Xie C , Zhen XG , Yang J .

NS45383 NINDS NIH HHS , R01 NS045383 NINDS NIH HHS

	Figure 1. Pore-lining residues in S6. Amino acid sequence alignment of the four S6 segments in the Cav2.1 subunit and the M2/S6 segment of KcsA and Shaker K+ channels. Amino acid numbers are given on both sides. For Ca2+ channel S6 segments, the amino acid numbering defined in this study is shown on the top. Position 0 presumably represents the membrane/cytoplasm interface. Bold positions denote those that can be modified by internal MTSET. Underlined positions were studied in this work. For K+ channel M2/S6 segments, bold residues denote pore-lining positions defined either structurally or by MTS reagent accessibility. Arrow marks the M2/S6 bundle crossing.
	Figure 2. Closed-state modification of a cysteine above the putative membrane/cytoplasm interface. (A) Voltage protocol for closed-state modification by internal MTSET and representative current traces. Current was evoked by a 20-ms test pulse before and after MTSET (1 mM) application. Voltage was held at −80 mV when MTSET was applied for 2 min and then washed out for 1 min. (B) An exemplar time course of closed-state modification of IIIS6-V9C. Each point represents the peak current evoked by the 20-ms test pulse. Pulsing was stopped during MTSET application and washout. (C) Cartoon of S5 and S6 transmembrane helices and P-loop in the closed state. The thiol group of a pore-lining cysteine above the membrane/cytoplasm interface is shown. Dark gray spheres represent MTSET molecules.
	Figure 3. Open-state modification of a cysteine above the putative membrane/cytoplasm interface. (A) Voltage protocol for open-state modification by internal MTSET and representative current traces. Current was evoked every 6 s by a 500-ms test pulse. MTSET (1 mM) was applied continuously in both the open and closed states until steady-state inhibition was reached. (B) An exemplar time course of open-state modification of IIIS6-V9C. (C) Cartoon of S5 and S6 transmembrane helices and P-loop in the open state. The thiol group of a pore-lining cysteine above the membrane/cytoplasm interface is shown. Dark gray spheres represent MTSET molecules.
	Figure 4. Comparison of open- and closed-state modification of a cysteine above the putative membrane/cytoplasm interface. (A) Voltage protocol for closed-state modification using fast perfusion. Current was evoked every 20 s by a 20-ms test pulse. Between the test pulses MTSET (1 mM) was applied for 10 s and subsequently washed out for 10 s. (B and D) Time course of MTSET modification of a representative cysteine (IIS6-A4C) in closed state (B) and open state (D). (C and E) The MTSET inhibition phase in B and D plotted against the cumulative time in MTSET or the cumulative channel open time, respectively, superimposed with a single-exponential fitting curve.
	Figure 5. MTSET modification rates in open and closed states. (A) Apparent second-order rate constants for MTSET modification of selected pore-lining residues. Filled circles represent rate constants measured in the closed state (−80 mV) and open circles represent those measured in the open state (at +20 or +30 mV for different mutant channels). (B) Cartoon of the closed-state conformation of S5 and S6 helices, showing that cysteines below the membrane/cytoplasm interface can be accessed by MTSET (dark gray spheres) in the closed state.
	Figure 6. Voltage dependence of MTSET modification. Data were collected from IIS6-A4C. Filled squares and open circles represent, respectively, the mean modification rates (n = 3–5) and channel open probability measured at different voltages. The solid line is the Boltzmann fit to the voltage-activation curves obtained from 10 experiments (midpoint = −44.8 ± 0.6 mV and slope factor = 9.6 ± 0.5 mV).
	Figure 7. Trapping of MTSET in the inner pore in the closed state. (A) Two voltage protocols for measuring MTSET modification. Currents were evoked by a depolarizing pulse to +30 mV every 6 s from a holding potential of −80 mV. The duration of the depolarizing pulse was either 500 ms or 10 ms. (B) Time course of MTSET inhibition of currents evoked by the 500-ms test pulse (filled circles) or the 10-ms test pulse (open circles) in the IIS6-A4C mutant channel. (C) The MTSET inhibition phase in B plotted against the cumulative channel open time, superimposed with a single-exponential fitting curve with the indicated time constant.

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