KCNE1 is an auxiliary subunit of two distinct ion channel superfamilies.

Determination of what is the specificity of subunits composing a protein complex is essential when studying gene variants on human pathophysiology. The pore-forming α-subunit KCNQ1, which belongs to the voltage-gated ion channel superfamily, associates to its ß-auxiliary subunit KCNE1 to generate the slow cardiac potassium IKs current, whose dysfunction leads to cardiac arrhythmia. Using pharmacology, gene invalidation, and single-molecule fluorescence assays, we found that KCNE1 fulfils all criteria of a bona fide auxiliary subunit of the TMEM16A chloride channel, which belongs to the anoctamin superfamily. Strikingly, assembly with KCNE1 switches TMEM16A from a calcium-dependent to a voltage-dependent ion channel. Importantly, clinically relevant inherited mutations within the TMEM16A-regulating domain of KCNE1 abolish the TMEM16A modulation, suggesting that the TMEM16A-KCNE1 current may contribute to inherited pathologies. Altogether, these findings challenge the dogma of the specificity of auxiliary subunits regarding protein complexes and questions ion channel classification.

Allosteric inhibitors targeting the calmodulin-PIP2 interface of SK4 K+ channels for atrial fibrillation treatment.

The Ca2+-activated SK4 K+ channel is gated by Ca2+-calmodulin (CaM) and is expressed in immune cells, brain, and heart. A cryoelectron microscopy (cryo-EM) structure of the human SK4 K+ channel recently revealed four CaM molecules per channel tetramer, where the apo CaM C-lobe and the holo CaM N-lobe interact with the proximal carboxyl terminus and the linker S4-S5, respectively, to gate the channel. Here, we show that phosphatidylinositol 4-5 bisphosphate (PIP2) potently activates SK4 channels by docking to the boundary of the CaM-binding domain. An allosteric blocker, BA6b9, was designed to act to the CaM-PIP2-binding domain, a previously untargeted region of SK4 channels, at the interface of the proximal carboxyl terminus and the linker S4-S5. Site-directed mutagenesis, molecular docking, and patch-clamp electrophysiology indicate that BA6b9 inhibits SK4 channels by interacting with two specific residues, Arg191 and His192 in the linker S4-S5, not conserved in SK1-SK3 subunits, thereby conferring selectivity and preventing the Ca2+-CaM N-lobe from properly interacting with the channel linker region. Immunohistochemistry of the SK4 channel protein in rat hearts showed a widespread expression in the sarcolemma of atrial myocytes, with a sarcomeric striated Z-band pattern, and a weaker occurrence in the ventricle but a marked incidence at the intercalated discs. BA6b9 significantly prolonged atrial and atrioventricular effective refractory periods in rat isolated hearts and reduced atrial fibrillation induction ex vivo. Our work suggests that inhibition of SK4 K+ channels by targeting drugs to the CaM-PIP2-binding domain provides a promising anti-arrhythmic therapy.

M-Current Inhibition in Hippocampal Excitatory Neurons Triggers Intrinsic and Synaptic Homeostatic Responses at Different Temporal Scales.

