect of PKC on both channel ability to conduct current and voltage to activate the channel by measuring the maximal channel conductance (Gmax) and the voltage that activates half of the maximal current (V1/2), respectively. Our results shows that upon short acetylcholine stimulation, human IKs channels show an increase in Gmax and a shift in V1/2 whereas rat channels exhibit a shift in V1/2 without Gmax changes. Finally, prolonged acetylcholine stimulation abolished Gmax increase on human channels whereas decreases it on rat channels. Thus, understanding of the PKC regulation of human IKs current as compared with other species, may allow the development of specific drugs to control cardiac rhythm pathologies.

Biophysical Characterization of I ks Channels in Cardiomyocytes Derived from Human Embryonic Stem Cells Reveals Insights Into Stoichiometry of KCNQ1-KCNE1 Complex

The slowly activating IKs cardiac potassium channels are heteromultimers composed of four identical α-subunits (KCNQ1) which assemble with auxiliary -subunits (KCNE1) that confer distinct biophysical properties on the channels: a slowing of activation and deactivation kinetics and a large depolarizing shift in activation. Here we characterize the electrophysiological properties of endogenous IKs channels in single cardiomyocytes derived from human embryonic stem cells (hESC-CMs). IKs was identified as Chromanol 293B-sensitive current measured during prolonged depolarization. The mid-point of IKs activation occurred at 9.1±2.4 mV (n=11), between that of KCNQ1 alone (V1/2=-21.3±1.5 mV, n=4) and KCNQ1 expressed with a saturating amount of KCNE1 (V1/2=27.2±1.2 mV, n=5) in HEK293 cells. To determine whether ESC-CM background affects IKs biophysical properties, we engineered KCNQ1 channels to impart a sensitivity to charybdotoxin (CTX) and transiently expressed these channels (CTX-KCNQ1) in hESC-CMs with or without KCNE1. We used CTX to dissect the recombinant IKs channels from endogenous channels and found they shared similar properties as in HEK293 cells (CTX-KCNQ1: V1/2=-27.2±4.4 mV, n=4; CTX-KCNQ1 + KCNE1: V1/2=30.9±1.1 mV, n=5). This confirms that the mid-point of activation of endogenous hESC-CM IKs lies between KCNQ1 alone and KCNQ1/KCNE1 heteromultimers, suggesting that endogenous IKs is produced by KCNQ1 channels that are not saturated by KCNE1. We tested this hypothesis by overexpressing KCNE1 in hESC-CMs and found that endogenous IKs channels activated more slowly and at more positive voltages (V1/2=34.2±1.4 mV, n=4) than in non-transfected cells. This is the first comprehensive report of the biophysical properties of IKs in hESC-CMs, suggesting that endogenous IKs is produced by KCNQ1 channels that are assembling, but not saturated with, KCNE1 subunits.

Chronic PKC Activation Inhibits the Repolarizing Cardiac Current by Decreasing the Functional ion Channel Expression at the Plasma Membrane

Background: Cardiac arrhythmias are particularly common in heart failure patients. The slow delayed rectifier K+ current (IKs) is decreased in patients and in animal models of heart failure. However, the molecular mechanisms for IKs decrease are not known. PKC signaling is one of the most prominent pathways activated during heart failure, with both expression and activation of PKC being increased. Hypothesis: Chronic PKC activation inhibits IKs and delays cardiac repolarization, contributing to arrhythmogenic risk in heart failure. Methods: Human IKs channels, PKCalpha catalytic subunit (CA) and alpha1A-adrenoceptor (alpha1A-AR) were expressed in HEK293T cells. IKs current was measured by patch clamp. Subcellular localization of GFP-tagged IKs channels was observed by confocal microscopy. Results: IKs currents were strongly inhibited by either overexpression of PKCalphaCA or 2-hr alpha1A-AR stimulation by phenylephirne (Fig. 1A), although acute application of Ca2+-dependent PKC (cPKC) activator peptide or alpha1A-AR stimulation increased IKs. Plasma membrane localization of GFP-tagged IKs channels was significantly decreased by chronic alpha1A-AR-cPKC signaling activation (Fig.1B). Application of PKC inhibitors or mutation of a putative PKC phosphorylation site in the auxiliary KCNE1 subunit abolished this effect. Conclusion: Chronic PKC activation inhibits IKs currents by decreasing channel plasma membrane expression via direct phosphorylation of KCNE1 subunit. Our results suggest that cPKC-dependent phosphorylation of the IKs channel may contribute to QT prolongation and arrhythmogenesis in heart failure and implicate cPKC inhibitors as potential novel antiarrhythmic drugs.

