Molecular determinants of β₁ subunit-induced gating modulation in voltage-dependent Na⁺ channels

Recombinant brain, skeletal muscle, and heart voltage-gated Na⁺ channel α subunits differ in their functional responses to an accessory β₁ subunit when coexpressed in Xenopus oocytes. We exploited the distinct β₁ subunit responses observed for the human heart (hH1) and human skeletal muscle (hSkM1) isoforms to identify determinants of this response. Chimeric α subunits were constructed by exchanging the S5-S6 interhelical loops of each domain between hilt and hSkM1 and then examined for effects on inactivation induced by coexpressed β₁ subunit in oocytes. Substitution of single S5-S6 loops in either domain 1 (D1/S5-S6) or domain 4 (D4/S5-S6) of hSkM1 by the corresponding segments of hill produced channels that exhibited an attenuated response to coexpressed β₁ subunit. Substitutions of both D1/S5-S6 and D4/S5-S6 in hSkM1 by the corresponding loops from hilt completely abolished the effects of the β₁ subunit on inactivation. The reciprocal chimera in which both D1/S5-S6 and D4/S5-S6 from hSkM1 were transplanted into hill exhibited significant β₁ responsiveness (accelerated inactivation). The region within D4/S5-S6 that conferred β₁ responsiveness was determined to reside primarily within an extracellular loop between the putative pore- forming segment SS2 and D4/S6. Gating modulation was also demonstrated using a chimeric β subunit consisting of the extracellular domains of β₁ and the transmembrane and C-terminal domains of the rat brain β₂ subunit. These results suggest that the D1/S5-S6 and D4/S5-S6 loops in the α subunit and the extracellular domain of the β₁ subunit are important determinants of the β₁ subunit-induced gating modulation in Na⁺ channels.