Persistent alterations in neuronal activity elicit homeostatic plastic changes in synaptic transmission and/or intrinsic excitability. However, it is unknown whether these homeostatic processes operate in concert or at different temporal scales to maintain network activity around a set-point value. Here we show that chronic neuronal hyperactivity, induced by M-channel inhibition, triggered intrinsic and synaptic homeostatic plasticity at different timescales in cultured hippocampal pyramidal neurons from mice of either sex. Homeostatic changes of intrinsic excitability occurred at a fast timescale (1-4 h) and depended on ongoing spiking activity. This fast intrinsic adaptation included plastic changes in the threshold current and a distal relocation of FGF14, a protein physically bridging Nav1.6 and Kv7.2 channels along the axon initial segment. In contrast, synaptic adaptations occurred at a slower timescale (∼2 d) and involved decreases in miniature EPSC amplitude. To examine how these temporally distinct homeostatic responses influenced hippocampal network activity, we quantified the rate of spontaneous spiking measured by multielectrode arrays at extended timescales. M-Channel blockade triggered slow homeostatic renormalization of the mean firing rate (MFR), concomitantly accompanied by a slow synaptic adaptation. Thus, the fast intrinsic adaptation of excitatory neurons is not sufficient to account for the homeostatic normalization of the MFR. In striking contrast, homeostatic adaptations of intrinsic excitability and spontaneous MFR failed in hippocampal GABAergic inhibitory neurons, which remained hyperexcitable following chronic M-channel blockage. Our results indicate that a single perturbation such as M-channel inhibition triggers multiple homeostatic mechanisms that operate at different timescales to maintain network mean firing rate.SIGNIFICANCE STATEMENT Persistent alterations in synaptic input elicit homeostatic plastic changes in neuronal activity. Here we show that chronic neuronal hyperexcitability, induced by M-type potassium channel inhibition, triggered intrinsic and synaptic homeostatic plasticity at different timescales in hippocampal excitatory neurons. The data indicate that the fast adaptation of intrinsic excitability depends on ongoing spiking activity but is not sufficient to provide homeostasis of the mean firing rate. Our results show that a single perturbation such as M-channel inhibition can trigger multiple homeostatic processes that operate at different timescales to maintain network mean firing rate.

M-current inhibition rapidly induces a unique CK2-dependent plasticity of the axon initial segment.

Alterations in synaptic input, persisting for hours to days, elicit homeostatic plastic changes in the axon initial segment (AIS), which is pivotal for spike generation. Here, in hippocampal pyramidal neurons of both primary cultures and slices, we triggered a unique form of AIS plasticity by selectively targeting M-type K+ channels, which predominantly localize to the AIS and are essential for tuning neuronal excitability. While acute M-current inhibition via cholinergic activation or direct channel block made neurons more excitable, minutes to hours of sustained M-current depression resulted in a gradual reduction in intrinsic excitability. Dual soma-axon patch-clamp recordings combined with axonal Na+ imaging and immunocytochemistry revealed that these compensatory alterations were associated with a distal shift of the spike trigger zone and distal relocation of FGF14, Na+, and Kv7 channels but not ankyrin G. The concomitant distal redistribution of FGF14 together with Nav and Kv7 segments along the AIS suggests that these channels relocate as a structural and functional unit. These fast homeostatic changes were independent of l-type Ca2+ channel activity but were contingent on the crucial AIS protein, protein kinase CK2. Using compartmental simulations, we examined the effects of varying the AIS position relative to the soma and found that AIS distal relocation of both Nav and Kv7 channels elicited a decrease in neuronal excitability. Thus, alterations in M-channel activity rapidly trigger unique AIS plasticity to stabilize network excitability.

Competition of calcified calmodulin N lobe and PIP2 to an LQT mutation site in Kv7.1 channel.

Voltage-gated potassium 7.1 (Kv7.1) channel and KCNE1 protein coassembly forms the slow potassium current IKS that repolarizes the cardiac action potential. The physiological importance of the IKS channel is underscored by the existence of mutations in human Kv7.1 and KCNE1 genes, which cause cardiac arrhythmias, such as the long-QT syndrome (LQT) and atrial fibrillation. The proximal Kv7.1 C terminus (CT) binds calmodulin (CaM) and phosphatidylinositol-4,5-bisphosphate (PIP2), but the role of CaM in channel function is still unclear, and its possible interaction with PIP2 is unknown. Our recent crystallographic study showed that CaM embraces helices A and B with the apo C lobe and calcified N lobe, respectively. Here, we reveal the competition of PIP2 and the calcified CaM N lobe to a previously unidentified site in Kv7.1 helix B, also known to harbor an LQT mutation. Protein pulldown, molecular docking, molecular dynamics simulations, and patch-clamp recordings indicate that residues K526 and K527 in Kv7.1 helix B form a critical site where CaM competes with PIP2 to stabilize the channel open state. Data indicate that both PIP2 and Ca2+-CaM perform the same function on IKS channel gating by producing a left shift in the voltage dependence of activation. The LQT mutant K526E revealed a severely impaired channel function with a right shift in the voltage dependence of activation, a reduced current density, and insensitivity to gating modulation by Ca2+-CaM. The results suggest that, after receptor-mediated PIP2 depletion and increased cytosolic Ca2+, calcified CaM N lobe interacts with helix B in place of PIP2 to limit excessive IKS current inhibition.

