KCNQ1 Subunits Research Articles

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.

N and C Terminal Interactions Underlie Channel Gating of M Channels

Neuronal M-type K channels composed of KCNQ2 and KCNQ3 subunits regulate firing properties of neurons. Presynapticlly, KCNQ2 subunits were also found to regulate neurotransmitter release by directly influencing presynaptic function. Previously, we have showed the existence of constitutive interactions between the cytosolic N and C termini of homomeric KCNQ2 or KCNQ3 channels in living cells, and demonstrated that a modulation of the N-C interactions of KCNQ2, but not of KCNQ3, by two regulatory proteins, syntaxin 1A and calmodulin, accompany a reduction in single-channel open probability, suggesting that closer N-C termini proximity underlies gating downregulation. Furthermore, in KCNQ3, identified N terminal and C terminal structural determinants, employ the preclusion of the regulatory proteins effects on the N-C rearrangement and gating regulation. This strongly suggested that N and C structural determinants confer the isoform-specific gating downregulation. Notably, the N-C interactions of both subunits were found to consist of a basal interaction, similar in both channels, and an additional interaction in KCNQ3 formed by an NT distal-end module. Here, we show that the basal N-C interaction is common to both KCNQ2 and KCNQ3 subunits and is essential for proper channel gating of both types of channel. We demonstrate, using optical, biochemical, electrophysiological and molecular biology analyses, that mutations and truncations at specific locations at the N or C termini of the channels abolish, partially or totally, the channels N-C interaction, as well as single- channel gating. Further, we look into the involvement of calmodulin in the basal N-C interactions.

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.

Abstract 16820: Impaired KCNQ1/KCNE1 Channel Activation by Alpha-Adrenergic Receptor is Associated with Emotion/Arousal Triggered Events in Long QT Syndrome Type 1

Background: 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. IKs is strongly regulated by β-adrenergic receptor (AR) stimulation, but little is known about the role of α 1 -AR-mediated regulation and relation to the LQT1 phenotype. Methods: We co-expressed wild-type and mutant KCNQ1/KCNE1 subunits together in HEK293T cells. A single cardiomyocyte model adapted from the Flaim-Giles-McCulloch model was used to simulate the effect of α 1 -AR and β-AR activation in action potential duration (APD). Mutation specific adrenergic-effects on APD were correlated to the cumulative rate for exercise and stress-triggered cardiac events (syncope, sudden death and aborted cardiac arrest) by age 40 for patients carrying the mutation. Results: Acute α 1 -AR activation strongly facilitated voltage dependence of IKs activation independently of β-AR stimulation (V 1/2 shift @ - 20 mV) via activation of cPKC. We measured the effect of β-AR-PKA and α 1 -AR-cPKC activation for 8 LQT1 associated mutations. For each mutant we introduced the current activation measured in a single cardiomyocyte model to simulate the overall effect of (α 1 + β1) AR stimulation. Mutant specific α 1 - and β- AR-mediated APD showed stronger correlation to the cumulative rate of emotion/arousal triggered cardiac events than β-AR alone (α+β: R 2 =0.94, β: R 2 =0.58), while β-AR activation alone showed a better correlation with exercised triggered event rate (α+β: R 2 =0.34, β: R 2 =0.74) for patients carrying these mutations. Conclusion: Our study suggests that acute α 1 -AR regulation of the voltage activation of IKs may be important for QT shortening in response to strong adrenergic stimulation in the flight and fight response. In addition, impaired α 1 -AR regulation may be linked to increased risk of sudden emotion/arousal triggered cardiac events in LQT1patients.

