It is now well established that the voltage-sensing S4 segment in voltage-dependent ion channels undergoes a conformational change in response to varying membrane potential. However, the magnitude of the movement of S4 relative to the membrane and the rest of the protein remains controversial. Here, by using histidine scanning mutagenesis in the Shaker K channel, we identified mutants I241H (S1 segment) and I287H (S2 segment) that generate inward currents at hyperpolarized potentials, suggesting that these residues are part of a hydrophobic plug that separates the water-accessible crevices. Additional experiments with substituted cysteine residues showed that, at hyperpolarized potentials, both I241C and I287C can spontaneously form disulphide and metal bridges with R362C, the position of the first charge-carrying residue in S4. These results constrain unambiguously the closedstate positions of the S4 segment with respect to the S1 and S2 segments, which are known to undergo little or no movement during gating. To satisfy these constraints, the S4 segment must undergo an axial rotation of Ϸ180°and a transmembrane (vertical) movement of Ϸ6.5 Å at the level of R362 in going from the open to the closed state of the channel, moving the gating charge across a focused electric field.gating current ͉ metal bridge ͉ omega current ͉ S-S bridge V oltage-gated channels are crucial players in excitability and cell homeostasis. Voltage-dependent sodium and potassium conductances are responsible for the generation and propagation of the nerve impulse (1). The salient property of these channels is the steep membrane potential dependence of their open probability. This voltage dependence is conferred by the voltage sensor that has been identified with their first four transmembrane segments (S1-S4). It is now clear that the four most extracellular basic residues of the S4 segment and the most intracellular acidic residue in the S2 segment in the Shaker K channel are the gating charge-carrying residues that move in the field in response to changes in membrane potential (2, 3). The movement and conformational change of segments S1-S4 within the voltage-sensing module in response to membrane depolarization is somehow coupled to the intracellular gate of the pore domain (S5-S6) to open and close the channel. A multitude of biophysical experiments have attempted to delineate the conformation of the voltage sensor and the position of its charges in the open and closed states. The structural restraints deduced from those experiments have subsequently been shown to be consistent with the overall conformation of the open state from the crystallographic structure of the Kv1.2 channel. However, the experiments providing structural constraints about the closed state conformation of the voltage sensor are more uncertain. Although it is generally accepted that the S1 and S2 helical segments do not move extensively upon gating (4-8), there is disagreement concerning the magnitude of movements associated with the S3 and S4 segments. For exampl...
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.
SUMMARY Most action potentials are produced by the sequential activation of voltage-gated sodium (Nav) and potassium (Kv) channels. This is mainly achieved by the rapid conformational rearrangement of voltage-sensor (VS) modules in Nav channels, with activation kinetics up to 6-fold faster than Shaker-type Kv channels. Here, using mutagenesis and gating current measurements, we show that a 3-fold acceleration of the VS kinetics in Nav versus Shaker Kv channels is produced by the hydrophilicity of two “speed-control” residues located in the S2 and S4 segments in Nav domains I–III. An additional 2-fold acceleration of the Nav VS kinetics is provided by the coexpression of the β1 subunit, ubiquitously found in mammal tissues. This study uncovers the molecular bases responsible for the differential activation of Nav versus Kv channels, a fundamental prerequisite for the genesis of action potentials.
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.
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.
Voltage sensor domains (VSDs) regulate ion channels and enzymes by transporting electrically charged residues across a hydrophobic VSD constriction called the gating pore or hydrophobic plug. How the gating pore controls the gating charge movement presently remains debated. Here, using saturation mutagenesis and detailed analysis of gating currents from gating pore mutations in the Shaker Kv channel, we identified statistically highly significant correlations between VSD function and physicochemical properties of gating pore residues. A necessary small residue at position S240 in S1 creates a "steric gap" that enables an intracellular access pathway for the transport of the S4 Arg residues. In addition, the stabilization of the depolarized VSD conformation, a hallmark for most Kv channels, requires large side chains at positions F290 in S2 and F244 in S1 acting as "molecular clamps," and a hydrophobic side chain at position I237 in S1 acting as a local intracellular hydrophobic barrier. Finally, both size and hydrophobicity of I287 are important to control the main VSD energy barrier underlying transitions between resting and active states. Taken together, our study emphasizes the contribution of several gating pore residues to catalyze the gating charge transfer. This work paves the way toward understanding physicochemical principles underlying conformational dynamics in voltage sensors.energy landscape | kinetic model | global fit
Channelrhodopsin is a light-gated cation channel whose reaction cycle involves proton-transfer reactions. Understanding how channelrhodopsin works is 16a Sunday,
1 The voltage-dependent displacement of the scorpion Tityus serrulatus a-toxin Ts3 was investigated in native sodium channels of GH3 cells by examining the removal of its effects in toxin-free solution. 2 Toxin at saturating concentration was pulsed (B1 s) directly onto the cell, thus causing an eightfold increase of the slow component (t s ¼ 6 ms) of fast inactivation, and a three-fold increase of the time constant of its fast component. 3 At 0 mV, maximal conductance was achieved in cells before and after treatment with Ts3, and no displacement of the toxin could be detected. 4 Toxin displacement occurred if stronger depolarising pulses (4100 mV) were applied. The rate of displacement depended on the amplitude and duration of the pulses, and was not related with outward Na þ flux. 5 We propose a model in which activation does not require complete movement of segment S4 of domain IV (IVS4) and that a more extensive movement of this segment is needed for normal fast inactivation. A kinetic model is presented that can account for the typical effects of site 3 toxins.
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