Structural Insights into the M-Channel Proximal C-Terminus/Calmodulin Complex.

The Kv7 (KCNQ) channel family, comprising voltage-gated potassium channels, plays major roles in fine-tuning cellular excitability by reducing firing frequency and controlling repolarization. Kv7 channels have a unique intracellular C-terminal (CT) domain bound constitutively by calmodulin (CaM). This domain plays key functions in channel tetramerization, trafficking, and gating. CaM binds to the proximal CT, comprising helices A and B. Kv7.2 and Kv7.3 are expressed in neural tissues. Together, they form the heterotetrameric M channel. We characterized Kv7.2, Kv7.3, and chimeric Kv7.3 helix A-Kv7.2 helix B (Q3A-Q2B) proximal CT/CaM complexes by solution methods at various Ca(2+)concentrations and determined them all to have a 1:1 stoichiometry. We then determined the crystal structure of the Q3A-Q2B/CaM complex at high Ca(2+) concentration to 2.0 Å resolution. CaM hugs the antiparallel coiled coil of helices A and B, braced together by an additional helix. The structure displays a hybrid apo-Ca(2+) CaM conformation even though four Ca(2+) ions are bound. Our results pinpoint unique interactions enabling the possible intersubunit pairing of Kv7.3 helix A and Kv7.2 helix B while underlining the potential importance of Kv7.3 helix A's role in stabilizing channel oligomerization. Also, the structure can be used to rationalize various channelopathic mutants. Functional testing of the chimeric channel found it to have a voltage-dependence similar to the M channel, thereby demonstrating helix A's importance in imparting gating properties.

Long QT mutations at the interface between KCNQ1 helix C and KCNE1 disrupt I(KS) regulation by PKA and PIP2.

KCNQ1 and KCNE1 co-assembly generates the I(KS) K(+) current, which is crucial to the cardiac action potential repolarization. Mutations in their corresponding genes cause long QT syndrome (LQT) and atrial fibrillation. The A-kinase anchor protein, yotiao (also known as AKAP9), brings the I(KS) channel complex together with signaling proteins to achieve regulation upon ß1-adrenergic stimulation. Recently, we have shown that KCNQ1 helix C interacts with the KCNE1 distal C-terminus. We postulated that this interface is crucial for I(KS) channel modulation. Here, we examined the yet unknown molecular mechanisms of LQT mutations located at this intracellular intersubunit interface. All LQT mutations disrupted the internal KCNQ1-KCNE1 intersubunit interaction. LQT mutants in KCNQ1 helix C led to a decreased current density and a depolarizing shift of channel activation, mainly arising from impaired phosphatidylinositol-4,5-bisphosphate (PIP2) modulation. In the KCNE1 distal C-terminus, the LQT mutation P127T suppressed yotiao-dependent cAMP-mediated upregulation of the I(KS) current, which was caused by reduced KCNQ1 phosphorylation at S27. Thus, KCNQ1 helix C is important for channel modulation by PIP2, whereas the KCNE1 distal C-terminus appears essential for the regulation of IKS by yotiao-mediated PKA phosphorylation.

Maturation- and sex-sensitive depression of hippocampal excitatory transmission in a rat schizophrenia model.

Schizophrenia is associated with behavioral and brain structural abnormalities, of which the hippocampus appears to be one of the most consistent region affected. Previous studies performed on the poly I:C model of schizophrenia suggest that alterations in hippocampal synaptic transmission and plasticity take place in the offspring. However, these investigations yielded conflicting results and the neurophysiological alterations responsible for these deficits are still unclear. Here we performed for the first time a longitudinal study examining the impact of prenatal poly I:C treatment and of gender on hippocampal excitatory neurotransmission. In addition, we examined the potential preventive/curative effects of risperidone (RIS) treatment during the peri-adolescence period. Excitatory synaptic transmission was determined by stimulating Schaffer collaterals and monitoring fiber volley amplitude and slope of field-EPSP (fEPSP) in CA1 pyramidal neurons in male and female offspring hippocampal slices from postnatal days (PNDs) 18-20, 34, 70 and 90. Depression of hippocampal excitatory transmission appeared at juvenile age in male offspring of the poly I:C group, while it expressed with a delay in female, manifesting at adulthood. In addition, a reduced hippocampal size was found in both adult male and female offspring of poly I:C treated dams. Treatment with RIS at the peri-adolescence period fully restored in males but partly repaired in females these deficiencies. A maturation- and sex-dependent decrease in hippocampal excitatory transmission occurs in the offspring of poly I:C treated pregnant mothers. Pharmacological intervention with RIS during peri-adolescence can cure in a gender-sensitive fashion early occurring hippocampal synaptic deficits.

