Glutamine Substitution at Alanine¹⁶⁴⁹ in the S4–S5 Cytoplasmic Loop of Domain 4 Removes the Voltage Sensitivity of Fast Inactivation in the Human Heart Sodium Channel

Normal activation–inactivation coupling in sodium channels insures that inactivation is slow at small but rapid at large depolarizations. M1651Q/M1652Q substitutions in the cytoplasmic loop connecting the fourth and fifth transmembrane segments of Domain 4 (S4–S5/D4) of the human heart sodium channel subtype 1 (hH1) affect the kinetics and voltage dependence of inactivation (Tang, L., R.G. Kallen, and R. Horn. 1996. J. Gen. Physiol. 108:89–104.). We now show that glutamine substitutions NH₂-terminal to the methionines (L¹⁶⁴⁶, L¹⁶⁴⁷, F¹⁶⁴⁸, A¹⁶⁴⁹, L¹⁶⁵⁰) also influence the kinetics and voltage dependence of inactivation compared with the wild-type channel. In contrast, mutations at the COOH-terminal end of the S4–S5/D4 segment (L¹⁶⁵⁴, P¹⁶⁵⁵, A¹⁶⁵⁶) are without significant effect. Strikingly, the A1649Q mutation renders the current decay time constants virtually voltage independent and decreases the voltage dependences of steady state inactivation and the time constants for the recovery from inactivation. Single-channel measurements show that at negative voltages latency times to first opening are shorter and less voltage dependent in A1649Q than in wild-type channels; peak open probabilities are significantly smaller and the mean open times are shorter. This indicates that the rate constants for inactivation and, probably, activation are increased at negative voltages by the A1649Q mutation reminiscent of Y1494Q/ Y1495Q mutations in the cytoplasmic loop between the third and fourth domains (O'Leary, M.E., L.Q. Chen, R.G. Kallen, and R. Horn. 1995. J. Gen. Physiol. 106:641–658.). Other substitutions, A1649S and A1649V, decrease but fail to eliminate the voltage dependence of time constants for inactivation, suggesting that the decreased hydrophobicity of glutamine at either residues A¹⁶⁴⁹ or Y¹⁴⁹⁴Y¹⁴⁹⁵ may disrupt a linkage between S4–S5/D4 and the interdomain 3–4 loop interfering with normal activation–inactivation coupling.