Molecular Determinants of Na+ Channel Function in the Extracellular Domain of the β1 Subunit

Abstract

The rat brain voltage-gated Na؉ channel is composed of three glycoprotein subunits: the pore-forming ␣ subunit and two auxiliary subunits, ␤1 and ␤2, which contain immunoglobulin (Ig)-like folds in their extracellular domains. When expressed in Xenopus oocytes, ␤1 modulates the gating properties of the channel-forming type IIA ␣ 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 ␤1 subunit in the modulation of ␣ 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 ␤1 function. Mutations of hydrophobic residues in the core ␤ sheets of the Ig fold disrupted ␤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 ␤1 subunit in modulating the rate of inactivation but did not significantly affect the association of the mutant ␤1 subunit with the ␣ IIA subunit or its effect on recovery from inactivation. Our data suggest that the Ig fold of the ␤1 extracellular domain serves as a scaffold that presents the charged residues of the A/A strands for interaction with the pore-forming ␣ subunit.The rat brain voltage-gated sodium (Na ϩ ) channel is composed of three glycoprotein subunits: the pore-forming ␣ subunit with a relative molecular mass of 260 kDa, and two auxiliary subunits, ␤1 (36 kDa) and ␤2 (33 kDa) (for review, see Refs. 1 and 2). The ␣ subunit has four internally homologous domains, each containing six potential transmembrane-spanning regions and a pore-forming loop (3). The ␤1 and ␤2 subunits from rat brain are not closely related in terms of amino acid sequence, but each contains a single membrane-spanning segment that separates a large NH 2 -terminal extracellular domain from a smaller COOH-terminal intracellular domain (4, 5). In addition, the extracellular domains of the ␤1 and ␤2 subunits have sequences similar to those of proteins of the immunoglobulin (Ig) 1 superfamily and are proposed to contain an Ig-like motif (5, 6). The ␤2 subunit is linked covalently to the ␣ subunit by disulfide bonds, whereas the ␤1 subunit associates with the ␣ subunit in a noncovalent manner (7). Biochemical analyses of detergent-solubilized rat brain Na ϩ channels suggest that both ionic and hydrophobic interactions are important in association of ␤1 with the ␣⅐␤2 complex (7).Although the auxiliary subunits are not required for the formation of functional Na ϩ channels (8, 9), coexpression of the ␤1 subunit with the rat brain type IIA ␣ subunit in Xenopus oocytes increases the proportion of Na ϩ channels that function in a fast gating mode (4, 10). Na ϩ channel inactivation is accelerated 5-fol...

A novel locus for episodic ataxia:UBR4 the likely candidate

“…31 The deletion of the extracellular domain of the beta-1 prevents sodium channel modulation. 32 Beta-1 knockout mice develop an ataxic gait and frequent generalized seizures that are followed by periods of immobility. 33 Mutations in sodium channel alpha subunit genes have already been reported to cause complex neurological phenotypes that include ataxia including SCN2A 34 and SCN8A.…”

Modulatory Proteins Can Rescue a Trafficking Defective Epileptogenic Nav1.1 Na+Channel Mutant

“…To obtain the truncated h␤1 STOP mutant (McCormick et al, 1998;Meadows et al, 2001) with deleted C-terminal intracellular domain (see Fig. 5C), we substituted with Quick Change XL Kit a stop codon (TAA) Figure 1.…”

“…␤2 and ␤4 subunits are covalently linked to the ␣ subunit by disulfide bridges (dashed-dotted line), but the cysteine residues implicated have not been identified yet. ␤1 and ␤3 subunits have noncovalent intracellular and extracellular interactions with the ␣ subunit (dashed lines) (McCormick et al, 1998;Qu et al, 1999;Meadows et al, 2001;Spampanato et al, 2004). M1841T lies in a 41-residue area, highlighted in the inset, comprising a cytoplasmic binding domain for ␤1 and, probably, ␤3 (Spampanato et al, 2004).…”

“…In particular, ␤1 can associate with the ␣ subunit both through intracellular and the extracellular domains ( Fig. 1) (McCormick et al, 1998Qu et al, 1999;Meadows et al, 2001;Spampanato et al, 2004). To determine which domain of ␤1 is more important for rescuing hNa v 1.1-M1841T, we studied the effect of ␤1 subunits with defective extracellular domain or with deleted intracellular domain.…”

“…The ␤1 STOP is a truncated protein with a deletion of the whole intracellular domain (the last 34 aa) (Fig. 5C), but it is not a complete loss-of-function mutant, because it shows reduced association with Na v 1.2 ␣ subunit when expressed in Chinese hamster lung cells (Meadows et al, 2001) but not when expressed in Xenopus oocytes, where it is fully functional (McCormick et al, 1998). Coexpression in tsA-201 cells of hNa v 1.1-M1841T and ␤1 STOP resulted in a 2.3-fold increase in current density (Fig.…”