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Unformatted text preview: Inhibition of Sodium Channel Gating by Trapping the Domain II Voltage Sensor with Protoxin II Stanislav Sokolov, Richard L. Kraus, Todd Scheuer, and William A. Catterall Department of Pharmacology, University of Washington, Seattle, Washington (S.S., T.S., W.A.C.) and Merck Research Laboratories, West Point, Pennsylvania (R.L.K.) Received August 17, 2007; accepted December 21, 2007 ABSTRACT ProTx-II, an inhibitory cysteine knot toxin from the tarantula Thrixopelma pruriens , inhibits voltage-gated sodium channels. Using the cut-open oocyte preparation for electrophysiological recording, we show here that ProTx-II impedes movement of the gating charges of the sodium channel voltage sensors and reduces maximum activation of sodium conductance. At a concentration of 1 m M, the toxin inhibits 65.3 6 4.1% of the sodium conductance and 24.6 6 6.8% of the gating current of brain Na v 1.2a channels, with a specific effect on rapidly moving gating charge. Strong positive prepulses can reverse the inhib- itory effect of ProTx-II, indicating voltage-dependent dissocia- tion of the toxin. Voltage-dependent reversal of the ProTx-II effect is more rapid for cardiac Na v 1.5 channels, suggesting subtype-specific action of this toxin. Voltage-dependent bind- ing and block of gating current are hallmarks of gating modifier toxins, which act by binding to the extracellular end of the S4 voltage sensors of ion channels. The mutation L833C in the S3S4 linker in domain II reduces affinity for ProTx-II, and mutation of the outermost two gating-charge-carrying arginine residues in the IIS4 voltage sensor to glutamine abolishes volt- age-dependent reversal of toxin action and toxin block of gat- ing current. Our results support a voltage-sensor-trapping model for ProTx-II action in which the bound toxin impedes the normal outward gating movement of the IIS4 transmembrane segment, traps the domain II voltage sensor module in its resting state, and thereby inhibits channel activation. Voltage-gated sodium channels are responsible for the in- crease in sodium permeability that initiates action potentials in electrically excitable cells and are the molecular targets for several groups of neurotoxins that bind to different receptor sites and alter voltage-dependent activation, conductance, and inactivation (Catterall, 1980; Ceste `le and Catterall, 2000). Sodium channels are composed of one pore-forming a subunit of approximately 2000 amino acid residues associated with one or two smaller auxiliary subunits, b 1 to b 4 (Catterall, 2000). The a subunit consists of four homologous domains (IIV), each containing six transmembrane segments (S1S6), and a re- entrant pore loop (P) between S5 and S6 (Catterall, 2000). The S4 transmembrane segments are positively charged and serve as voltage sensors to initiate channel activation (Armstrong, 1981; Catterall, 1986; Stu hmer et al., 1989; Yang and Horn, 1995; Chanda and Bezanilla, 2002). The sliding helix (Catter- all, 1986) or helical screw (Guy and Seetharamulu, 1986)...
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