Differential regulation of β1-dependent voltage shifts and kinetic modulation by an extracellular glutamate in Na_V1.6 VSDIV

Auxiliary β1 subunits play an important role in modulating voltage-gated Na⁺ (Na_V) channel gating, thereby influencing their voltage-dependent behavior and overall function. Here, we examine β1 modulation of human Na_V1.6 channels expressed in Xenopus laevis oocytes and identify a glutamate in the extracellular S3–S4 loop of domain IV (VSDIV; E1607) as a key determinant of β1-induced voltage shifts. Co-expression of β1 produces hyperpolarizing shifts in activation and steady-state inactivation (SSI) and accelerates macroscopic inactivation kinetics. Substitution of E1607 with lysine or alanine, but not the negatively charged aspartate, selectively attenuates β1-induced shifts in voltage dependence while preserving effects on slope factor, inactivation kinetics, and persistent current. Pharmacological perturbation of VSDIV with the α-scorpion toxin AaHII attenuates β1-dependent shifts in SSI without abolishing β1 effects on gating kinetics. Together, these findings identify an extracellular VSDIV element that contributes to β1-dependent control of voltage dependence and support a model in which β1 modulates Na_V1.6 gating through VSDIV-linked transitions.