Voltage-gated ion channels open and close in response to changes in membrane potential, thereby enabling electrical signaling in excitable cells. The voltage sensitivity is conferred through four voltage-sensor domains (VSDs) where positively charged residues in the fourth transmembrane segment (S4) sense the potential. While an open state is known from the Kv1.2/2.1 X-ray structure, the conformational changes underlying voltage sensing have not been resolved. We present 20 additional interactions in one open and four different closed conformations based on metal-ion bridges between all four segments of the VSD in the voltage-gated Shaker K channel. A subset of the experimental constraints was used to generate Rosetta models of the conformations that were subjected to molecular simulation and tested against the remaining constraints. This achieves a detailed model of intermediate conformations during VSD gating. The results provide molecular insight into the transition, suggesting that S4 slides at least 12 Å along its axis to open the channel with a 3 10 helix region present that moves in sequence in S4 in order to occupy the same position in space opposite F290 from open through the three first closed states. V oltage-gated ion channels are critical for biological signaling, and they are able to regulate ion flux on a millisecond time scale. To sense changes in membrane voltage, each ion channel is equipped with four voltage-sensor domains (VSDs) connected to a central ion-conducting pore domain. The fourth transmembrane segment (S4) of each VSD carries several positively charged amino-acid residues responsible for VSD gating (1). At least three elementary charges per VSD must traverse outwards through the membrane electric field to open a channel that corresponds to a considerable displacement of the S4 helix ( Fig. 1A) (2-4). The positive charges in S4 make salt bridges with negative countercharges on their move through the VSD (4-8). It has even been proposed that the VSD undergoes a conformational alteration following the opening, when the channel relaxes to an inactivated, that is closed, state (9). In addition to conferring voltage dependence to ion channels, VSDs also regulate enzymes (10), act as voltage-gated proton channels (11,12), are susceptible to disease-causing mutations (13,14), and serve as targets for drugs and toxins (1,(15)(16)(17)(18). Therefore, it is of crucial interest to understand the details underlying voltage sensing by VSDs.Few, if any, segments of membrane proteins have received more attention than the S4 helix of voltage sensors. In addition to their paramount biological importance, they can help us understand fundamental biophysical problems such as why some membrane protein segments can be hydrophilic (19), how charges effectively move through a membrane, or how a potential triggers structural changes on a microsecond time scale. These questions are inherently linked to transient conformations and contacts that can be difficult to capture in a single structure. Although active...

The scaling relationship between the size of an appendage or organ and that of the body as a whole is tightly regulated during animal development. If a structure grows at a different rate than the rest of the body, this process is termed allometric growth. The zebrafish another longfin (alf) mutant shows allometric growth resulting in proportionally enlarged fins and barbels. We took advantage of this mutant to study the regulation of size in vertebrates. Here, we show that alf mutants carry gain-of-function mutations in kcnk5b, a gene encoding a two-pore domain potassium (K+) channel. Electrophysiological analysis in Xenopus oocytes reveals that these mutations cause an increase in K+ conductance of the channel and lead to hyperpolarization of the cell. Further, somatic transgenesis experiments indicate that kcnk5b acts locally within the mesenchyme of fins and barbels to specify appendage size. Finally, we show that the channel requires the ability to conduct K+ ions to increase the size of these structures. Our results provide evidence for a role of bioelectric signaling through K+ channels in the regulation of allometric scaling and coordination of growth in the zebrafish.

Abstract-Physical and emotional stress is accompanied by release of stress hormones such as the glucocorticoid cortisol. This hormone upregulates the serum-and glucocorticoid-inducible kinase (SGK)1, which in turn stimulates I Ks , a slow delayed rectifier potassium current that mediates cardiac action potential repolarization. Mutations in I Ks channel ␣ (KCNQ1, KvLQT1, Kv7.1) or ␤ (KCNE1, IsK, minK) subunits cause long QT syndrome (LQTS), an inherited cardiac arrhythmia associated with increased risk of sudden death. Together with the GTPases RAB5 and RAB11, SGK1 facilitates membrane recycling of KCNQ1 channels. Here, we show altered SGK1-dependent regulation of LQTS-associated mutant I Ks channels. Whereas some mutant KCNQ1 channels had reduced basal activity but were still activated by SGK1, currents mediated by KCNQ1(Y111C) or KCNQ1(L114P) were paradoxically reduced by SGK1. Heteromeric channels coassembled of wild-type KCNQ1 and the LQTS-associated KCNE1(D76N) mutant were similarly downregulated by SGK1 because of a disrupted RAB11-dependent recycling. Mutagenesis experiments indicate that stimulation of I Ks channels by SGK1 depends on residues H73, N75, D76, and P77 in KCNE1. Identification of the I Ks recycling pathway and its modulation by stress-stimulated SGK1 provides novel mechanistic insight into potentially fatal cardiac arrhythmias triggered by physical or psychological stress.

