Abstract
The position of the voltage-sensing transmembrane segment, S4, in voltage-gated ion channels as a function of voltage remains incompletely elucidated. Site-3 toxins bind primarily to the extracellular loops connecting transmembrane helical segments S1-S2 and S3-S4 in Domain 4 (D4) and S5-S6 in Domain 1 (D1) and slow fast-inactivation of voltage-gated sodium channels. As S4 of the human skeletal muscle voltage-gated sodium channel, hNav1.4, moves in response to depolarization from the resting to the inactivated state, two D4S4 reporters (R2C and R3C, Arg1451Cys and Arg1454Cys, respectively) move from internal to external positions as deduced by reactivity to internally or externally applied sulfhydryl group reagents, methane thiosulfonates (MTS). The changes in reporter reactivity, when cycling rapidly between hyperpolarized and depolarized voltages, enabled determination of the positions of the D4 voltage-sensor and of its rate of movement. Scorpion α-toxin binding impedes D4S4 segment movement during inactivation since the modification rates of R3C in hNav1.4 with methanethiosulfonate (CH3SO2SCH2CH2R, where R = -N(CH3)3 + trimethylammonium, MTSET) and benzophenone-4-carboxamidocysteine methanethiosulfonate (BPMTS) were slowed ~10-fold in toxin-modified channels. Based upon the different size, hydrophobicity and charge of the two reagents it is unlikely that the change in reactivity is due to direct or indirect blockage of access of this site to reagent in the presence of toxin (Tx), but rather is the result of inability of this segment to move outward to the normal extent and at the normal rate in the toxin-modified channel. Measurements of availability of R3C to internally applied reagent show decreased access (slower rates of thiol reaction) providing further evidence for encumbered D4S4 movement in the presence of toxins consistent with the assignment of at least part of the toxin binding site to the region of D4S4 region of the voltage-sensor module.
Highlights
Voltage-gated ion channels (Nav) are tetradomain proteins containing six (S1-S6) transmembrane segments per domain (D) with four domains that cluster around a central pore
Electrophilic methane thiosulfonates (MTS) reagents are attacked by the cysteine thiolate anion to form a mixed disulfide with the addition of a side-chain determined by the nature of the reagent [44]
Normalized FS values as a function of time progress from zero to unity monoexponentially providing the rate of modification. This is illustrated by the reaction of R3C with MTSET (20 μM), which slows the decay of current simultaneously with the appearance of a pedestal or residual current (Figure 2C), similar to the action of scorpion α-toxins such as LqhαIT (Figure 2B) when compared with unmodified R1454C (Figure 2A)
Summary
Voltage-gated ion channels (Nav) are tetradomain proteins containing six (S1-S6) transmembrane segments per domain (D) with four domains that cluster around a central pore. Navs are characterized by three main processes: (i) opening (activation) from a resting state when the membrane is depolarized allowing current flow; (ii) closing to the non-conducting resting state upon hyperpolarization (deactivation); and (iii) closing to a non-conducting refractory state (inactivation) in which the channel remains nonconducting in response to continuing depolarization [6]. The various states represent different conformations of the channel protein that are controlled, in large part, by the highly charged S4 segments of the “voltagesensor”, which contain 4 to 8 cationic residues in the various domains with each basic group generally separated by two hydrophobic amino acids [7,8]. While the S4 movement communicates the change in membrane potential to the remainder of the protein, the nature of the conformational alteration of the voltage-sensor remains a matter of some uncertainty
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