Regulation of Neuronal M-Channel Gating in an Isoform-Specific Manner: Functional Interplay between Calmodulin and Syntaxin 1A

Whereas neuronal M-type K(+) channels composed of KCNQ2 and KCNQ3 subunits regulate firing properties of neurons, presynaptic KCNQ2 subunits were demonstrated to regulate neurotransmitter release by directly influencing presynaptic function. Two interaction partners of M-channels, syntaxin 1A and calmodulin, are known to act presynaptically, syntaxin serving as a major protein component of the membrane fusion machinery and calmodulin serving as regulator of several processes related to neurotransmitter release. Notably, both partners specifically modulate KCNQ2 but not KCNQ3 subunits, suggesting selective presynaptic targeting to directly regulate exocytosis without interference in neuronal firing properties. Here, having first demonstrated in Xenopus oocytes, using analysis of single-channel biophysics, that both modulators downregulate the open probability of KCNQ2 but not KCNQ3 homomers, we sought to resolve the channel structural determinants that confer the isoform-specific gating downregulation and to get insights into the molecular events underlying this mechanism. We show, using optical, biochemical, electrophysiological, and molecular biology analyses, the existence of constitutive interactions between the N and C termini in homomeric KCNQ2 and KCNQ3 channels in living cells. Furthermore, rearrangement in the relative orientation of the KCNQ2 termini that accompanies reduction in single-channel open probability is induced by both regulators, strongly suggesting that closer N-C termini proximity underlies gating downregulation. Different structural determinants, identified at the N and C termini of KCNQ3, prevent the effects by syntaxin 1A and calmodulin, respectively. Moreover, we show that the syntaxin 1A and calmodulin effects can be additive or blocked at different concentration ranges of calmodulin, bearing physiological significance with regard to presynaptic exocytosis.

Fasting and 17β-Estradiol Differentially Modulate the M-Current in Neuropeptide Y Neurons

Multiple K(+) conductances are targets for many peripheral and central signals involved in the control of energy homeostasis. Potential K(+) channel targets are the KCNQ subunits that form the channels underlying the M-current, a subthreshold, non-inactivating K(+) current that is a common target for G-protein-coupled receptors. Whole-cell recordings were made from GFP (Renilla)-tagged neuropeptide Y (NPY) neurons from the arcuate nucleus of the hypothalamus using protocols to isolate and characterize the M-current in these orexigenic neurons. We recorded robust K(+) currents in the voltage range of the M-current, which were inhibited by the selective KCNQ channel blocker 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride (XE991) (40 μm), in both intact males and ovariectomized, 17β-estradiol (E2)-treated females. Since NPY neurons are orexigenic and are active during fasting, the M-current was measured in fed and fasted male mice. Fasting attenuated the XE991-sensitive current by threefold, which correlated with decreased expression of the KCNQ2 and KCNQ3 subunits as measured with quantitative real-time PCR. Furthermore, E2 treatment augmented the XE991-sensitive M-current by threefold in ovariectomized (vs oil-treated) female mice. E2 treatment increased the expression of the KCNQ5 subunit in females but not KCNQ2 or KCNQ3 subunits. Fasting in females abrogated the effects of E2 on M-current activity, at least in part, by decreasing KCNQ2 and KCNQ3 expression. In summary, these data suggest that the M-current plays a pivotal role in the modulation of NPY neuronal excitability and may be an important cellular target for neurotransmitter and hormonal signals in the control of energy homeostasis in both males and females.

Partial restoration of the long QT syndrome associated KCNQ1 A341V mutant by the KCNE1 β-subunit