Mechanisms underlying the cardiac pacemaker: the role of SK4 calcium-activated potassium channels.

The proper expression and function of the cardiac pacemaker is a critical feature of heart physiology. The sinoatrial node (SAN) in human right atrium generates an electrical stimulation approximately 70 times per minute, which propagates from a conductive network to the myocardium leading to chamber contractions during the systoles. Although the SAN and other nodal conductive structures were identified more than a century ago, the mechanisms involved in the generation of cardiac automaticity remain highly debated. In this short review, we survey the current data related to the development of the human cardiac conduction system and the various mechanisms that have been proposed to underlie the pacemaker activity. We also present the human embryonic stem cell-derived cardiomyocyte system, which is used as a model for studying the pacemaker. Finally, we describe our latest characterization of the previously unrecognized role of the SK4 Ca(2+)-activated K(+) channel conductance in pacemaker cells. By exquisitely balancing the inward currents during the diastolic depolarization, the SK4 channels appear to play a crucial role in human cardiac automaticity.

SK4 Ca2+ activated K+ channel is a critical player in cardiac pacemaker derived from human embryonic stem cells.

Proper expression and function of the cardiac pacemaker is a critical feature of heart physiology. Two main mechanisms have been proposed: (i) the "voltage-clock," where the hyperpolarization-activated funny current If causes diastolic depolarization that triggers action potential cycling; and (ii) the "Ca(2+) clock," where cyclical release of Ca(2+) from Ca(2+) stores depolarizes the membrane during diastole via activation of the Na(+)-Ca(2+) exchanger. Nonetheless, these mechanisms remain controversial. Here, we used human embryonic stem cell-derived cardiomyocytes (hESC-CMs) to study their autonomous beating mechanisms. Combined current- and voltage-clamp recordings from the same cell showed the so-called "voltage and Ca(2+) clock" pacemaker mechanisms to operate in a mutually exclusive fashion in different cell populations, but also to coexist in other cells. Blocking the "voltage or Ca(2+) clock" produced a similar depolarization of the maximal diastolic potential (MDP) that culminated by cessation of action potentials, suggesting that they converge to a common pacemaker component. Using patch-clamp recording, real-time PCR, Western blotting, and immunocytochemistry, we identified a previously unrecognized Ca(2+)-activated intermediate K(+) conductance (IK(Ca), KCa3.1, or SK4) in young and old stage-derived hESC-CMs. IK(Ca) inhibition produced MDP depolarization and pacemaker suppression. By shaping the MDP driving force and exquisitely balancing inward currents during diastolic depolarization, IK(Ca) appears to play a crucial role in human embryonic cardiac automaticity.

Promiscuous gating modifiers target the voltage sensor of K(v)7.2, TRPV1, and H(v)1 cation channels.

Some of the fascinating features of voltage-sensing domains (VSDs) in voltage-gated cation channels (VGCCs) are their modular nature and adaptability. Here we examined the VSD sensitivity of different VGCCs to 2 structurally related nontoxin gating modifiers, NH17 and NH29, which stabilize K(v)7.2 potassium channels in the closed and open states, respectively. The effects of NH17 and NH29 were examined in Chinese hamster ovary cells transfected with transient receptor potential vanilloid 1 (TRPV1) or K(v)7.2 channels, as well as in dorsal root ganglia neurons, using the whole-cell patch-clamp technique. NH17 and NH29 exert opposite effects on TRPV1 channels, operating, respectively, as an activator and a blocker of TRPV1 currents (EC50 and IC50 values ranging from 4 to 40 µM). Combined mutagenesis, electrophysiology, structural homology modeling, molecular docking, and molecular dynamics simulation indicate that both compounds target the VSDs of TRPV1 channels, which, like vanilloids, are involved in π-π stacking, H-bonding, and hydrophobic interactions. Reflecting their promiscuity, the drugs also affect the lone VSD proton channel mVSOP. Thus, the same gating modifier can promiscuously interact with different VGCCs, and subtle differences at the VSD-ligand interface will dictate whether the gating modifier stabilizes channels in either the closed or the open state.

