MOLECULAR DETERMINANTS OF NA-1 SUBUNIT( CHANNEL FUNCTION IN THE EXTRACELLULAR DOMAIN OF THE BETA)

The rat brain voltage-gated Na+ channel is composed of three glycoprot ein subunits: the pore-forming cu subunit and two auxiliary subunits, beta 1 and beta 2, which contain immunoglobulin (Ig)-like folds in the ir extracellular domains, When expressed in Xenopus oocytes, beta 1 mo dulates the gating properties of the channel-forming type IIA alpha su bunit, resulting in an acceleration of inactivation, We have used a co mbination of deletion, alanine-scanning, site-directed, and chimeric m utagenesis strategies to examine the importance of different structura l features of the beta 1 subunit in the modulation of alpha(IIA), func tion, with an emphasis on the extracellular domain, Deletion analysis revealed that the extracellular domain is required for function, but t he intracellular domain is not, The mutation of four putative sites of N-linked glycosylation showed that they are not required for beta 1 f unction. Mutations of hydrophobic residues in the core beta sheets of the Ig fold disrupted beta 1 function, whereas substitution of amino a cid 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 n ot significantly affect the association of the mutant beta 1 subunit w ith 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' s trands for interaction with the pore-forming alpha subunit.