Background The A341V mutation in the pore-forming KCNQ1 subunit of the slowly activating delayed-rectifier potassium current (IKs) underlies a common form of the long QT syndrome, and is associated with an unusually severe phenotype. However, there is controversy regarding the underlying mechanism responsible for the clinically observed phenotype. We investigated the biophysical characteristics of A341V in a cardiac environment by utilizing a cardiac cell line, and in particular the impact of the KCNE1 β-subunit. Methods Whole-cell current were recorded from transiently transfected HL-1 cells, a cardiac cell line. Mutant KCNQ1 and KCNE1 were constructed by site-directed mutagenesis. Results The A341V mutant resulted in a non-functional channel when expressed alone. When co-expressed with wild type KCNE1, A341V produced a slowly activating current, with a smaller current density, slower rates of activation, and a depolarized shift in its activation curve compared to the wild type KCNQ1 + KCNE1. Confocal microscopy confirmed the surface expression of GFP-tagged A341V, suggesting a functionally defective protein. A T58A mutation in KCNE1 abolished functional restoration of A341V. Under heterozygous conditions, the expression of A341V + KCNQ1 + KCNE1 reduced but did not abolish the electrophysiological changes observed in A341V + KCNE1. A dominant negative effect of A341V was also observed. Action potential simulations revealed that the A341V mutation is arrhythmogenic. Conclusions The KCNE1 β-subunit partially rescued the non-functional A341V mutant, with electrophysiological properties distinct from the wild type IKs. General significance The severity of the A341V phenotype may be due to a combination of a significant suppression of the IKs with altered biophysical characteristics.

KCNQ5/K(v)7.5 potassium channel expression and subcellular localization in primate retinal pigment epithelium and neural retina.

Previous studies identified in retinal pigment epithelial (RPE) cells an M-type K(+) current, which in many other cell types is mediated by channels encoded by KCNQ genes. The aim of this study was to assess the expression of KCNQ genes in the monkey RPE and neural retina. Application of the specific KCNQ channel blocker XE991 eliminated the M-type current in freshly isolated monkey RPE cells, indicating that KCNQ subunits contribute to the underlying channels. RT-PCR analysis revealed the expression of KCNQ1, KCNQ4, and KCNQ5 transcripts in the RPE and all five KCNQ transcripts in the neural retina. At the protein level, KCNQ5 was detected in the RPE, whereas both KCNQ4 and KCNQ5 were found in neural retina. In situ hybridization in frozen monkey retinal sections revealed KCNQ5 gene expression in the ganglion cell layer and the inner and outer nuclear layers of the neural retina, but results in the RPE were inconclusive due to the presence of melanin. Immunohistochemistry revealed KCNQ5 in the inner and outer plexiform layers, in cone and rod photoreceptor inner segments, and near the basal membrane of the RPE. The data suggest that KCNQ5 channels contribute to the RPE basal membrane K(+) conductance and, thus, likely play an important role in active K(+) absorption. The distribution of KCNQ5 in neural retina suggests that these channels may function in the shaping of the photoresponses of cone and rod photoreceptors and the processing of visual information by retinal neurons.

Distribution of M-channel subunits KCNQ2 and KCNQ3 in rat hippocampus

Neuronal M-channels are low threshold, slowly activating and non-inactivating, voltage dependent K(+) channels that play a crucial role in controlling neuronal excitability. The native M-channel is composed of heteromeric or homomeric assemblies of subunits belonging to the Kv7/KCNQ family, with KCNQ2/3 heteromers being the most abundant form. KCNQ2 and KCNQ3 subunits have been found to be expressed in various neurons in the central and peripheral nervous system of rodents and humans. Previous evidence shows preferential localization of both subunits to axon initial segments, somata and nodes of Ranvier. In this work, we show the distribution and co-localization of KCNQ2 and KCNQ3 subunits throughout the hippocampal formation, via immunostaining experiments on unfixed rat brain slices and confocal microscopy. We find intense localization and colocalization to the axonal initial segment in several regions of the hippocampus, as well as staining for non-neuronal cells in the area of the lateral ventricle. We did not observe colocalization of KCNQ2 or KCNQ3 with the presynaptic protein, synaptophysin.

