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Voltage sensor domains (VSDs) regulate ion channels and enzymes by undergoing conformational changes depending on membrane electrical signals. The molecular mechanisms underlying the VSD transitions are not fully understood. Here, we show that some mutations of I241 in the S1 segment of the Shaker Kv channel positively shift the voltage dependence of the VSD movement and alter the functional coupling between VSD and pore domains. Among the I241 mutants, I241W immobilized the VSD movement during activation and deactivation, approximately halfway between the resting and active states, and drastically shifted the voltage activation of the ionic conductance. This phenotype, which is consistent with a stabilization of an intermediate VSD conformation by the I241W mutation, was diminished by the charge-conserving R2K mutation but not by the charge-neutralizing R2Q mutation. Interestingly, most of these effects were reproduced by the F244W mutation located one helical turn above I241. Electrophysiology recordings using nonnatural indole derivatives ruled out the involvement of cation-Π interactions for the effects of the Trp inserted at positions I241 and F244 on the channel’s conductance, but showed that the indole nitrogen was important for the I241W phenotype. Insight into the molecular mechanisms responsible for the stabilization of the intermediate state were investigated by creating in silico the mutations I241W, I241W/R2K, and F244W in intermediate conformations obtained from a computational VSD transition pathway determined using the string method. The experimental results and computational analysis suggest that the phenotype of I241W may originate in the formation of a hydrogen bond between the indole nitrogen atom and the backbone carbonyl of R2. This work provides new information on intermediate states in voltage-gated ion channels with an approach that produces minimum chemical perturbation.

Several naturally occurring polypeptide neurotoxins target specific sites on the voltage-gated sodium channels. Of these, the gating modifier toxins alter the behavior of the sodium channels by stabilizing transient intermediate states in the channel gating pathway. Here we have used an integrated approach that combines electrophysiological and spectroscopic measurements to determine the structural rearrangements modified by the β-scorpion toxin Ts1. Our data indicate that toxin binding to the channel is restricted to a single binding site on domain II voltage sensor. Analysis of Cole-Moore shifts suggests that the number of closed states in the activation sequence prior to channel opening is reduced in the presence of toxin. Measurements of charge–voltage relationships show that a fraction of the gating charge is immobilized in Ts1-modified channels. Interestingly, the charge–voltage relationship also shows an additional component at hyperpolarized potentials. Site-specific fluorescence measurements indicate that in presence of the toxin the voltage sensor of domain II remains trapped in the activated state. Furthermore, the binding of the toxin potentiates the activation of the other three voltage sensors of the sodium channel to more hyperpolarized potentials. These findings reveal how the binding of β-scorpion toxin modifies channel function and provides insight into early gating transitions of sodium channels.

In nerves and muscles, action potentials are initiated by the rapid activation of voltage-gated sodium (Nav) channels and terminated by the delayed activation of voltage-gated potassium (Kv) channels. This sequential activation, which is the prerequisite for the genesis of the action potential, requires faster activation kinetics of the voltage-sensor domains (VSD) in Nav as compared to Kv channels. Despite on decades of investigations, the molecular determinants and mechanisms underlying this phenomenon remain elusive. Here, we show that this differential gating is mostly imparted by six conserved hydrophilic residues Thr or Ser located in the S2 and S4 segments of the VSD in domains I-III of Nav channels while these positions are commonly occupied by hydrophobic residues in the VSD of Kv channels. Hydrophilic substitutions at the S2 position in the Shaker Kv channel accelerate the gating charge transfer by decreasing its energy barrier while hydrophilic substitutions at the S4 position speed up VSD activation by destabilizing its resting conformation. Interestingly, these hydrophilic residues are present in a Nav-related gene expressed in an evolutionary-distant unicellular choanoflagellate, suggesting that rapidly-gated Nav channels evolved before the emergence of metazoans and their nervous systems. We also show that the physiological co-expression of the ubiquitous regulatory β1 subunit further accelerates VSD movement in both a neuronal and a muscular Nav, providing a molecular basis for the β1-dependent fast gating mode previously detected from the ionic conductance. Our study uncovers the fundamental molecular determinants and possible mechanisms that enabled the differential gating in sodium and potassium channels and the emergence of the action potential. This work was supported by NIH grant GM030376.