Opposite effects of acute and chronic IGF1 on rat dorsal root ganglion neuron excitability.

Insulin-like growth factor-1 (IGF-1) is a polypeptide hormone with a ubiquitous distribution in numerous tissues and with various functions in both neuronal and non-neuronal cells. IGF-1 provides trophic support for many neurons of both the central and peripheral nervous systems. In the central nervous system (CNS), IGF-1R signaling regulates brain development, increases neuronal firing and modulates synaptic transmission. IGF-1 and IGF-IR are not only expressed in CNS neurons but also in sensory dorsal root ganglion (DRG) nociceptive neurons that convey pain signals. DRG nociceptive neurons express a variety of receptors and ion channels that are essential players of neuronal excitability, notably the ligand-gated cation channel TRPV1 and the voltage-gated M-type K+ channel, which, respectively, triggers and dampens sensory neuron excitability. Although many lines of evidence suggest that IGF-IR signaling contributes to pain sensitivity, its possible modulation of TRPV1 and M-type K+ channel remains largely unexplored. In this study, we examined the impact of IGF-1R signaling on DRG neuron excitability and its modulation of TRPV1 and M-type K+ channel activities in cultured rat DRG neurons. Acute application of IGF-1 to DRG neurons triggered hyper-excitability by inducing spontaneous firing or by increasing the frequency of spikes evoked by depolarizing current injection. These effects were prevented by the IGF-1R antagonist NVP-AEW541 and by the PI3Kinase blocker wortmannin. Surprisingly, acute exposure to IGF-1 profoundly inhibited both the TRPV1 current and the spike burst evoked by capsaicin. The Src kinase inhibitor PP2 potently depressed the capsaicin-evoked spike burst but did not alter the IGF-1 inhibition of the hyperexcitability triggered by capsaicin. Chronic IGF-1 treatment (24 h) reduced the spike firing evoked by depolarizing current injection and upregulated the M-current density. In contrast, chronic IGF-1 markedly increased the spike burst evoked by capsaicin. In all, our data suggest that IGF-1 exerts complex effects on DRG neuron excitability as revealed by its dual and opposite actions upon acute and chronic exposures.

The SK4 channel allosteric blocker, BA6b9, reduces atrial fibrillation substrate in rats with reduced ejection fraction.

Atrial fibrillation (AF), the most common cardiac arrhythmia, is strongly associated with several comorbidities including heart failure (HF). AF in general, and specifically in the context of HF, is progressive in nature and associated with poor clinical outcomes. Current therapies for AF are limited in number and efficacy and do not target the underlying causes of atrial remodeling such as inflammation or fibrosis. We previously identified the calcium-activated SK4 K+ channels, which are preferentially expressed in the atria relative to the ventricles in both rat and human hearts, as attractive druggable target for AF treatment. Here, we examined the ability of BA6b9, a novel allosteric inhibitor of SK4 channels that targets the specific calmodulin-PIP2 binding domain, to alter AF susceptibility and atrial remodeling in a systolic HF rat postmyocardial infarction (post-MI) model. Daily BA6b9 injection (20 mg/kg/day) for 3 weeks starting 1-week post-MI prolonged the atrial effective refractory period, reduced AF induction and duration, and dramatically prevented atrial structural remodeling. In the post-MI left atrium (LA), pronounced upregulation of the SK4 K+ channel was observed, with corresponding increases in collagen deposition, α-SMA levels, and NLRP3 inflammasome expression. Strikingly, BA6b9 treatment reversed these changes while also significantly reducing the lateralization of the atrial connexin Cx43 in the LA of post-MI rats. Our findings indicate that the blockade of SK4 K+ channels using BA6b9 not only favors rhythm control but also remarkably reduces atrial structural remodeling, a property that is highly desirable for novel AF therapies, particularly in patients with comorbid HF.