Role of neuronal potassium M-channels in sympathetic regulation of cardiac function

Role of neuronal potassium M-channels in sympathetic regulation of cardiac function

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,

KCNE1 enhances phosphatidylinositol 4,5-bisphosphate (PIP 2 ) sensitivity of I Ks to modulate channel activity

Phosphatidylinositol 4,5-bisphosphate (PIP(2)) is necessary for the function of various ion channels. The potassium channel, I(Ks), is important for cardiac repolarization and requires PIP(2) to activate. Here we show that the auxiliary subunit of I(Ks), KCNE1, increases PIP(2) sensitivity 100-fold over channels formed by the pore-forming KCNQ1 subunits alone, which effectively amplifies current because native PIP(2) levels in the membrane are insufficient to activate all KCNQ1 channels. A juxtamembranous site in the KCNE1 C terminus is a key structural determinant of PIP(2) sensitivity. Long QT syndrome associated mutations of this site lower PIP(2) affinity, resulting in reduced current. Application of exogenous PIP(2) to these mutants restores wild-type channel activity. These results reveal a vital role of PIP(2) for KCNE1 modulation of I(Ks) channels that may represent a common mechanism of auxiliary subunit modulation of many ion channels.

AKAP79/150 Signal Complexes in G-Protein Modulation of Neuronal Ion Channels

Voltage-gated M-type (KCNQ) K+ channels play a critical role in modulation of neuronal excitability and action potential firing. A-kinase-anchoring protein (AKAP)79/150 mediated PKC phosphorylation of M channels is involved in M current (IM) suppression by muscarinic M1, but not bradykinin B2 receptors. In this study, we first explored if purinergic and angiotensin suppression of IM in superior cervical ganglion (SCG) sympathetic neurons involves AKAP79/150. Transfection into rat SCG neurons of ΔA-AKAP79, which lacks the A-domain necessary for PKC binding, or the absence of AKAP150 in AKAP150-/- mice, did not affect IM suppression by the purinergic agonist UTP, nor by bradykinin, but did reduce IM suppression by muscarinic agonists and by angiotensin II. Transfection of AKAP79, but not ΔA-AKAP79 or AKAP15, “rescued” the muscarinic suppression of IM in AKAP150-/- neurons. We also tested association of AKAP79 or KCNQ channels with M1, B2, P2Y6 and AT1 receptors via fluorescence resonance energy transfer (FRET) experiments on CHO cells under total internal refection fluorescence microscopy, which revealed substantial FRET between AKAP79 with M1 and AT1 receptors, but only weak FRET with P2Y6 or B2 receptors. Similarly, we observed strong FRET between KCNQ2 with M1 and AT1, but not P2Y6 or B2, receptors. The involvement of AKAP79/150 in the regulation of N- and L-type Ca2+ channels in SCG neurons by Gq/11-coupled muscarinic receptors and by cAMP/PKA was also studied. We found AKAP79/150 to not play a role in the former, but to be necessary for forskolin-induced up-regulation of the L-type current. Our data suggest that AKAP79/150 orchestrates signal complexes that include PKC, KCNQ subunits and M1 or AT1 receptors, which correlates with the PIP2-depletion mode of neuronal M current suppression, but does not generalize to Gq/11-mediated inhibition of N- or L-type Ca2+ channels.

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.

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.

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.

The C-terminal domain of βIV-spectrin is crucial for KCNQ2 aggregation and excitability at nodes of Ranvier

