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Voltage sensors regulate the conformations of voltage-dependent ion channels and enzymes. Their nearly switchlike response as a function of membrane voltage comes from the movement of positively charged amino acids, arginine or lysine, across the membrane field. We used mutations with natural and unnatural amino acids, electrophysiological recordings, and x-ray crystallography to identify a charge transfer center in voltage sensors that facilitates this movement. This center consists of a rigid cyclic \"cap\" and two negatively charged amino acids to interact with a positive charge. Specific mutations induce a preference for lysine relative to arginine. By placing lysine at specific locations, the voltage sensor can be stabilized in different conformations, which enables a dissection of voltage sensor movements and their relation to ion channel opening.

We report two structures of the human voltage-gated potassium channel (Kv) Kv1.3 in immune cells alone (apo-Kv1.3) and bound to an immunomodulatory drug called dalazatide (dalazatide–Kv1.3). Both the apo-Kv1.3 and dalazatide–Kv1.3 structures are in an activated state based on their depolarized voltage sensor and open inner gate. In apo-Kv1.3, the aromatic residue in the signature sequence (Y447) adopts a position that diverges 11 Å from other K⁺ channels. The outer pore is significantly rearranged, causing widening of the selectivity filter and perturbation of ion binding within the filter. This conformation is stabilized by a network of intrasubunit hydrogen bonds. In dalazatide–Kv1.3, binding of dalazatide to the channel’s outer vestibule narrows the selectivity filter, Y447 occupies a position seen in other K⁺ channels, and this conformation is stabilized by a network of intersubunit hydrogen bonds. These remarkable rearrangements in the selectivity filter underlie Kv1.3’s transition into the drug-blocked state.

Temperature-sensitive transient receptor potential (TRP) ion channels are members of the large tetrameric cation channels superfamily but are considered to be uniquely sensitive to heat, which has been presumed to be due to the existence of an unidentified temperature-sensing domain. Here we report that the homologous voltage-gated potassium (Kv) channels also exhibit high temperature sensitivity comparable to that of TRPV1, which is detectable under specific conditions when the voltage sensor is functionally decoupled from the activation gate through either intrinsic mechanisms or mutations. Interestingly, mutations could tune Shaker channel to be either heat-activated or heat-deactivated. Therefore, high temperature sensitivity is intrinsic to both TRP and Kv channels. Our findings suggest important physiological roles of heat-induced variation in Kv channel activities. Mechanistically our findings indicate that temperature-sensing TRP channels may not contain a specialized heat-sensor domain; instead, non-obligatory allosteric gating permits the intrinsic heat sensitivity to drive channel activation, allowing temperature-sensitive TRP channels to function as polymodal nociceptors. If you touch something too hot, it can cause you pain and damage your skin. Sensing the heat given off by an object or the temperature of the environment is possible, at least in part, because of proteins called temperature-sensitive TRP ion channels. These proteins are found in the cell membranes of nerve endings that are underneath the skin; and they open in response to heat, allowing ions to flow into the nerve cell. This in turn triggers a nerve impulse that is sent to our central nervous system and is perceived as heat and/or pain. The ability to sense heat was thought to be unique to these TRP ion channels, and it was believed that these ion channels contained an as-yet unidentified temperature-sensing domain. However, Yang and Zheng now report that similar ion channels, which open in response to changes in the voltage that exists across a cell's membrane, are also sensitive to changes in temperature. The temperature response of these ‘voltage-gated channels’ had largely eluded the attention of researchers in the past. This is because parts of the ion channel—which act like a ‘voltage sensor’ and only shift when the membrane voltage changes—normally keep the channel closed and directly open the channel when they move. Like all other proteins, ion channels are made from smaller building blocks called amino acids; and by changing some of the amino acids in the voltage-gated channel Yang and Zheng could decouple these normally linked actions. The changes to the channel meant that it did not immediately open when the voltage sensor moved; and decreasing the concentration of calcium ions inside the cell had the same effect as changing these amino acids. Both approaches revealed that, after a change in membrane voltage caused the voltage sensor to move, the ion channel remained closed until a high temperature caused it to open. Yang and Zheng revealed that the response of the modified voltage-gated channel to temperature was comparable to that of a typical heat-sensitive TRP ion channel. Further experiments showed that replacing some of the amino acids in the voltage-gated potassium ion channel with different amino acids could cause the channel to be either opened or closed by heat. The findings of Yang and Zheng indicate that temperature-sensing TRP channels may not contain a specialized heat-sensor domain. Instead, as these TRP ion channels do not require other parts of the protein to move in order to open the channel, they can be activated by their own inherent sensitivity to heat.

