Excitatory and inhibitory hippocampal neurons differ in their homeostatic adaptation to chronic M-channel modulation.

A large body of studies has investigated bidirectional homeostatic plasticity both in vitro and in vivo using numerous pharmacological manipulations of activity or behavioral paradigms. However, these experiments rarely explored in the same cellular system the bidirectionality of the plasticity and simultaneously on excitatory and inhibitory neurons. M-channels are voltage-gated potassium channels that play a crucial role in regulating neuronal excitability and plasticity. In cultured hippocampal excitatory neurons, we previously showed that chronic exposure to the M-channel blocker XE991 leads to adaptative compensations, thereby triggering at different timescales intrinsic and synaptic homeostatic plasticity. This plastic adaptation barely occurs in hippocampal inhibitory neurons. In this study, we examined whether this homeostatic plasticity induced by M-channel inhibition was bidirectional by investigating the acute and chronic effects of the M-channel opener retigabine on hippocampal neuronal excitability. Acute retigabine exposure decreased excitability in both excitatory and inhibitory neurons. Chronic retigabine treatment triggered in excitatory neurons homeostatic adaptation of the threshold current and spontaneous firing rate at a time scale of 4-24 h. These plastic changes were accompanied by a substantial decrease in the M-current density and by a small, though significant, proximal relocation of Kv7.3-FGF14 segment along the axon initial segment. Thus, bidirectional homeostatic changes were observed in excitatory neurons though not symmetric in kinetics and mechanisms. Contrastingly, in inhibitory neurons, the compensatory changes in intrinsic excitability barely occurred after 48 h, while no homeostatic normalization of the spontaneous firing rate was observed. Our results indicate that excitatory and inhibitory hippocampal neurons differ in their adaptation to chronic alterations in neuronal excitability induced by M-channel bidirectional modulation.

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.

Personalized treatment with retigabine for pharmacoresistant epilepsy arising from a pathogenic variant in the KCNQ2 selectivity filter.

Mutations in the KCNQ2 gene, encoding the voltage-gated potassium channel, Kv7.2, cause neonatal epilepsies. The potassium channel opener, retigabine, may improve epilepsy control in cases with loss-of-function mutations, but exacerbate seizures in cases with gain-of-function mutations. Our aim was to describe a patient with a KCNQ2 mutation within the K+-selectivity filter and illustrate how electrophysiological analysis helped us to implement personalized treatment. Medical history of a patient with severe neonatal epileptic encephalopathy was recorded. Diagnosis was reached by whole-exome-sequencing. The pathogenic variant was expressed in Chinese hamster ovary cells, and patch-clamp studies were performed, directing therapy. A seven-year-old male presented with neonatal seizures, progressing to hundreds of seizures/day without developmental milestones. Whole-exome sequencing revealed a pathogenic variant, p.Gly281Arg, in the KCNQ2 gene, located within the ion selectivity filter of the pore, predicted to cause loss-of-function of Kv7.2, not affected by retigabine. Patch-clamp analysis revealed no current with the mutant homomer and reduced current with heterotetramer (KCNQ2WT/KCNQ2G281R/KCNQ3WT) channels, consistent with a dominant-negative effect. Addition of 5 µM retigabine did not produce a current with the mutant homomer, but increased current with the heterotetramer (V50: -30.4 mV vs. -51.3 mV). Following these results, retigabine at 15 mg/kg was administered off-label, prompting a 90% seizure reduction. Drug withdrawal, imposed by revocation of marketing authorisation for retigabine, caused 50% increase in seizure burden. Retigabine may be used for precision therapy in patients with KCNQ2-related epilepsy due to loss-of-function variants. It is imperative to reintroduce safe marketing of retigabine for selected patients as personalized treatment.

A unique mechanism of inactivation gating of the Kv channel family member Kv7.1 and its modulation by PIP2 and calmodulin.