The spectrin cytoskeleton has an important function in the targeting of proteins to excitable membrane domains. In axons, βIV-spectrin stabilizes voltage-gated sodium (Nav) channel clusters at nodes of Ranvier and axon initial segments, two regions crucial for the generation and conduction of action potentials. Here, I investigated the physiology of the neuromuscular junction and peripheral nerves in quivering-3J mice, which show a frame-shift base insertion in the Spnb4 gene and lack the C-terminus of βIV-spectrin. The quivering-3J mice show prominent spontaneous and evoked hyperactivities at diaphragm neuromuscular junctions. These neuromyotonic and myokymic discharges were more prominent in adult animals when tremors and ataxia were pronounced. Recordings of sciatic and phrenic nerves showed that the hyperactivities originate in myelinated axons distally from nerve terminals. Axon and myelin structure in the PNS were unaffected in quivering-3J mice. Of interest, KCNQ2 subunit aggregates were undetectable at PNS and CNS nodes, whereas Nav and Kv1.1/Kv1.2 channels were properly concentrated at nodal and juxtaparanodal regions, respectively. The protein level of KCNQ2 subunits was normal in mutant animals, suggesting that KCNQ2 subunit absence stems from clustering or trafficking defects in axons. The quivering-3J nodes also presented high densities of ankyrin-G and CK2α, two cytosolic molecules involved with aggregating Nav and KCNQ2/3 channels in axons. Because βIV-spectrin does not interact with KCNQ2/3 subunits, it is suspected that βIV-spectrin regulates the distribution of KCNQ2/3 subunits in axonal subdomains via regulatory partners. Retigabine, an activator of KCNQ2/3 channels, attenuated the repetitive activities in quivering-3J mice, suggesting that depletion of KCNQ2 subunits at nodes initiates neuromyotonic/myokymic discharges. These findings demonstrate that spectrin cytoskeleton finely regulates ion channel distribution and implicates KCNQ2/3 subunits in axonal excitability and in myokymia aetiology.

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.

A Pore Residue of the KCNQ3 Potassium M-Channel Subunit Controls Surface Expression

KCNQ2 (Kv7.2) and KCNQ3 (Kv7.3) are the principal subunits underlying the potassium M-current, which exerts a strong control on neuronal excitability. KCNQ3 subunits coassemble with KCNQ2 to form functional heteromeric channels that are specifically transported to the axonal initial segment and nodes of Ranvier. In contrast, there is no evidence for functional homomeric KCNQ3 channels in neurons, and it appears that these are inefficiently trafficked to the plasma membrane. Among eukaryotic potassium channels, the KCNQ3 subunit is unusual because it has an alanine in place of a threonine at the pore inner vestibule, three residues upstream of the GYG signature sequence of the selectivity filter. This residue is critical for the potentiation of the current after heteromerization, but the mechanism is unknown. We report that the presence of this uncommon residue at position 315 has a strong impact on the stability of the homotetramers and on channel trafficking. Wild-type KCNQ3 expressed alone is retained within the endoplasmic reticulum, and this mechanism is overcome by the substitution of threonine for Ala315. KCNQ3 subunits require assembly with KCNQ2 to exit this compartment, whereas KCNQ3-A315T is no longer dependent on KCNQ2 to form channels that are efficiently trafficked to the plasma membrane. The presence of this alanine, therefore, plays an important role in regulating the subunit composition of functional M-channels expressed at the surface of neurons.

Identification of a protein–protein interaction between KCNE1 and the activation gate machinery of KCNQ1

KCNQ1 channels assemble with KCNE1 transmembrane (TM) peptides to form voltage-gated K+ channel complexes with slow activation gate opening. The cytoplasmic C-terminal domain that abuts the KCNE1 TM segment has been implicated in regulating KCNQ1 gating, yet its interaction with KCNQ1 has not been described. Here, we identified a protein–protein interaction between the KCNE1 C-terminal domain and the KCNQ1 S6 activation gate and S4–S5 linker. Using cysteine cross-linking, we biochemically screened over 300 cysteine pairs in the KCNQ1–KCNE1 complex and identified three residues in KCNQ1 (H363C, P369C, and I257C) that formed disulfide bonds with cysteine residues in the KCNE1 C-terminal domain. Statistical analysis of cross-link efficiency showed that H363C preferentially reacted with KCNE1 residues H73C, S74C, and D76C, whereas P369C showed preference for only D76C. Electrophysiological investigation of the mutant K+ channel complexes revealed that the KCNQ1 residue, H363C, formed cross-links not only with KCNE1 subunits, but also with neighboring KCNQ1 subunits in the complex. Cross-link formation involving the H363C residue was state dependent, primarily occurring when the KCNQ1–KCNE1 complex was closed. Based on these biochemical and electrophysiological data, we generated a closed-state model of the KCNQ1–KCNE1 cytoplasmic region where these protein–protein interactions are poised to slow activation gate opening.