Donepezil as a new therapeutic potential in KCNQ2- and KCNQ3-related autism.

Introduction: The KCNQ2/KCNQ3 genes encode the voltage-gated K channel underlying the neuronal M-current, regulating neuronal excitability. Loss-of-function (LoF) variants cause neonatal epilepsy, treatable with the M-current-opener retigabine, which is no longer marketed due to side effects. Gain-of-function (GoF) variants cause developmental encephalopathy and autism that could be amenable to M-current, but such therapies are not clinically available. In this translational project, we investigated whether donepezil, a cholinergic drug used in Alzheimer's, suppresses M currents in vitro and improves cognitive symptoms in patients with GoF variants. Methods: (1) The effect of 1 µM donepezil on the amplitude of the M-current was measured in excitatory and inhibitory neurons of mouse primary cultured hippocampal cells. M-current was measured using the standard deactivation protocol (holding at 0 mV and deactivation at -60 mV) in the voltage-clamp configuration of the whole-cell patch clamp technique. The impact of donepezil was also examined on the spontaneous firing activity of hippocampal neurons in the current-clamp configuration. (2) Four children with autism, aged 2.5-8 years, with the following GoF variants were enrolled: KCNQ2 (p. Arg144Gln) and KCNQ 3 (p.Arg227Gln, p.Arg230Cys). Patients were treated off-label with donepezil 2.5-5 mg/d for 12 months and assessed with: clinical Global Impression of Change (CGI-c), Childhood Autism Rating Scale 2 (CARS-2), Adaptive Behavior Assessment System-II (ABAS-II), and Child Development Inventory (CDI). Results: (1) Application of donepezil for at least 6 min produced a significant inhibition of the M-current with an IC50 of 0.4 µM. At 1 µM, donepezil reduced by 67% the M-current density of excitatory neurons (2.4 ± 0.46 vs. 0.89 ± 0.15 pA/pF, p < 0.05*). In inhibitory neurons, application of 1 µM donepezil produced a lesser inhibition of 59% of the M-current density (1.39 ± 0.43 vs. 0.57 ± 0.21, p > 0.05). Donepezil (1 µM) potently increased by 2.6-fold the spontaneous firing frequency, which was prevented by the muscarinic receptor antagonist atropine (10 µM). (2) The CARS-2 decreased by 3.8 ± 4.9 points (p > 0.05), but in two patients with KCNQ3 variants, the improvement was over the 4.5 clinically relevant threshold. The global clinical change was also clinically significant in these patients (CGI-c = 1). The CDI increased by 65% (p < 0.05*), while the ABAS-II remained unchanged. Discussion: Donepezil should be repurposed as a novel alternative treatment for GoF variants in KCNQ2/KCNQ3 encephalopathy.

Dual Kv7.2/3-TRPV1 modulators inhibit nociceptor hyperexcitability and alleviate pain without target-related side effects.

ABSTRACT: Persistent or chronic pain is the primary reason people seek medical care, yet current therapies are either limited in efficacy or cause intolerable side effects. Diverse mechanisms contribute to the basic phenomena of nociceptor hyperexcitability that initiates and maintains pain. Two prominent players in the modulation of nociceptor hyperexcitability are the transient receptor potential vanilloid type 1 (TRPV1) ligand-gated ion channel and the voltage-gated potassium channel, Kv7.2/3, that reciprocally regulate neuronal excitability. Across many drug development programs targeting either TRPV1 or Kv7.2/3, significant evidence has been accumulated to support these as highly relevant targets; however, side effects that are poorly separated from efficacy have limited the successful clinical translation of numerous Kv7.2/3 and TRPV1 drug development programs. We report here the pharmacological profile of 3 structurally related small molecule analogues that demonstrate a novel mechanism of action (MOA) of dual modulation of Kv7.2/3 and TRPV1. Specifically, these compounds simultaneously activate Kv7.2/3 and enable unexpected specific and potent inhibition of TRPV1. This in vitro potency translated to significant analgesia in vivo in several animal models of acute and chronic pain. Importantly, this specific MOA is not associated with any previously described Kv7.2/3 or TRPV1 class-specific side effects. We suggest that the therapeutic potential of this MOA is derived from the selective and specific targeting of a subpopulation of nociceptors found in rodents and humans. This efficacy and safety profile supports the advancement of dual TRPV1-Kv7.2/3 modulating compounds into preclinical and clinical development for the treatment of chronic pain.