Combination In Vitro and in Silico Methodology for Risk Assessment of Long QT Type 1 Patients

There is a long history of simulating the effects of channelopathies on the cardiac action potential (AP) in diseases like Long QT (LQT) Syndrome. While an important proof of concept, studies have been limited because the number of mutants considered was small and correlation with the phenotype was anecdotal. This study seeks to address such limitations by including 17 Long QT type 1 (LQT 1) mutations for which both in vitro characterizations have been performed and detailed clinical outcome data are known. The IKs channel (KCNQ1 and KCNE1 subunits) were expressed in both Xenopus oocytes and HEK-293T cells, and Gmax, Vhalf and the τ of activation and deactivation were measured. The properties were then incorporated into a humanized version of the Flaim-Giles-McCulloch reconstruction of canine cardiomyocytes. The model also included beta-adrenergic stimulation that is known to modulate IKs and is thought to contribute to exercise-induced sudden death in some LQT patients. Both single cell and 1-D cable models were investigated. The simulated QT prolongation in the cable model correlated well with the QTc in patients carrying the 17 mutations studied. The cable model including beta-adrenergic stimulation showed early after-depolarizations (EADs) and T-wave alternans for mutants. The presence of T-wave alternans correlated with cardiac risk for these patients. Correlation of electrophysiology and clinical phenotype showed that slow activation rates for LQT1 mutations are risk factors for these patients independent from QT prolongation. Incorporation of slow IKs activation into the model produced a more severe to response to β-adrenergic-mediated EADs as compared to responses with reduced IKs conductance only. Our results suggest that cellular electrophysiology in combination with 1-D cable models is a good methodology to predict cardiac risk associated with Long QT1 mutations.

A Spectrum of Functional Phenotypes Associated with LQT1 Mutations Identified in Patients with Early-Onset Atrial Fibrillation

Mutations in KCNQ1, the gene encoding the voltage-gated K+ channel α-subunit that underlies the slowly activating delayed rectifier K+ current (IKs) in the heart, are linked to Type I Long QT Syndrome (LQT1) and Familial Atrial Fibrillation (FAF). Compared to the background prevalence of 0.1%, early-onset atrial fibrillation (patients < 50 years) was observed in ∼2% of LQT1 patients (Johnson et al., Heart Rhythm, 2008). We expressed several LQT1 mutations (P7S, R231H, and T322A) identified in patients with early-onset AF in HEK293 to better understand their functional phenotype. Cells were transfected with cDNA for the auxiliary K+ channel subunit KCNE1 (E1) and WT-, P7S-, R231H-, or T322A-KCNQ (Q1). Whole-cell Q1E1 currents (IQ1E1) were measured using the patch-clamp technique and holding potential of −80mV. We measured I-V relations from these cells by prepulsing from a holding potential of −80 mV to 70 mV in 10-mV increments for 5 seconds, followed by a test-pulse to −50mV for 5 seconds. The peak IQ1E1 measured during the test-pulse was plotted as function of the pre-pulse and the data were described using the Boltzmann equation to calculate the maximally activated IQ1E1 (IMAX), the midpoint potential (V1/2), and slope factor (k). Each mutation generated different functional phenotypes. Compared to cells expressing WT (n=7), cells expressing P7S (n=10) did not alter IMAX (WT=99±9 pA/pF, P7S=93±17 pA/pF), V1/2 (WT=26±3mV, P7S=24±3mV), or k (WT=16±1mV/e-fold ΔI, P7S=16±1mV/e-fold ΔI ). Cells expressing R231H (n=9) generated constitutively active IQ1E1 (R231H=64.07±27.48 pA/pF) at −80 mV that was similar to previously described FAF-linked mutations, and IMAX was not altered (80.09±15.58 pA/pF). In contrast, cells expressing T322A (n=7) generated no IQ1E1. These data demonstrate there is no characteristic IQ1E1 phenotype for LQT1 mutations identified in patients with early-onset AF.

