Omega Mutations along S4 in Nav1.2 Channels Give Insight into Domain Specific Contribution to Activation and Steady State Inactivation

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We previously identified the resting state positions of the voltage sensor S4 for each domain of Nav1.2 by means of omega mutations. We found that a double gap is needed to open the omega pore (narrow part of the gating pore) resulting in detectable omega current, also known as gating pore current. At hyperpolarizing conditions, the resting state of S4 was found for double gap RR1,2QQ in domain I, II and IV and for double gap RR2,3QQ in domain III. In this work we evaluated additional conformational states of the voltage sensor S4 moving through the gating pore by further double gap mutations along S4 (second double gap 2,3QQ and third double gap 3,4QQ). Two electrode voltage clamping on X. laevis oocytes expressing rat brain sodium channels Nav1.2 was used to measure macroscopic ionic current through the alpha pore and, if present, omega current through the omega pore. In DI and DII we detected clear outward omega current for S4 mutants RK3,4QQ at depolarizing conditions. Furthermore, we found that activation of sodium currents was right shifted by about 30 mV towards higher potentials compared to the first and second double gap mutants or wild-type sodium channel. These findings suggest two sequential gating steps of S4 between resting and activated state in both domains DI and DII. In DIII and DIV no clear outward omega current could be detected at depolarized potentials either for the second or for the third double gap mutant. However steady state inactivation was strongly left shifted by about 50 to 100 mV to more hyperpolarized potentials for the second and third double gap mutant in both domains, consistent with involvement in recovery from inactivation and immobilization, respectively.