KCNQ1 channels do not undergo concerted but sequential gating transitions in both the absence and the presence of KCNE1 protein.

The co-assembly of KCNQ1 with KCNE1 produces I(KS), a K(+) current, crucial for the repolarization of the cardiac action potential. Mutations in these channel subunits lead to life-threatening cardiac arrhythmias. However, very little is known about the gating mechanisms underlying KCNQ1 channel activation. Shaker channels have provided a powerful tool to establish the basic gating mechanisms of voltage-dependent K(+) channels, implying prior independent movement of all four voltage sensor domains (VSDs) followed by channel opening via a last concerted cooperative transition. To determine the nature of KCNQ1 channel gating, we performed a thermodynamic mutant cycle analysis by constructing a concatenated tetrameric KCNQ1 channel and by introducing separately a gain and a loss of function mutation, R231W and R243W, respectively, into the S4 helix of the VSD of one, two, three, and four subunits. The R231W mutation destabilizes channel closure and produces constitutively open channels, whereas the R243W mutation disrupts channel opening solely in the presence of KCNE1 by right-shifting the voltage dependence of activation. The linearity of the relationship between the shift in the voltage dependence of activation and the number of mutated subunits points to an independence of VSD movements, with each subunit incrementally contributing to channel gating. Contrary to Shaker channels, our work indicates that KCNQ1 channels do not experience a late cooperative concerted opening transition. Our data suggest that KCNQ1 channels in both the absence and the presence of KCNE1 undergo sequential gating transitions leading to channel opening even before all VSDs have moved.

Multifaceted modulation of K+ channels by protein-tyrosine phosphatase ε tunes neuronal excitability.

Non-receptor-tyrosine kinases (protein-tyrosine kinases) and non-receptor tyrosine phosphatases (PTPs) have been implicated in the regulation of ion channels, neuronal excitability, and synaptic plasticity. We previously showed that protein-tyrosine kinases such as Src kinase and PTPs such as PTPα and PTPε modulate the activity of delayed-rectifier K(+) channels (I(K)). Here we show cultured cortical neurons from PTPε knock-out (EKO) mice to exhibit increased excitability when compared with wild type (WT) mice, with larger spike discharge frequency, enhanced fast after-hyperpolarization, increased after-depolarization, and reduced spike width. A decrease in I(K) and a rise in large-conductance Ca(2+)-activated K(+) currents (mBK) were observed in EKO cortical neurons compared with WT. Parallel studies in transfected CHO cells indicate that Kv1.1, Kv1.2, Kv7.2/7.3, and mBK are plausible molecular correlates of this multifaceted modulation of K(+) channels by PTPε. In CHO cells, Kv1.1, Kv1.2, and Kv7.2/7.3 K(+) currents were up-regulated by PTPε, whereas mBK channel activity was reduced. The levels of tyrosine phosphorylation of Kv1.1, Kv1.2, Kv7.3, and mBK potassium channels were increased in the brain cortices of neonatal and adult EKO mice compared with WT, suggesting that PTPε in the brain modulates these channel proteins. Our data indicate that in EKO mice, the lack of PTPε-mediated dephosphorylation of Kv1.1, Kv1.2, and Kv7.3 leads to decreased I(K) density and enhanced after-depolarization. In addition, the deficient PTPε-mediated dephosphorylation of mBK channels likely contributes to enhanced mBK and fast after-hyperpolarization, spike shortening, and consequent increase in neuronal excitability observed in cortical neurons from EKO mice.

Intracellular domains interactions and gated motions of I(KS) potassium channel subunits.