Inactivation of voltage-gated K+ (Kv) channels mostly occurs by fast N-type or/and slow C-type mechanisms. Here, we characterized a unique mechanism of inactivation gating comprising two inactivation states in a member of the Kv channel superfamily, Kv7.1. Removal of external Ca2+ in wild-type Kv7.1 channels produced a large, voltage-dependent inactivation, which differed from N- or C-type mechanisms. Glu295 and Asp317 located, respectively, in the turret and pore entrance are involved in Ca2+ coordination, allowing Asp317 to form H-bonding with the pore helix Trp304, which stabilizes the selectivity filter and prevents inactivation. Phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+-calmodulin prevented Kv7.1 inactivation triggered by Ca2+-free external solutions, where Ser182 at the S2-S3 linker relays the calmodulin signal from its inner boundary to the external pore to allow proper channel conduction. Thus, we revealed a unique mechanism of inactivation gating in Kv7.1, exquisitely controlled by external Ca2+ and allosterically coupled by internal PIP2 and Ca2+-calmodulin.

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.

The Hyperpolarization-Activated Cyclic-Nucleotide-Gated Channel Blocker Ivabradine Does Not Prevent Arrhythmias in Catecholaminergic Polymorphic Ventricular Tachycardia.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited, stressed-provoked ventricular arrhythmia. CPVT is treated by ß-adrenergic receptor blockers, Na+ channel inhibitors, sympathetic denervation, or by implanting a defibrillator. We showed recently that blockers of SK4 Ca2+-activated K+ channels depolarize the maximal diastolic potential, reduce the heart rate, and attenuate ventricular arrhythmias in CPVT. The aim of the present study was to examine whether the pacemaker channel inhibitor, ivabradine could demonstrate anti-arrhythmic properties in CPVT like other bradycardic agents used in this disease and to compare them with those of the SK4 channel blocker, TRAM-34. The effects of ivabradine were examined on the arrhythmic beating of human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) from CPVT patients, on sinoatrial node (SAN) calcium transients, and on ECG measurements obtained from transgenic mice model of CPVT. Ivabradine did neither prevent the arrhythmic pacing of hiPSC-CMs derived from CPVT patients, nor preclude the aberrant SAN calcium transients. In contrast to TRAM-34, ivabradine was unable to reduce in vivo the ventricular premature complexes and ventricular tachyarrhythmias in transgenic CPVT mice. In conclusion, ivabradine does not exhibit anti-arrhythmic properties in CPVT, which indicates that this blocker cannot be used as a plausible treatment for CPVT ventricular arrhythmias.

Inactivation gating of Kv7.1 channels does not involve concerted cooperative subunit interactions.

Inactivation is an intrinsic property of numerous voltage-gated K+ (Kv) channels and can occur by N-type or/and C-type mechanisms. N-type inactivation is a fast, voltage independent process, coupled to activation, with each inactivation particle of a tetrameric channel acting independently. In N-type inactivation, a single inactivation particle is necessary and sufficient to occlude the pore. C-type inactivation is a slower process, involving the outermost region of the pore and is mediated by a concerted, highly cooperative interaction between all four subunits. Inactivation of Kv7.1 channels does not exhibit the hallmarks of N- and C-type inactivation. Inactivation of WT Kv7.1 channels can be revealed by hooked tail currents that reflects the recovery from a fast and voltage-independent inactivation process. However, several Kv7.1 mutants such as the pore mutant L273F generate an additional voltage-dependent slow inactivation. The subunit interactions during this slow inactivation gating remain unexplored. The goal of the present study was to study the nature of subunit interactions along Kv7.1 inactivation gating, using concatenated tetrameric Kv7.1 channel and introducing sequentially into each of the four subunits the slow inactivating pore mutation L273F. Incorporating an incremental number of inactivating mutant subunits did not affect the inactivation kinetics but slowed down the recovery kinetics from inactivation. Results indicate that Kv7.1 inactivation gating is not compatible with a concerted cooperative process. Instead, adding an inactivating subunit L273F into the Kv7.1 tetramer incrementally stabilizes the inactivated state, which suggests that like for activation gating, Kv7.1 slow inactivation gating is not a concerted process.

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.

Ca2+-Calmodulin and PIP2 interactions at the proximal C-terminus of Kv7 channels.