Gemcitabine is an antineoplastic drug commonly used in the treatment of several types of cancers including pancreatic cancer and non–small cell lung cancer. Although gemcitabine-induced cardiotoxicity is widely recognized, the exact mechanism of cardiac dysfunction causing arrhythmias remains unclear. The objective of this study was to electrophysiologically evaluate the proarrhythmic cardiotoxicity of gemcitabine focusing on the human rapid delayed rectifier potassium channel, hERG channel. In heterologous hERG expressing HEK293 cells (hERG-HEK cells), hERG channel current ( I hERG ) was reduced by gemcitabine when applied for 24 h but not immediately after the application. Gemcitabine modified the activation gating properties of the hERG channel toward the hyperpolarization direction, while inactivation, deactivation or reactivation gating properties were unaffected by gemcitabine. When gemcitabine was applied to hERG-HEK cells in combined with tunicamycin, an inhibitor of N-acetylglucosamine phosphotransferase, gemcitabine was unable to reduce I hERG or shift the activation properties toward the hyperpolarization direction. While a mannosidase I inhibitor kifunensine alone reduced I hERG and the reduction was even larger in combined with gemcitabine, kifunensine was without effect on I hERG when hERG-HEK cells were pretreated with gemcitabine for 24 h. In addition, gemcitabine down-regulated fluorescence intensity for hERG potassium channel protein in rat neonatal cardiomyocyte, although hERG mRNA was unchanged. Our results suggest the possible mechanism of arrhythmias caused by gemcitabine revealing a down-regulation of I hERG through the post-translational glycosylation disruption possibly at the early phase of hERG channel glycosylation in the endoplasmic reticulum that alters the electrical excitability of cells.

Recently, Conorfamide-Sr3 (CNF-Sr3) was isolated from the venom of Conus spurius and was demonstrated to have an inhibitory concentration-dependent effect on the Shaker K+ channel. The voltage-gated potassium channels play critical functions on cellular signaling, from the regeneration of action potentials in neurons to the regulation of insulin secretion in pancreatic cells, among others. In mammals, there are at least 40 genes encoding voltage-gated K+ channels and the process of expression of some of them may include alternative splicing. Given the enormous variety of these channels and the proven use of conotoxins as tools to distinguish different ligand- and voltage-gated ion channels, in this work, we explored the possible effect of CNF-Sr3 on four human voltage-gated K+ channel subtypes homologous to the Shaker channel. CNF-Sr3 showed a 10 times higher affinity for the Kv1.6 subtype with respect to Kv1.3 (IC50 = 2.7 and 24 μM, respectively) and no significant effect on Kv1.4 and Kv1.5 at 10 µM. Thus, CNF-Sr3 might become a novel molecular probe to study diverse aspects of human Kv1.3 and Kv1.6 channels.

KCNQ2 and KCNQ3 channels are associated with multiple neurodevelopmental disorders and are also therapeutic targets for neurological and neuropsychiatric diseases. For more than two decades, it has been thought that most KCNQ channels in the brain are either KCNQ2/3 or KCNQ3/5 heteromers. Here, we investigated the potential heteromeric compositions of KCNQ2-containing channels. We applied split-intein protein trans-splicing to form KCNQ2/5 tandems and coexpressed these with and without KCNQ3. Unexpectedly, we found that KCNQ2/5 tandems form functional channels independent of KCNQ3 in heterologous cells. Using mass spectrometry, we went on to demonstrate that KCNQ2 associates with KCNQ5 in native channels in the brain, even in the absence of KCNQ3. Additionally, our functional heterologous expression data are consistent with the formation of KCNQ2/3/5 heteromers. Thus, the composition of KCNQ channels is more diverse than has been previously recognized, necessitating a re-examination of the genotype/phenotype relationship of KCNQ2 pathogenic variants.

Potassium channels encoded by human Ether-à-go-go-Related Gene ( hERG ) are inhibited by diverse cardiac and non-cardiac drugs. Disopyramide is a chiral Class Ia antiarrhythmic that inhibits hERG at clinical concentrations. This study evaluated effects of disopyramide enantiomers on hERG current (I hERG ) from hERG expressing HEK 293 cells at 37 °C. S(+) and R(−) disopyramide inhibited wild-type (WT) I hERG with IC 50 values of 3.9 µM and 12.9 µM respectively. The attenuated-inactivation mutant N588K had little effect on the action of S(+) disopyramide but the IC 50 for the R(−) enantiomer was ~ 15-fold that for S(+) disopyramide. The enhanced inactivation mutant N588E only slightly increased the potency of R(−) disopyramide. S6 mutation Y652A reduced S(+) disopyramide potency more than that of R(−) disopyramide (respective IC 50 values ~ 49-fold and 11-fold their WT controls). The F656A mutation also exerted a stronger effect on S(+) than R(−) disopyramide, albeit with less IC 50 elevation. A WT-Y652A tandem dimer exhibited a sensitivity to the enantiomers that was intermediate between that of WT and Y652A, suggesting Y652 groups on adjacent subunits contribute to the binding. Moving the Y (normally at site 652) one residue in the N- terminal (up) direction in N588K hERG markedly increased the blocking potency of R(−) disopyramide. Molecular dynamics simulations using a hERG pore model produced different binding modes for S(+) and R(−) disopyramide consistent with the experimental observations. In conclusion, S(+) disopyramide interacts more strongly with S6 aromatic binding residues on hERG than does R(−) disopyramide, whilst optimal binding of the latter is more reliant on intact inactivation.