Background: Cardiac action potential repolarisation is determined by K⁺ currents including I_Ks. I_Ks channels are heteromeric channels composed of KCNQ1 and KCNE E-subunits. Mutations in KCNQ1 are associated with sinus bradycardia, familial atrial fibrillation (fAF) and/or short QT syndrome as a result of gain-of-function, and long QT syndrome (LQTS) due to loss-of-function in the ventricles. Here, we report that the missense mutation R231C located in S4 voltage sensor domain is associated with a combined clinical phenotype of sinus bradycardia, fAF and LQTS. We aim to understand the molecular basis of the complex clinical phenotype. Methods: We expressed and functionally analyzed the respective channels kinetics in Xenopus laevis oocytes. The molecular nature of the residue R231 was studied by homology modeling and molecular dynamics simulation. Results: As a result, the mutation reduced voltage sensitivity of channels, possibly due to neutralization of the positive charge of the arginine side chain substituted by cysteine. Modeling suggested that the charge carrying side chain of R231 is positioned suitably to transfer transmembrane voltages into conformational energy. Further, the mutation altered the functional interactions with KCNE subunits. Conclusion: The mutation acted in a E-subunit dependent manner, suggesting I_Ks function altered by the presence of different KCNE subunits in sinus node, atria and ventricles as the molecular basis of sinus bradycardia, fAF and LQTS in mutation carriers.

Infections with coxsackieviruses of type B (CVBs), which are known to induce severe forms of acute and chronic myocarditis, are often accompanied by ventricular arrhythmias and sudden cardiac death. The mechanisms underlying the development of virus-induced, life-threatening arrhythmias, which are phenotypically similar to those observed in patients having functionally impaired cardiac ion channels, remain, however, enigmatic. In the present study, we show, for the first time, modulating time-dependent effects of CVB3 on the cardiac ion channels KCNQ1, hERG1, and Cav1.2 in heterologous expression. Channel protein abundance in cellular plasma membrane and patterns of their subcellular distribution were altered in infected murine hearts. The antiviral compound AG7088 did not prevent these effects on channels. In silico analyses of infected human myocytes suggest pronounced alterations of electrical and calcium signaling and increased risk of arrhythmogenesis. These modifications are attenuated by the common Asian polymorphism KCNQ1 P448R, a genetic determinant preventing coxsackievirus-induced effects in vitro. This study provides a previously unknown explanation for the development of arrhythmias in enteroviral myocarditis, which will help to develop therapeutic strategies for arrhythmia treatment.

Our results suggest that the schizophrenia-linked mutation of the kinase results in reduced KCNQ channel function and thereby might explain the loss of dopaminergic control in schizophrenic patients. Moreover, the addictive potential of dopaminergic drugs often observed in schizophrenic patients might be explained by this mechanism. At least, the insufficiency of (N251S)-PIP5K2A to stimulate neuronal M channels may contribute to the clinical phenotype of schizophrenia.

The plateau phase of the ventricular action potential is the result of balanced Ca²⁺ influx and K⁺ efflux. The action potential is finally terminated by repolarising K⁺ currents. Under β-adrenergic stimulation the slowly activating component of the human cardiac delayed rectifier K⁺ current I_Ks provides the major repolarising component. I_Ks channels are heteromeric channels composed of KCNQ1 and KCNE1. Mutations in the voltage sensor S4 of KCNQ1 are associated with long-QT syndrome 1 (LQTS1). Here, we study the effects of the mutations S225L, I235N and L239P located in S4. The respective channels were expressed in Xenopus oocytes and analyzed by dual electrode voltage clamp. As a result all mutants shifted the voltage dependence of activation to the right and reduced the voltage dependence of deactivation kinetics. The activation kinetics were differently affected in homomeric mutant channels compared to wild type KCNQ1. All three mutations reduced KCNQ1/KCNE1 channel currents in a dominant-negative manner when the mutants were coexpressed with wt subunits suggesting reduced I_Ks as the molecular basis of LQT1.

Rationale - The plateau phase of the ventricular action potential is the result of balanced Ca²⁺ influx and K⁺ efflux. The action potential is terminated by repolarizing K⁺ currents. Under β-adrenergic stimulation, both the Ca²⁺-influx and the delayed rectifier K⁺ currents I_K are stimulated to adjust the cardiac action potential duration to the enhanced heart rate and to ascertain adequate increase in net Ca²⁺ influx. Intracellularly, a Calsequestrin2 (CASQ2)-ryanodine receptor complex serves as the most effective Ca²⁺ reservoir/release system to aid the control of intracellular Ca²⁺ levels. Currently, it is unclear if disease-associated CASQ2 gene variants alter intracellular free Ca²⁺ concentrations and if cardiac ion channels are affected by it. Objective – The goal of this study is to test if CASQ2 determines intracellular free Ca²⁺ concentrations and to identify cardiac ion channels that are affected by it. Further, we aim to study disease-associated CASQ2 gene variants in this context. Methods and Results - Here, we study the effects of the CASQ2 mutations R33Q, F189L, and D307H, located in highly conserved regions, on the functions of cardiac potassium channels in Xenopus oocytes using two electrode voltage clamp. As a result, CASQ2 wild type and CASQ2-mutants modulated hERG functions differently. Free Ca²⁺ measurements and molecular dynamics simulations imply alterations in Ca²⁺ buffer capacity paralled by changes in the dynamic behavior of the CASQ2-mutants compared to CASQ2 wild type. Conclusions - These in vitro and in silico data suggest a regulatory role of CASQ2 on cytosolic Ca²⁺ and hERG channels which may contribute to the etiology of CPVT.