In the heart, co-assembly of Kv7.1 with KCNE1 produces the slow IKS potassium current, which repolarizes the cardiac action potential and mutations in human Kv7.1 and KCNE1 genes cause cardiac arrhythmias. The proximal Kv7.1 C-terminus binds calmodulin (CaM) and phosphatidylinositol-4,5-bisphosphate (PIP2) and recently we revealed the competition of PIP2 with the calcified CaM N-lobe to a previously unidentified site in Kv7.1 helix B, also known to harbor a LQT mutation. Data indicated that PIP2 and Ca2+-CaM perform the same function on IKS channel gating to stabilize the channel open state. Here we show that similar features were observed for Kv7.1 currents expressed alone. We also find that conservation of homologous residues in helix B of other Kv7 subtypes confer similar competition of Ca2+-CaM with PIP2 binding to their proximal C-termini and suggest that PIP2-CaM interactions converge to Kv7 helix B to modulates channel activity in a Kv7 subtype-dependent manner.

SK4 K+ channels are therapeutic targets for the treatment of cardiac arrhythmias.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a stress-provoked ventricular arrhythmia, which also manifests sinoatrial node (SAN) dysfunction. We recently showed that SK4 calcium-activated potassium channels are important for automaticity of cardiomyocytes derived from human embryonic stem cells. Here SK4 channels were identified in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from healthy and CPVT2 patients bearing a mutation in calsequestrin 2 (CASQ2-D307H) and in SAN cells from WT and CASQ2-D307H knock-in (KI) mice. TRAM-34, a selective blocker of SK4 channels, prominently reduced delayed afterdepolarizations and arrhythmic Ca2+ transients observed following application of the ß-adrenergic agonist isoproterenol in CPVT2-derived hiPSC-CMs and in SAN cells from KI mice. Strikingly, in vivo ECG recording showed that intraperitoneal injection of the SK4 channel blockers, TRAM-34 or clotrimazole, greatly reduced the arrhythmic features of CASQ2-D307H KI and CASQ2 knockout mice at rest and following exercise. This work demonstrates the critical role of SK4 Ca2+-activated K+ channels in adult pacemaker function, making them promising therapeutic targets for the treatment of cardiac ventricular arrhythmias such as CPVT.

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.

Zinc transport and the inhibition of the L-type calcium channel are two separable functions of ZnT-1.

Traditionally, proteins are considered to perform a single role, be it as an enzyme, a channel, a transporter or as a structural scaffold. However, recent studies have described moonlighting proteins that perform distinct and independent functions; for example, TRPM7 is both an ion channel and a kinase. ZnT-1 is a member of the Carrier Diffusion Facilitator family that is expressed throughout the phylogenetic tree from bacteria to humans. Since its cloning in 1995, ZnT-1 is considered a major extruder of Zn2+ based on its capability to protect cells against zinc toxicity. Recently, we reported that ZnT-1 inhibits the L-type calcium channel (LTCC), a major Zn2+ and Ca2+ entry pathway. Here we show that ZnT-1 is a dual-function protein by demonstrating that its abilities to exchange Zn2+/H+ and to inhibit the LTCC are independent of each other and are mediated by different parts of the protein. Specifically, mutations in the membrane-spanning helices that render ZnT-1 unable to transport zinc do not prevent it from inhibiting the LTCC. Moreover, a fragment consisting of the intracellular ZnT-1 C-terminal, which lacks all ion-transfer segments, inhibits the LTCC as efficiently as wild-type ZnT-1. Our data therefore indicates that ZnT-1 performs two structurally independent functions related to zinc homeostasis.

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.

Maternal immune activation produces neonatal excitability defects in offspring hippocampal neurons from pregnant rats treated with poly I:C.

Maternal immune activation (MIA) resulting from prenatal exposure to infectious pathogens or inflammatory stimuli is increasingly recognized to play an important etiological role in neuropsychiatric disorders with neurodevelopmental features. MIA in pregnant rodents induced by injection of the synthetic double-stranded RNA, Poly I:C, a mimic of viral infection, leads to a wide spectrum of behavioral abnormalities as well as structural and functional defects in the brain. Previous MIA studies using poly I:C prenatal treatment suggested that neurophysiological alterations occur in the hippocampus. However, these investigations used only juvenile or adult animals. We postulated that MIA-induced alterations could occur earlier at neonatal/early postnatal stages. Here we examined the neurophysiological properties of cultured pyramidal-like hippocampal neurons prepared from neonatal (P0-P2) offspring of pregnant rats injected with poly I:C. Offspring neurons from poly I:C-treated mothers exhibited significantly lower intrinsic excitability and stronger spike frequency adaptation, compared to saline. A similar lower intrinsic excitability was observed in CA1 pyramidal neurons from hippocampal slices of two weeks-old poly I:C offspring. Cultured hippocampal neurons also displayed lower frequency of spontaneous firing, higher charge transfer of IPSCs and larger amplitude of miniature IPSCs. Thus, maternal immune activation leads to strikingly early neurophysiological abnormalities in hippocampal neurons.

