The second and third basic residues of the S4 segment of domain 4 (D4:R2 and D4:R3) of the human skeletal muscle Na+ channel are known to be translocated from a cytoplasmic to an extracellular position during depolarization. Accessibilities of individual S4 residues were assayed by alteration of inactivation kinetics during modification of cysteine mutants by hydrophilic methanethiosulfonate reagents. The voltage dependences of the reaction rates are identical for extracellular application of cationic methanethiosulfonate-ethyltrimethylammonium (MTSET) and anionic methanethiosulfonate-ethylsulfonate (MTSES), suggesting that D4:R3C is situated outside the membrane electric field at depolarized voltages. The absolute rate of R3C modification is 281-fold greater for MTSET than for MTSES, however, suggesting that at depolarized voltages this S4 thiol resides in a negatively charged hydrophilic crevice. The two hydrophobic residues between D4:R2C and D4:R3C in the primary sequence (L1452 and A1453) are not externally exposed at any voltage. An α-helical representation of D4/S4 shows that the basic residues D4:R2 and D4:R3 are on the face opposite that of L1452 and A1453. We propose that in the depolarized conformation, the hydrophobic face of this portion of D4/S4 remains in contact with a hydrophobic region of the extracellular vestibule of the S4 channel.
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