The voltage-sensitive sodium channel confers electrical excitability on neurons, a fundamental property required for higher processes including cognition. The ion-conducting ␣-subunit of the channel is regulated by two known auxiliary subunits, ␤1 and ␤2. We have identified rat and human forms of an additional subunit, ␤3. It is most closely related to ␤1 and is the product of a separate gene localized to human chromosome 11q23.3. When expressed in Xenopus oocytes, ␤3 inactivates sodium channel opening more slowly than ␤1 does. Structural modeling has identified an amino acid residue in the putative ␣-subunit binding site of ␤3 that may play a role in this difference. The expression of ␤3 within the central nervous system differs significantly from ␤1. Our results strongly suggest that ␤3 performs a distinct neurophysiological function.T he voltage-sensitive sodium channel plays a fundamental role in excitable cells, transiently increasing the sodium permeability of the plasma membrane in response to changes in membrane potential and thus propagating the action potential (1, 2). Not surprisingly, mutations in sodium channel genes are implicated in several pathologies, including epilepsy and cardiac arrhythmias (3-5), and therapeutic drugs, including antiepileptics, local anesthetics, and anticonvulsants (6), act on the channel.In the central nervous system, the channel is conventionally described as a heterotrimer composed of a 260-kDa ␣-subunit, a noncovalently associated 36-kDa ␤1-subunit, and a disulfidelinked 33-kDa ␤2-subunit (2). The ␣-subunit forms the ion pore and is responsible for the voltage-sensitive characteristics of the complex. There are multiple isoforms of the ␣-subunit expressed in different regions of the brain and peripheral nervous system that differ in their kinetic properties (1). The ␤-subunits are auxiliary components acting in a regulatory capacity (7). ␤1 increases the fraction of ␣-subunits operating in a fast gating mode, thus accelerating the activation and inactivation kinetics of the channel and modulating the frequency with which neurons fire (8). The ␤2-subunit is required for the efficient assembly of the channel but has minor effects on gating kinetics. These two ␤-subunits are distantly related by sequence (9).We now report the cloning and analysis of the rat and human forms of a previously uncharacterized sequence that we call ␤3. It is homologous to ␤1, but differs from ␤1 both in its distribution within the brain and in some of its kinetic properties. The discovery of this subunit increases the complexity of the sodium channel and raises further questions about the role of these auxiliary subunits. Materials and MethodsCloning Methodology. We isolated a variant of the rat pheochromocytoma cell line PC12 (termed A35C), which lacks typical neuronal properties (10). To discover previously unidentified neuroendocrine-specific genes, subtractive cloning was used to identify transcripts expressed at a level in the variant cells lower than that in normal PC12 cells. Total RNA wa...

1. We have compared the mRNA distribution of sodium channel alpha subunits known to be expressed during development with the known auxiliary subunits Nab1.1 and Nab2.1 and the novel, recently cloned subunit, b3. 2.In situ hybridisation studies demonstrated high levels of Nav1.2, Nav1.3, Nav1.6 and b3 mRNA at embryonic stages whilst Nab1.1 and Nab2.1 mRNA was absent throughout this period.3. Nab1.1 and Nab2.1 expression occurred after postnatal day 3 (P3), increasing steadily in most brain regions until adulthood. b3 expression differentially decreased after P3 in certain areas but remained high in the hippocampus and striatum.4. Emulsion-dipped slides showed co-localisation of b3 with Nav1.3 mRNA in areas of the CNS suggesting that these subunits may be capable of functional interaction.5. Co-expression in Xenopus oocytes revealed that b3 could modify the properties of Nav1.3; b3 changed the equilibrium of Nav1.3 between the fast and slow gating modes and caused a negative shift in the voltage dependence of activation and inactivation.6. In conclusion, b3 is shown to be the predominant b subunit expressed during development and is capable of modulating the kinetic properties of the embryonic Nav1.3 subunit. These findings provide new information regarding the nature and properties of voltage-gated sodium channels during development.