α-Scorpion toxins bind in a voltage-dependent way to site 3 of the sodium channels, which is partially formed by the loop connecting S3 and S4 segments of domain IV, slowing down fast inactivation. We have used Ts3, an α-scorpion toxin from the Brazilian scorpion Tityus serrulatus, to analyze the effects of this family of toxins on the muscle sodium channels expressed in Xenopus oocytes. In the presence of Ts3 the total gating charge was reduced by 30% compared with control conditions. Ts3 accelerated the gating current kinetics, decreasing the contribution of the slow component to the ON gating current decay, indicating that S4-DIV was specifically inhibited by the toxin. In addition, Ts3 accelerated and decreased the fraction of charge in the slow component of the OFF gating current decay, which reflects an acceleration in the recovery from the fast inactivation. Site-specific fluorescence measurements indicate that Ts3 binding to the voltage-gated sodium channel eliminates one of the components of the fluorescent signal from S4-DIV. We also measured the fluorescent signals produced by the movement of the first three voltage sensors to test whether the bound Ts3 affects the movement of the other voltage sensors. While the fluorescence–voltage (F-V) relationship of domain II was only slightly affected and the F-V of domain III remained unaffected in the presence of Ts3, the toxin significantly shifted the F-V of domain I to more positive potentials, which agrees with previous studies showing a strong coupling between domains I and IV. These results are consistent with the proposed model, in which Ts3 specifically impairs the fraction of the movement of the S4-DIV that allows fast inactivation to occur at normal rates.

Voltage-sensors (VS) are protein domains which transduce changes in membrane potential into conformational changes thereby controlling the opening of a pore or the activity of a phosphatase domain. This operation creates measurable gating currents (Ig) by a local rearrangement of charged groups in the VS. Although there is high conservation of these gating charges among VS, the kinetics of Ig varies greatly, spreading over several orders of magnitudes. These kinetic differences explain the temporal separation of Na+ and K+ conductances in excitable cells, critical for the generation and propagation of the action potential. Despite its fundamental importance, the molecular mechanism underlying this phenomenon is not well understood. Here, we identified in the Shaker K+ channel two hydrophobic residues Ile241 and Ile287 located in S1 and S2 respectively which directly interact with S4 arginines during gating charge movement. We show that Shaker gating kinetics can be speeded up ∼ 3 fold by mutating Ile287 to more hydrophilic residues threonine or serine. Interestingly, voltage-dependent sodium channel (Nav) possesses threonines in domains I-III at positions homologous to Ile287 and exhibit Ig ∼ 10 times faster than that of Shaker. Mutating these threonines to isoleucines in Nav1.4 slows down Ig 2-3 times. Moreover, the sensing currents of the voltage-sensitive phosphatase Ci-VSP, which are ∼ 8 times slower than that of Shaker, can be speeded 3-4 fold by substituting Ile126, (homologous to Ile241 in Shaker) to threonine. Taken together, our data indicate that the side chains of these two key positions form a hydrophobic plug separating the external and internal media. This plug constitutes the main energy barrier for charge crossing and its amplitude is modulated by its degree of hydrophobicity, thus controlling the kinetics of the sensor. Support: NIH-GM030376.

The Kv7 family of voltage-gated K+ channels consists of five members (Kv7.1- Kv7.5), each showing specific tissue distribution and physiological role. Mutations in Kv7.1 cause the long QT syndrome, whereas Kv7.4 mutations underlie a rare form of slowly progressive deafness (DFNA2); finally, mutations in Kv7.2 and Kv7.3 genes have been identified in families affected by Benign Familial Neonatal Seizures. Disease-causing mutations often affect residues in the voltage-sensing domain of Kv7 subunits. In the present study, we have characterized the ionic and gating current of homomeric neuronally-expressed Kv7 channels (Kv7.2-Kv7.5) using the cut-open oocyte voltage clamp. The ionic and gating current were recorded at 18°C and also at 28°C to speed up gating charge movement. Increasing the recording temperature from 18°C to 28°C caused an accelerated activation/deactivation kinetics of the ionic currents in all homomeric Kv7 channels (the Q10 for the activation kinetics at 0 mV was 3.8, 4.1, 8.3, and 2.8 for Kv7.2, Kv7.3, Kv7.4 and Kv7.5). Moreover, currents carried by Kv7.4 channels (and less so Kv7.2) also showed a significant increase in their maximal value. Gating currents were only resolved in Kv7.4 and Kv7.5 channels, possibly because of an higher membrane channel density; ON gating charges at saturated potential (+40mV) amounted to 1.34±0.34 (Kv7.4) and 0.79±0.20 nC (Kv7.5). At 28°C, Kv7.4 gating currents had the following salient properties: 1) similar time integral of ON and OFF, indicating charge conservation; 2) a left-shift in the V1/2 of the ON gating charge when compared to ionic currents; 3) a rising phase in the OFF gating charge after depolarizations to values >0 mV. These results represent the first description of Kv7.4 and Kv7.5 gating currents and may help to clarify the molecular consequence of disease-causing mutations affecting channel gating. Supported by NIH GM30376 and Telethon GGP07125.