Activation of KNCQ1/KCNE1 Channel by Classic PKC is Impaired in Long QT Syndrome Type 1

Stimulation of the cardiac K+ channel IKs (KCNQ1/KCNE1) is crucial in mediating the QT shortening at high adrenergic stimulation states, with mutations in the KCNQ1 subunit being linked to a high risk of cardiac events triggered by adrenergic stimulation. In addition to β-adrenergic receptors (AR), α1-AR is also activated upon adrenergic stimulation in the heart. IKs is strongly regulated by β-AR stimulation, but little is known about the role of α1-AR-mediated regulation. In this study, we investigated the molecular mechanism underlying α1-AR-mediated regulation of human IKs channel and mutant channels found in Long QT syndrome type 1 (LQT1). We overexpressed wild-type and mutant KCNQ1/KCNE1 subunits together with the α1A-AR in HEK293T cells. α1-AR activation strongly facilitated voltage dependence of IKs activation independently of β-AR stimulation (V1/2 shift ≅ - 20 mV). This effect was blocked by pretreatment of cell-permeable classic PKC (cPKC) inhibitory peptide and selective cPKC activation by cell-permeable cPKC activator peptide mimicked this effect. A mutation in a putative PKC phosphorylation site in the auxiliary subunit KCNE1 (S102A) specifically abolished the voltage shift by α1-AR stimulation or cPKC activation, suggesting cPKC phosphorylation of KCNE1(S102) increases IKs current through facilitation in voltage dependence of activation. LQT1 associated mutations at the voltage activation domain (S1-S5) of KCNQ1 subunit, also impaired cPKC regulation of the IKs channel independently of β-AR regulation, suggesting that the interactions between the KCNE1 and the voltage-gating domain of KCNQ1 is crucial for cPKC regulation. In conclusion, our study indicates that the voltage activation of IKs by cPKC isoform is important in the IKs channel regulation under adrenergic stimulation and that impairment of this regulation may be linked to LQT1 clinical phenotype.

Is Rapid Atrial Pacing Associated with Thrombogenesis in Normal Hearts?

that irregular ventricular activation alters ion channel expression and cellular electrophysiology. Methods:mRNA expression of ion channels and accessory subunits was examined in neonatal rat ventricular myocytes (NVCMs) paced either regularly or irregularly for 24 hours to simulate ventricular activation in sinus rhythm (SR) and AF respectively. Relevant transmembrane currents were measured after 96 hours of regular vs irregular pacing using whole cell patch clamp approach. LV myocardium from end-stage heart failure (ESHF) patients in SR and chronic AF undergoing cardiac transplantation also underwent mRNA expression studies as guided by results from the NVCMmodel. Results: Irregular pacing induced a 48% increase in NVCMmRNA expression of KCNA4. However, there was no difference in Ito current density across pacing strategies. Irregular pacing induced trivial changes in NVCM mRNAexpression for theNa channel SCN5A (11% reduction), and the Ca channels CACNA1C (19% increase), and CACNA1H (8% decrease). No differences were observed for ion channels CACNA1G, KCNQ1, KCNH2, KCND3, KCNJ2, and KCNJ11; accessory subunits KCNE1, KCNE2, KCNIP2, and ABCC9; and gap junction subunits GJA1 and GJA5. There were no differences in mRNA expression of SCN5A, CACNA1C, CACNA1H, or KCNA4 in LV myocardium from ESHF patients in AF vs SR. Conclusion: In an acute model, irregular ventricular a i a a

KCNQ1 Channels Voltage Dependence through a Voltage-dependent Binding of the S4-S5 Linker to the Pore Domain

Voltage-dependent potassium (Kv) channels are tetramers of six transmembrane domain (S1-S6) proteins. Crystallographic data demonstrate that the tetrameric pore (S5-S6) is surrounded by four voltage sensor domains (S1-S4). One key question remains: how do voltage sensors (S4) regulate pore gating? Previous mutagenesis data obtained on the Kv channel KCNQ1 highlighted the critical role of specific residues in both the S4-S5 linker (S4S5(L)) and S6 C terminus (S6(T)). From these data, we hypothesized that S4S5(L) behaves like a ligand specifically interacting with S6(T) and stabilizing the closed state. To test this hypothesis, we designed plasmid-encoded peptides corresponding to portions of S4S5(L) and S6(T) of the voltage-gated potassium channel KCNQ1 and evaluated their effects on the channel activity in the presence and absence of the ancillary subunit KCNE1. We showed that S4S5(L) peptides inhibit KCNQ1, in a reversible and state-dependent manner. S4S5(L) peptides also inhibited a voltage-independent KCNQ1 mutant. This inhibition was competitively prevented by a peptide mimicking S6(T), consistent with S4S5(L) binding to S6(T). Val(254) in S4S5(L) is known to contact Leu(353) in S6(T) when the channel is closed, and mutations of these residues alter the coupling between the two regions. The same mutations introduced in peptides altered their effects, further confirming S4S5(L) binding to S6(T). Our results suggest a mechanistic model in which S4S5(L) acts as a voltage-dependent ligand bound to its receptor on S6 at rest. This interaction locks the channel in a closed state. Upon plasma membrane depolarization, S4 pulls S4S5(L) away from S6(T), allowing channel opening.

