Sodium channels isolated from mammalian brain are composed of ␣, ␤1, and ␤2 subunits. The auxiliary ␤ subunits do not form the ion conducting pore, yet play important roles in channel modulation and plasma membrane expression. ␤1 and ␤2 are transmembrane proteins with one extracellular V-set immunoglobulin (Ig) protein domain. It has been shown recently that ␤1 and ␤2 interact with the extracellular matrix proteins tenascin-C and tenascin-R. In the present study we show that rat brain ␤1 and ␤2, but not ␣IIA, subunits interact in a trans-homophilic fashion, resulting in recruitment of the cytoskeletal protein ankyrin to sites of cell-cell contact in transfected Drosophila S2 cells. Whereas ␣IIA subunits expressed alone do not cause cellular aggregation, ␤ subunits co-expressed with ␣IIA retain the ability to adhere and recruit ankyrin. Truncated ␤ subunits lacking cytoplasmic domains interact homophilically to produce cell aggregation but do not recruit ankyrin. Thus, the cytoplasmic domains of ␤1 and ␤2 are required for cytoskeletal interactions. It is hypothesized that sodium channel ␤ subunits serve as a critical communication link between the extracellular and intracellular environments of the neuron and may play a role in sodium channel placement at nodes of Ranvier.Control of the cell surface density and localization of voltagegated sodium channels are critical aspects of neuronal function. This is especially important at nodes of Ranvier of myelinated axons where high densities of sodium channels are needed for rapid and reliable saltatory conduction (1, 2). Sodium channels from mammalian brain are heterotrimeric structures composed of a central pore forming ␣ subunit and two auxiliary subunits, ␤1 and ␤2 (3, 4). Though the ␤ subunits do not form the ion conducting pore, they play critical roles in channel gating, voltage dependence of activation and inactivation, and channel plasma membrane expression levels (5-8). ␤1 and ␤2 contain Ig-like extracellular domains and are members of the V-set of the Ig superfamily, which includes many cell adhesion molecules (CAMs) 1 (9). ␤1 and ␤2 have recently been shown to interact with the extracellular matrix proteins tenascin-C and tenascin-R (10, 11). Transfected cells expressing ␤1 or ␤2 subunits initially bind to and then are repelled from a tenascin-R substrate (11). Purified sodium channels or the bacterially expressed ␤2 subunit extracellular domain bind tenascin-C and tenascin-R in an enzyme-linked immunosorbent type biochemical assay (10). These results suggested that sodium channel ␤ subunits function as CAMs. CAMs of the L1 family also bind directly to cytoskeletal elements such as ankyrin (12,13,(15)(16)(17). Sodium channels have been shown to colocalize with ankyrin G and spectrin at axon initial segments and nodes of Ranvier, and there is some evidence to show that sodium channels bind ankyrin directly in vitro (18 -22). However, it is not known to which sodium channel subunit ankyrin binds. Because ␤1 and ␤2 are structurally and functionally homologous ...

Sodium channel ␤-subunits modulate channel gating, assembly, and cell surface expression in heterologous cell systems. We generated ␤2 ؊/؊ mice to investigate the role of ␤2 in control of sodium channel density, localization, and function in neurons in vivo. Measurements of [ 3 H]saxitoxin (STX) binding showed a significant reduction in the level of plasma membrane sodium channels in ␤2 ؊/؊ neurons. The loss of ␤2 resulted in negative shifts in the voltage dependence of inactivation as well as significant decreases in sodium current density in acutely dissociated hippocampal neurons. The integral of the compound action potential in optic nerve was significantly reduced, and the threshold for action potential generation was increased, indicating a reduction in the level of functional plasma membrane sodium channels. In contrast, the conduction velocity, the number and size of axons in the optic nerve, and the specific localization of Na v1.6 channels in the nodes of Ranvier were unchanged. ␤2 ؊/؊ mice displayed increased susceptibility to seizures, as indicated by reduced latency and threshold for pilocarpine-induced seizures, but seemed normal in other neurological tests. Our observations show that ␤2-subunits play an important role in the regulation of sodium channel density and function in neurons in vivo and are required for normal action potential generation and control of excitability.auxiliary subunits ͉ gene targeting ͉ epilepsy ͉ action potential conduction

Auxiliary ␤1 subunits of voltage-gated sodium channels have been shown to be cell adhesion molecules of the Ig superfamily. Co-expression of ␣ and ␤1 subunits modulates channel gating as well as plasma membrane expression levels. We have cloned, sequenced, and expressed a splice variant of ␤1, termed ␤1A, that results from an apparent intron retention event. ␤1 and ␤1A are structurally homologous proteins with type I membrane topology; however, they contain little to no amino acid homology beyond the shared Ig loop region. ␤1A mRNA expression is developmentally regulated in rat brain such that it is complementary to ␤1. ␤1A mRNA is expressed during embryonic development, and then its expression becomes undetectable after birth, concomitant with the onset of ␤1 expression. In contrast, ␤1A mRNA is expressed in adult adrenal gland and heart. Western blot analysis revealed ␤1A protein expression in heart, skeletal muscle, and adrenal gland but not in adult brain or spinal cord. Immunocytochemical analysis of ␤1A expression revealed selective expression in brain and spinal cord neurons, with high expression in heart and all dorsal root ganglia neurons. Co-expression of ␣IIA and ␤1A subunits in Chinese hamster lung 1610 cells results in a 2.5-fold increase in sodium current density compared with cells expressing ␣IIA alone. This increase in current density reflected two effects of ␤1A: 1) an increase in the proportion of cells expressing detectable sodium currents and 2) an increase in the level of functional sodium channels in expressing cells. Sodium channels isolated from brain are composed of a central pore-forming ␣ subunit and two auxiliary subunits, ␤1 and ␤2, which do not form the pore yet play critical roles in channel modulation and expression. A mutation in the ␤1 gene (SCN1B) has been implicated to play a role in febrile seizures and generalized epilepsy, GEFSϩ (1). The primary structure of the ␤1 subunit predicts an integral membrane glycoprotein with type I transmembrane topology as well as an extracellular Ig fold (2, 3). ␤ 1 subunits can be classified as members of the V-set of the Ig superfamily, which includes many cell adhesion molecules. ␤1 and ␣ subunit co-expression has been well characterized in Xenopus oocytes and in mammalian cells. In oocytes, co-expression of type IIA (SCN2A) or I (SCN4A) ␣ subunits with ␤1 increases the proportion of sodium channels that function in a fast gating mode, accelerates the macroscopic rates of activation and inactivation, shifts the voltage dependence of inactivation in the hyperpolarizing direction, and increases the peak current amplitude consistent with increases in channel expression (4 -9). In Chinese hamster lung (CHL) 1 cells, stable coexpression of ␤1 with ␣IIA results in increased channel expression levels at the plasma membrane as well as moderate hyperpolarizing shifts in the voltage dependence of channel activation and inactivation (10).␤1 mRNA is expressed only after birth in the developing brain (5, 11). However, previous studies showing the devel...