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.

Armstrong, Time course of TEA(+)-induced anomalous rectification in squid giant axons. 1966, Pubmed

Armstrong, Time course of TEA(+)-induced anomalous rectification in squid giant axons. 1966, Pubmed
Armstrong, Interaction of tetraethylammonium ion derivatives with the potassium channels of giant axons. 1971, Pubmed
Armstrong, Ionic pores, gates, and gating currents. 1974, Pubmed
Bruening-Wright, Localization of the activation gate for small conductance Ca2+-activated K+ channels. 2002, Pubmed
Cha, Atomic scale movement of the voltage-sensing region in a potassium channel measured via spectroscopy. 1999, Pubmed
Chapman, Activation-dependent subconductance levels in the drk1 K channel suggest a subunit basis for ion permeation and gating. 1997, Pubmed , Xenbase
Doyle, The structure of the potassium channel: molecular basis of K+ conduction and selectivity. 1998, Pubmed
Enkvetchakul, ATP interaction with the open state of the K(ATP) channel. 2001, Pubmed
Flynn, Conformational changes in S6 coupled to the opening of cyclic nucleotide-gated channels. 2001, Pubmed , Xenbase
Glauner, Spectroscopic mapping of voltage sensor movement in the Shaker potassium channel. 1999, Pubmed , Xenbase
Holmgren, Trapping of organic blockers by closing of voltage-dependent K+ channels: evidence for a trap door mechanism of activation gating. 1997, Pubmed
Holmgren, The activation gate of a voltage-gated K+ channel can be trapped in the open state by an intersubunit metal bridge. 1998, Pubmed
Jiang, Crystal structure and mechanism of a calcium-gated potassium channel. 2002, Pubmed
Jiang, The open pore conformation of potassium channels. 2002, Pubmed
Jiang, X-ray structure of a voltage-dependent K+ channel. 2003, Pubmed
Jiang, The principle of gating charge movement in a voltage-dependent K+ channel. 2003, Pubmed
Kuo, Crystal structure of the potassium channel KirBac1.1 in the closed state. 2003, Pubmed
Larsson, Transmembrane movement of the shaker K+ channel S4. 1996, Pubmed , Xenbase
Lipkind, Molecular modeling of interactions of dihydropyridines and phenylalkylamines with the inner pore of the L-type Ca2+ channel. 2003, Pubmed
Liu, Change of pore helix conformational state upon opening of cyclic nucleotide-gated channels. 2000, Pubmed , Xenbase
Liu, Structure of the KcsA channel intracellular gate in the open state. 2001, Pubmed
Liu, Gated access to the pore of a voltage-dependent K+ channel. 1997, Pubmed
Loussouarn, Flexibility of the Kir6.2 inward rectifier K(+) channel pore. 2001, Pubmed
Mannuzzu, Independence and cooperativity in rearrangements of a potassium channel voltage sensor revealed by single subunit fluorescence. 2000, Pubmed , Xenbase
Mannuzzu, Direct physical measure of conformational rearrangement underlying potassium channel gating. 1996, Pubmed , Xenbase
Obejero-Paz, Y3+ block demonstrates an intracellular activation gate for the alpha1G T-type Ca2+ channel. 2004, Pubmed
Perozo, Structural rearrangements underlying K+-channel activation gating. 1999, Pubmed
Phillips, Gating dependence of inner pore access in inward rectifier K(+) channels. 2003, Pubmed
Rothberg, Voltage-controlled gating at the intracellular entrance to a hyperpolarization-activated cation channel. 2002, Pubmed
Rothberg, Movements near the gate of a hyperpolarization-activated cation channel. 2003, Pubmed
Shin, Blocker state dependence and trapping in hyperpolarization-activated cation channels: evidence for an intracellular activation gate. 2001, Pubmed
Stauffer, Electrostatic potential of the acetylcholine binding sites in the nicotinic receptor probed by reactions of binding-site cysteines with charged methanethiosulfonates. 1994, Pubmed
Webster, Intracellular gate opening in Shaker K+ channels defined by high-affinity metal bridges. 2004, Pubmed
Xiao, Localization of PIP2 activation gate in inward rectifier K+ channels. 2003, Pubmed , Xenbase
Yang, Molecular determinants of Ca2+ selectivity and ion permeation in L-type Ca2+ channels. 1993, Pubmed , Xenbase
Yang, Evidence for voltage-dependent S4 movement in sodium channels. 1995, Pubmed
Zhen, Functional architecture of the inner pore of a voltage-gated Ca2+ channel. 2005, Pubmed , Xenbase
Zheng, Selectivity changes during activation of mutant Shaker potassium channels. 1997, Pubmed , Xenbase
Zheng, Intermediate conductances during deactivation of heteromultimeric Shaker potassium channels. 1998, Pubmed , Xenbase
del Camino, Blocker protection in the pore of a voltage-gated K+ channel and its structural implications. 2000, Pubmed
del Camino, Tight steric closure at the intracellular activation gate of a voltage-gated K(+) channel. 2001, Pubmed