Impact of KCNE subunits on KCNQ1 (Kv7.1) channel membrane surface targeting

The KCNQ1 (Kv7.1) channel plays an important role in cardiovascular physiology. Cardiomyocytes co-express KCNQ1 with KCNE1-5 proteins. KCNQ1 may co-associate with multiple KCNE regulatory subunits to generate different biophysically and pharmacologically distinct channels. Increasing evidence indicates that the location and targeting of channels are important determinants of their function. In this context, the presence of K(+) channels in sphingolipid-cholesterol-enriched membrane microdomains (lipid rafts) is under investigation. Lipid rafts are important for cardiovascular functioning. We aimed to determine whether KCNE subunits modify the localization and targeting of KCNQ1 channels in lipid rafts microdomains. HEK-293 cells were transiently transfected with KCNQ1 and KCNE1-5, and their traffic and presence in lipid rafts were analyzed. Only KCNQ1 and KCNE3, when expressed alone, co-localized in raft fractions. In addition, while KCNE2 and KCNE5 notably stained the cell surface, KCNQ1 and the rest of the KCNEs showed strong intracellular retention. KCNQ1 targets multiple membrane surface microdomains upon association with KCNE peptides. Thus, while KCNQ1/KCNE1 and KCNQ1/KCNE2 channels target lipid rafts, KCNQ1 associated with KCNE3-5 did not. Channel membrane dynamics, analyzed by fluorescence recovery after photobleaching (FRAP) experiments, further supported these results. In conclusion, the trafficking and targeting pattern of KCNQ1 can be influenced by its association with KCNEs. Since KCNQ1 is crucial for cardiovascular physiology, the temporal and spatial regulations that different KCNE subunits may confer to the channels could have a dramatic impact on membrane electrical activity and putative endocrine regulation.

The cardiac I Ks channel, complex indeed

The cardiac IKs channel is a major repolarization current in the heart that responds rapidly and robustly to sympathetic nervous system stimulation to ensure adequate diastolic filling time in the face of accompanying accelerated heart rate. In cardiac myocytes, the IKs channel is a macromolecular complex composed of a pore-forming α (KCNQ1) subunit and modulatory β (KCNE1) subunit, as well as intercellular proteins critical for controlling the phosphorylation state of the complex (1). Although KCNQ1 alone assembles to form a voltage-gated potassium channel, the presence of KCNE1 is required to reproduce the kinetic properties of the native IKs channel, and KCNE1 coassembly is necessary to mediate the characteristic IKs response to sympathetic stimulation (2). The importance of the KCNE1 subunit is evidenced by its role in IKs channelopathies: mutation in either KCNQ1 or KCNE1 can underlie congenital long QT syndrome, and the presence of the β subunit is reported to be required for the gain-of-function phenotype in two KCNQ1 mutations implicated in congenital atrial fibrillation (3, 4). Among the questions surrounding the nature of the KCNQ1–KCNE1 association is that of the stoichiometry of the α and β subunits. Because of its homology with the remainder of the Kv channel superfamily, KCNQ1 (Kv7.1) is thought to form channels consisting of four α subunits (5). The number …

Stoichiometry of the KCNQ1 - KCNE1 ion channel complex

The KCNQ1 voltage-gated potassium channel and its auxiliary subunit KCNE1 play a crucial role in the regulation of the heartbeat. The stoichiometry of KCNQ1 and KCNE1 complex has been debated, with some results suggesting that the four KCNQ1 subunits that form the channel associate with two KCNE1 subunits (a 42 stoichiometry), while others have suggested that the stoichiometry may not be fixed. We applied a single molecule fluorescence bleaching method to count subunits in many individual complexes and found that the stoichiometry of the KCNQ1-KCNE1 complex is flexible, with up to four KCNE1 subunits associating with the four KCNQ1 subunits of the channel (a 44 stoichiometry). The proportion of the various stoichiometries was found to depend on the relative expression densities of KCNQ1 and KCNE1. Strikingly, both the voltage-dependence and kinetics of gating were found to depend on the relative densities of KCNQ1 and KCNE1, suggesting the heart rhythm may be regulated by the relative expression of the auxiliary subunit and the resulting stoichiometry of the channel complex.