Molecular determinants of Na+ channel function in the extracellular domain of the beta1 subunit

The rat brain voltage-gated Na+ channel is composed of three glycoprotein subunits: the pore-forming alpha subunit and two auxiliary subunits, beta1 and beta2, which contain immunoglobulin (Ig)-like folds in their extracellular domains. When expressed in Xenopus oocytes, beta1 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 beta1 subunit in the modulation of alphaIIA 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 beta1 function. Mutations of hydrophobic residues in the core beta sheets of the Ig fold disrupted beta1 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 beta1 subunit in modulating the rate of inactivation but did not significantly affect the association of the mutant beta1 subunit with the alphaIIA subunit or its effect on recovery from inactivation. Our data suggest that the Ig fold of the beta1 extracellular domain serves as a scaffold that presents the charged residues of the A/A' strands for interaction with the pore-forming alpha subunit.