Retigabine is a recently approved anticonvulsant that acts by potentiating neuronal M-current generated by KCNQ2–5 channels, interacting with a conserved Trp residue in the channel pore domain. Using unnatural amino-acid mutagenesis, we subtly altered the properties of this Trp to reveal specific chemical interactions required for retigabine action. Introduction of a non-natural isosteric H-bond-deficient Trp analogue abolishes channel potentiation, indicating that retigabine effects rely strongly on formation of a H-bond with the conserved pore Trp. Supporting this model, substitution with fluorinated Trp analogues, with increased H-bonding propensity, strengthens retigabine potency. In addition, potency of numerous retigabine analogues correlates with the negative electrostatic surface potential of a carbonyl/carbamate oxygen atom present in most KCNQ activators. These findings functionally pinpoint an atomic-scale interaction essential for effects of retigabine and provide stringent constraints that may guide rational improvement of the emerging drug class of KCNQ channel activators. The antiepileptic drug retigabine potentiates neuronal KCNQ potassium channels. Here, the authors use a combination of unnatural amino acid mutagenesis and electrophysiology to show that retigabine acts by hydrogen bonding with a tryptophan indole nitrogen in the channel pore.

Vandetanib, a multi-kinase inhibitor used for the treatment of various cancers, has been reported to induce several adverse cardiac effects. However, the underlying mechanisms of vandetanib-induced cardiotoxicity are unclear. This study aimed to investigate the mechanism of vandetanib-induced cardiotoxicity using intracellular electrophysiological recordings on human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), rabbit Purkinje fibers, and HEK293 cells transiently expressing human ether-a-go-go-related gene (hERG; the rapidly activating delayed rectifier K+ channel, IKr), KCNQ1/KCNE1 (the slowly activating delayed rectifier K+ current, IKs), KCNJ2 (the inwardly rectifying K+ current, IK1) or SCN5A (the inward Na+ current, INa). Purkinje fiber assays and ion channel studies showed that vandetanib at concentrations of 1 and 3 μM inhibited the hERG currents and prolonged the action potential duration. Alanine scanning and in silico hERG docking studies demonstrated that Y652 and F656 in the hERG S6 domain play critical roles in vandetanib binding. In hiPSC-CMs, vandetanib markedly reduced the maximum rate of depolarization during the AP upstroke. Ion channel studies revealed that hiPSC-CMs were more sensitive to inhibition of the INa by vandetanib than in a heterogeneously expressed HEK293 cell model, consistent with the changes in the AP parameters of hiPSC-CMs. The subclasses of Class I antiarrhythmic drugs inhibited INa currents in a dose-dependent manner in hiPSC-CMs and SCN5A-encoded HEK293 cells. The inhibitory potency of vandetanib for INa was much higher in hiPSC-CMs (IC50: 2.72 μM) than in HEK293 cells (IC50: 36.63 μM). These data suggest that AP and INa assays using hiPSC-CMs are useful electrophysiological models for prediction of drug-induced cardiotoxicity.

Myo-inositol is an important cellular osmolyte in autoregulation of cell volume and fluid balance, particularly for mammalian brain and kidney cells. We find it also regulates excitability. Myo-inositol is the precursor of phosphoinositides, key signaling lipids including phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂]. However, whether myo-inositol accumulation during osmoregulation affects signaling and excitability has not been fully explored. We found that overexpression of the Na⁺/myo-inositol cotransporter (SMIT1) and myoinositol supplementation enlarged intracellular PI(4,5)P₂ pools, modulated several PI(4,5)P₂-dependent ion channels including KCNQ2/3 channels, and attenuated the action potential firing of superior cervical ganglion neurons. Further experiments using the rapamycin-recruitable phosphatase Sac1 to hydrolyze PI(4)P and the P4M probe to visualize PI(4)P suggested that PI(4)P levels increased after myoinositol supplementation with SMIT1 expression. Elevated relative levels of PIP and PIP₂ were directly confirmed using mass spectrometry. Inositol trisphosphate production and release of calcium from intracellular stores also were augmented after myo-inositol supplementation. Finally, we found that treatment with a hypertonic solution mimicked the effect we observed with SMIT1 overexpression, whereas silencing tonicity-responsive enhancer binding protein prevented these effects. These results show that ion channel function and cellular excitability are under regulation by several “physiological” manipulations that alter the PI(4,5)P₂ setpoint. We demonstrate a previously unrecognized linkage between extracellular osmotic changes and the electrical properties of excitable cells.