Molecular determinants of beta 1 subunit-induced gating modulation in voltage-dependent Na+ channels

Recombinant brain, skeletal muscle, and heart voltage-gated Na+ channel alpha subunits differ in their functional responses to an accessory beta 1 subunit when coexpressed in Xenopus oocytes. We exploited the distinct beta 1 subunit responses observed for the human heart (hH1) and human skeletal muscle (hSkM1) isoforms to identify determinants of this response. Chimeric alpha subunits were constructed by exchanging the S5-S6 interhelical loops of each domain between hH1 and hSkM1 and then examined for effects on inactivation induced by coexpressed beta 1 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 hH1 produced channels that exhibited an attenuated response to coexpressed beta 1 subunit. Substitutions of both D1/S5-S6 and D4/S5-S6 in hSkM1 by the corresponding loops from hH1 completely abolished the effects of the beta 1 subunit on inactivation. The reciprocal chimera in which both D1/S5-S6 and D4/S5-S6 from hSkM1 were transplanted into hH1 exhibited significant beta 1 responsiveness (accelerated inactivation). The region within D4/S5-S6 that conferred beta 1 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 beta subunit consisting of the extracellular domains of beta 1 and the transmembrane and C-terminal domains of the rat brain beta 2 subunit. These results suggest that the D1/S5-S6 and D4/S5-S6 loops in the alpha subunit and the extracellular domain of the beta 1 subunit are important determinants of the beta 1 subunit-induced gating modulation in Na+ channels.