Voltage-gated K(+) channels co-assemble with auxiliary beta subunits to form macromolecular complexes. In heart, assembly of Kv7.1 pore-forming subunits with KCNE1 beta subunits generates the repolarizing K(+) current I(KS). However, the detailed nature of their interface remains unknown. Mutations in either Kv7.1 or KCNE1 produce the life-threatening long or short QT syndromes. Here, we studied the interactions and voltage-dependent motions of I(KS) channel intracellular domains, using fluorescence resonance energy transfer combined with voltage-clamp recording and in vitro binding of purified proteins. The results indicate that the KCNE1 distal C-terminus interacts with the coiled-coil helix C of the Kv7.1 tetramerization domain. This association is important for I(KS) channel assembly rules as underscored by Kv7.1 current inhibition produced by a dominant-negative C-terminal domain. On channel opening, the C-termini of Kv7.1 and KCNE1 come close together. Co-expression of Kv7.1 with the KCNE1 long QT mutant D76N abolished the K(+) currents and gated motions. Thus, during channel gating KCNE1 is not static. Instead, the C-termini of both subunits experience molecular motions, which are disrupted by the D76N causing disease mutation.

Targeting the voltage sensor of Kv7.2 voltage-gated K+ channels with a new gating-modifier.

The pore and gate regions of voltage-gated cation channels have been often targeted with drugs acting as channel modulators. In contrast, the voltage-sensing domain (VSD) was practically not exploited for therapeutic purposes, although it is the target of various toxins. We recently designed unique diphenylamine carboxylates that are powerful Kv7.2 voltage-gated K(+) channel openers or blockers. Here we show that a unique Kv7.2 channel opener, NH29, acts as a nontoxin gating modifier. NH29 increases Kv7.2 currents, thereby producing a hyperpolarizing shift of the activation curve and slowing both activation and deactivation kinetics. In neurons, the opener depresses evoked spike discharges. NH29 dampens hippocampal glutamate and GABA release, thereby inhibiting excitatory and inhibitory postsynaptic currents. Mutagenesis and modeling data suggest that in Kv7.2, NH29 docks to the external groove formed by the interface of helices S1, S2, and S4 in a way that stabilizes the interaction between two conserved charged residues in S2 and S4, known to interact electrostatically, in the open state of Kv channels. Results indicate that NH29 may operate via a voltage-sensor trapping mechanism similar to that suggested for scorpion and sea-anemone toxins. Reflecting the promiscuous nature of the VSD, NH29 is also a potent blocker of TRPV1 channels, a feature similar to that of tarantula toxins. Our data provide a structural framework for designing unique gating-modifiers targeted to the VSD of voltage-gated cation channels and used for the treatment of hyperexcitability disorders.

Contextual fear response is modulated by M-type K+ channels and is associated with subtle structural changes of the axon initial segment in hippocampal GABAergic neurons.

Background: In the fear memory network, the hippocampus modulates contextual aspects of fear learning while mutual connections between the amygdala and the medial prefrontal cortex are widely involved in fear extinction. G-protein-coupled receptors (GPCRs) are involved in the regulation of fear and anxiety, so the regulation of GPCRs in fear signaling pathways can modulate the mechanisms of fear memory acquisition, consolidation and extinction. Various studies suggested a role of M-type K+ channels in modulating fear expression and extinction, although conflicting data prevented drawing of clear conclusions. In the present work, we examined the impact of M-type K+ channel blockade or activation on contextual fear acquisition and extinction. In addition, regarding the pivotal role of the hippocampus in contextual fear conditioning (CFC) and the involvement of the axon initial segment (AIS) in neuronal plasticity, we investigated whether structural alterations of the AIS in hippocampal neurons occurred during contextual fear memory acquisition and short-time extinction in mice in a behaviorally relevant context. Results: When a single systemic injection of the M-channel blocker XE991 (2 mg/kg, IP) was carried out 15 minutes before the foot shock session, fear expression was significantly reduced. Expression of c-Fos was increased following CFC, mostly in GABAergic neurons at day 1 and day 2 post-fear training in CA1 and dentate gyrus hippocampal regions. A significantly longer AIS segment was observed in GABAergic neurons of the CA1 hippocampal region at day 2. Conclusions: Our results underscore the role of M-type K + channels in CFC and the importance of hippocampal GABAergic neurons in fear expression.