The principal ␣ subunit of voltage-gated sodium channels is associated with auxiliary ␤ subunits that modify channel function and mediate protein-protein interactions. We have identified a new ␤ subunit termed ␤4. Like the ␤1-␤3 subunits, ␤4 contains a cleaved signal sequence, an extracellular Ig-like fold, a transmembrane segment, and a short intracellular C-terminal tail. Using TaqMan reverse transcription-PCR analysis, in situ hybridization, and immunocytochemistry, we show that ␤4 is widely distributed in neurons in the brain, spinal cord, and some sensory neurons. ␤4 is most similar to the ␤2 subunit (35% identity), and, like the ␤2 subunit, the Ig-like fold of ␤4 contains an unpaired cysteine that may interact with the ␣ subunit. Under nonreducing conditions, ␤4 has a molecular mass exceeding 250 kDa because of its covalent linkage to Na v 1.2a, whereas on reduction, it migrates with a molecular mass of 38 kDa, similar to the mature glycosylated forms of the other ␤ subunits. Coexpression of ␤4 with brain Na v 1.2a and skeletal muscle Na v 1.4 ␣ subunits in tsA-201 cells resulted in a negative shift in the voltage dependence of channel activation, which overrode the opposite effects of ␤1 and ␤3 subunits when they were present. This novel, disulfide-linked ␤ subunit is likely to affect both protein-protein interactions and physiological function of multiple sodium channel ␣ subunits.

Background-Voltage-gated sodium channels composed of pore-forming ␣ and auxiliary ␤ subunits are responsible for the rising phase of the action potential in cardiac muscle, but their localizations have not yet been clearly defined. Methods and Results-Immunocytochemical studies show that the principal cardiac ␣ subunit isoform Na v 1.5 and the ␤2 subunit are preferentially localized in intercalated disks, identified by immunostaining of connexin 43, the major protein of cardiac gap junctions. The brain ␣ subunit isoforms Na v 1.1, Na v 1.3, and Na v 1.6 are preferentially localized with ␤1 and ␤3 subunits in the transverse tubules, identified by immunostaining of ␣-actinin, a cardiac z-line protein. The ␤1 subunit is also present in a small fraction of intercalated disks. The recently cloned ␤4 subunit, which closely resembles ␤2 in amino acid sequence, is also expressed in ventricular myocytes and is localized in intercalated disks as are ␤2 and Na v 1.5. Conclusions-Our results suggest that the primary sodium channels present in ventricular myocytes are composed of Na v 1.5 plus ␤2 and/or ␤4 subunits in intercalated disks and Na v 1.1, Na v 1.3, and Na v 1.6 plus ␤1 and/or ␤3 subunits in the transverse tubules. Key Words: ion channels Ⅲ sodium Ⅲ myocytes Ⅲ ventricles Ⅲ immunohistochemistry V oltage-gated sodium channels are responsible for the initiation of action potentials in most excitable cells. They are composed of a pore-forming ␣ subunit and 1 or 2 auxiliary ␤ subunits. 1 Ten genes encoding ␣ subunits have been identified, and 9 have been functionally expressed. 2 The different ␣ subunit isoforms have distinct patterns of development and localization in the nervous system and skeletal and cardiac muscle, and they have different physiological and pharmacological properties. Isoforms preferentially expressed in the central nervous system (Na v 1.1, Na v 1.2, Na v 1.3, Na v 1.6) are inhibited by nanomolar concentrations of tetrodotoxin, as is the isoform present in adult skeletal muscle (Na v 1.4). In contrast, the primary cardiac isoform (Na v 1.5) requires micromolar concentrations of tetrodotoxin for inhibition because of the presence of a cysteine instead of an aromatic residue in the pore region of domain I. 3 Recent studies show that the brain-type isoforms Na v 1.1, Na v 1.3, and Na v 1.6 are expressed in ventricular myocytes and have distinct subcellular localization and function. 4 The 4 known ␤ subunits of sodium channels divide into 2 groups: ␤1 and ␤3 are most similar in sequence and are noncovalently associated with ␣ subunits, 5,6 while the ␤2 and ␤4 subunits are also closely related in amino acid sequence and are disulfide-linked to ␣ subunits. 7,8 The ␤ subunits are multifunctional because they modulate channel gating, regulate the level of expression at the plasma membrane, and function as cell adhesion molecules through interaction with the cytoskeleton, extracellular matrix, and other cell adhesion molecules that regulate cell migration and aggregation. 9 mRNA for the ␤1 subunit has been...

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...

Voltage-gated sodium channels in brain neurons were found to associate with receptor protein tyrosine phosphatase beta (RPTPbeta) and its catalytically inactive, secreted isoform phosphacan, and this interaction was regulated during development. Both the extracellular domain and the intracellular catalytic domain of RPTPbeta interacted with sodium channels. Sodium channels were tyrosine phosphorylated and were modulated by the associated catalytic domains of RPTPbeta. Dephosphorylation slowed sodium channel inactivation, positively shifted its voltage dependence, and increased whole-cell sodium current. Our results define a sodium channel signaling complex containing RPTPbeta, which acts to regulate sodium channel modulation by tyrosine phosphorylation.