The S4 transmembrane segments of voltage-gated ion channels move outward on depolarization, initiating a conformational change that opens the pore, but the mechanism of S4 movement is unresolved. favored compared with single cysteine mutants, and mutant cycle CCND2 analysis revealed strong free-energy coupling between these residues, further supporting interaction of R3 and D60 during gating. Our results demonstrate voltage-dependent formation of an ion pair during activation of the voltage sensor in real time and suggest that this interaction catalyzes S4 movement and channel activation. (22) is a small, 274-residue homotetrameric channel, which is a likely ancestor of the larger (2,000 residue) eukaryotic sodium and calcium channels that contain four covalently linked homologous domains. Despite its structural simplicity, NaChBac resembles individual domains of sodium and calcium channels having six transmembrane segments with a voltage-sensing module consisting of S1CS4 segments and a pore-forming module of S5 and S6 segments (22). Its activation is steeply voltage-dependent, but its kinetics of activation and inactivation are slower than eukaryotic sodium channels (6, 22). Its relatively small size and homotetrameric structure make it an ideal model for analysis of the molecular mechanisms of voltage-dependent gating. Disulfide bond formation between substituted cysteine residues has proven to be a powerful tool to analyze the structures of intermediates in protein-folding reactions (23, 24). Such disulfide bonds between substituted cysteine residues have been shown to lock intermediates in protein-folding pathways (23). Here, we have adapted this method to LY317615 (Enzastaurin) manufacture analyze the interaction of R3 in the S4 segment of NaChBac with D60 in the S2 segment in real-time voltage clamp experiments by measurements of state-dependent disulfide locking of substituted cysteine residues. Our results with this technique demonstrate a rapid, state-dependent interaction between these residues during voltage-dependent LY317615 (Enzastaurin) manufacture activation and suggest an important role for this interaction in catalyzing the transmembrane movement of the S4 gating charges. Results and Discussion Disulfide Locking of the NaChBac Voltage Sensor. In NaChBac, the first four arginine gating charges of the S4 transmembrane segment are in equivalent positions to those in the voltage-sensing modules in most mammalian voltage-gated ion channels (Scheme 1, residues indicated in bold). Scheme 1. Positions of LY317615 (Enzastaurin) manufacture S2 and S4 gating charges are conserved in NaChBac In addition, the first unfavorable charge in the S2 segment of NaChBac (D60) is in an analogous position to negatively charged aspartate or glutamate residues or hydrophilic asparagine residues that have a partially negatively charged carbonyl group in other voltage-gated sodium and potassium channels (Scheme 1). We developed a structural model for activation LY317615 (Enzastaurin) manufacture of the voltage sensor of NaChBac by using the Rosetta-Membrane algorithm, and the approach we described for KV1.2 channels (15). The model is based on the X-ray structure of the activated state of Kv1.2 (25), modeling of Kv1.2 in its resting and activated states (14, 15), and extension of those methods to NaChBac. In the resting state, the first arginine gating charge (R1) of the S4 segment is predicted to interact with D60 in the S2 segment [Fig. 1and supporting information (SI) and and for calculations of the energy and force of voltage sensor movement). Therefore, we hypothesized that a hyperpolarizing pulse to ?200 mV would have sufficient energy to break the disulfide bond formed between the S2 and S4 segments and release the disulfide-locked voltage sensor. To test this hypothesis, we hyperpolarized disulfide-locked D60C:R3C channels to increasingly unfavorable membrane potentials and then depolarized to measure the recovery of INa. Under control conditions, hyperpolarization to potentials more unfavorable than ?150 mV caused progressive recovery of INa (Fig. 2= 8) is similar to the time constant for slow inactivation of WT ( = 120 13 ms; = 9). The small difference between these values may reflect the incomplete compensation for the more unfavorable voltage dependence of activation of the double mutant. The similarity of these kinetics is consistent with the.