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.

Structural basis of a Kv7.1 potassium channel gating module: studies of the intracellular c-terminal domain in complex with calmodulin.

Kv7 channels tune neuronal and cardiomyocyte excitability. In addition to the channel membrane domain, they also have a unique intracellular C-terminal (CT) domain, bound constitutively to calmodulin (CaM). This CT domain regulates gating and tetramerization. We investigated the structure of the membrane proximal CT module in complex with CaM by X-ray crystallography. The results show how the CaM intimately hugs a two-helical bundle, explaining many channelopathic mutations. Structure-based mutagenesis of this module in the context of concatemeric tetramer channels and functional analysis along with in vitro data lead us to propose that one CaM binds to one individual protomer, without crosslinking subunits and that this configuration is required for proper channel expression and function. Molecular modeling of the CT/CaM complex in conjunction with small-angle X-ray scattering suggests that the membrane proximal region, having a rigid lever arm, is a critical gating regulator.

Capturing distinct KCNQ2 channel resting states by metal ion bridges in the voltage-sensor domain.

Although crystal structures of various voltage-gated K(+) (Kv) and Na(+) channels have provided substantial information on the activated conformation of the voltage-sensing domain (VSD), the topology of the VSD in its resting conformation remains highly debated. Numerous studies have investigated the VSD resting state in the Kv Shaker channel; however, few studies have explored this issue in other Kv channels. Here, we investigated the VSD resting state of KCNQ2, a K(+) channel subunit belonging to the KCNQ (Kv7) subfamily of Kv channels. KCNQ2 can coassemble with the KCNQ3 subunit to mediate the IM current that regulates neuronal excitability. In humans, mutations in KCNQ2 are associated with benign neonatal forms of epilepsy or with severe epileptic encephalopathy. We introduced cysteine mutations into the S4 transmembrane segment of the KCNQ2 VSD and determined that external application of Cd(2+) profoundly reduced the current amplitude of S4 cysteine mutants S195C, R198C, and R201C. Based on reactivity with the externally accessible endogenous cysteine C106 in S1, we infer that each of the above S4 cysteine mutants forms Cd(2+) bridges to stabilize a channel closed state. Disulfide bonds and metal bridges constrain the S4 residues S195, R198, and R201 near C106 in S1 in the resting state, and experiments using concatenated tetrameric constructs indicate that this occurs within the same VSD. KCNQ2 structural models suggest that three distinct resting channel states have been captured by the formation of different S4-S1 Cd(2+) bridges. Collectively, this work reveals that residue C106 in S1 can be very close to several N-terminal S4 residues for stabilizing different KCNQ2 resting conformations.

Recent molecular insights from mutated IKS channels in cardiac arrhythmia.

Co-assembly of KCNQ1 with KCNE1 generates the IKS potassium current that is vital for the proper repolarization of the cardiac action potential. Mutations in either KCNQ1 or KCNE1 genes lead to life-threatening cardiac arrhythmias causing long QT syndrome, short QT syndrome, sinus bradycardia and atrial fibrillation. Findings emerging from recent studies are beginning to provide a picture of how gain-of-function and loss-of-function mutations are associated with pleiotropic cardiac phenotypes in the clinics. In this review, we discuss recent molecular insights obtained from mutations altering different structural modules of the channel complex that are essential for proper IKS function. We present the possible molecular mechanisms underlying mutations impairing the voltage sensing functions, as well as those altering the channel regulation by phosphatidylinositol-4,5-bisphosphate, calmodulin and protein kinase A. We also discuss the significance of diseased IKS channels for adequate pharmacological targeting of cardiac arrhythmias.

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.

Channel gating pore: a new therapeutic target.

Each subunit of voltage-gated cation channels comprises a voltage-sensing domain and a pore region. In a paper recently published in Cell Research, Li et al. showed that the gating charge pathway of the voltage sensor of the KCNQ2 K+ channel can accommodate small opener molecules and offer a new target to treat hyperexcitability disorders.