Molecular determinants of Na+ channel function in the extracellular domain of the β1 subunit

The rat brain voltage-gated Na+ channel is composed of three glycoprotein subunits: the pore-forming cu subunit and two auxiliary subunits, beta 1 and beta 2, which contain immunoglobulin (Ig)-like folds in their extracellular domains, When expressed in Xenopus oocytes, beta 1 modulates the gating properties of the channel-forming type IIA alpha subunit, resulting in an acceleration of inactivation, We have used a combination of deletion, alanine-scanning, site-directed, and chimeric mutagenesis strategies to examine the importance of different structural features of the beta 1 subunit in the modulation of alpha(IIA), function, with an emphasis on the extracellular domain, Deletion analysis revealed that the extracellular domain is required for function, but the intracellular domain is not, The mutation of four putative sites of N-linked glycosylation showed that they are not required for beta 1 function. Mutations of hydrophobic residues in the core beta sheets of the Ig fold disrupted beta 1 function, whereas substitution of amino acid residues in connecting segments had no effect, Mutations of acidic residues in the A/A' strand of the Ig fold reduced the effectiveness of the beta 1 subunit in modulating the rate of inactivation hut did not significantly affect the association of the mutant beta 1 subunit with the alpha(IIA) subunit or its effect on recovery from inactivation, Our data suggest that the Ig fold of the beta 1 extracellular domain serves as a scaffold that presents the charged residues of the A/A' strands for interaction with the pore-forming alpha subunit.