Adult dorsal root ganglia (DRG) have been shown to express a wide range of voltage-gated sodium channel alpha-subunits. However, of the auxiliary subunits, beta1 is expressed preferentially in only large- and medium-diameter neurons of the DRG while beta2 is absent in all DRG cells. In view of this, we have compared the distribution of beta1 in rat DRG and spinal cord with a novel, recently cloned beta1-like subunit, beta3. In situ hybridization studies demonstrated high levels of beta3 mRNA in small-diameter c-fibres, while beta1 mRNA was virtually absent in these cell types but was expressed in 100% of large-diameter neurons. In the spinal cord, beta3 transcript was present specifically in layers I/II (substantia gelatinosa) and layer X, while beta1 mRNA was expressed in all laminae throughout the grey matter. Since the pattern of beta3 expression in DRG appears to correlate with the TTX-resistant voltage-gated sodium channel subunit PN3, we co-expressed the two subunits in Xenopus oocytes. In this system, beta3 caused a 5-mV hyperpolarizing shift in the threshold of activation of PN3, and a threefold increase in the peak current amplitude when compared with PN3 expressed alone. On the basis of these results, we examined the expression of beta-subunits in the chronic constriction injury model of neuropathic pain. Results revealed a significant increase in beta3 mRNA expression in small-diameter sensory neurons of the ipsilateral DRG. These results show that beta3 is the dominant auxiliary sodium channel subunit in small-diameter neurons of the rat DRG and that it is significantly upregulated in a model of neuropathic pain.

The upregulation of voltage-gated sodium channel Na v 1.3 has been linked to hyperexcitability of axotomized dorsal root ganglion (DRG) neurons, which underlies neuropathic pain. However, factors that regulate delivery of Na v 1.3 to the cell surface are not known. Contactin/ F3, a cell adhesion molecule, has been shown to interact with and enhance surface expression of sodium channels Na v 1.2 and Na v 1.9. In this study we show that contactin coimmunoprecipitates with Na v 1.

Although activation of G protein-coupled inward rectifying K+ (GIRK) channels by Gi/Go-coupled receptors has been shown to be important in postsynaptic inhibition in the central nervous system, there is also evidence to suggest that inhibition of GIRK channels by Gq-coupled receptors is involved in postsynaptic excitation. In the present study we addressed whether the Gq-coupled receptors of the bombesin family can couple to GIRK channels and examined the mechanism by which this process occurs. Different combinations of GIRK channel subunits (Kir3.1, Kir3.2, and Kir3.4) and bombesin receptors (BB1 and BB2) were expressed in Xenopus oocytes. In all combinations tested GIRK currents were reversibly inhibited upon application of the bombesin-related peptides, neuromedin B or gastrin-releasing peptide in a concentration-dependent manner. Incubation of oocytes in the phospholipase C inhibitor U73122 or the protein kinase C (PKC) inhibitors chelerythrine and staurosporine significantly reduced the inhibition of GIRK currents by neuromedin B, whereas the Ca2+ chelator, BAPTA-AM had no effect. The involvement of PKC was further demonstrated by direct inhibition of GIRK currents by the phorbol esters, phorbol-12,13-dibutyrate and phorbol-12-myristate-13-acetate. In contrast, the inactive phorbol ester 4alpha-phorbol and protein kinase A activators, forskolin and 8-bromo cAMP did not inhibit GIRK currents. At the single-channel level, direct activation of PKC using phorbol ester phorbol-12, 13-dibutyrate caused a dramatic reduction in open probability of GIRK channels due to an increase in duration of the interburst interval.

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This study investigated the distribution of beta3 in human tissues and the functional effects of the human beta3 subunit on the gating properties of brain and skeletal muscle alpha subunits. Using RT-PCR of human cDNA panels, beta3 message was detected in brain, heart, kidney, lung, pancreas and skeletal muscle. Both alphaIIA and SkM1 expressed in Xenopus oocytes inactivated with a time course described by two exponential components representing fast and slow gating modes, while co-expression of human beta3 with alphaIIA or SkM1 significantly increased the proportion of channels operating by the fast gating mode. In the presence of beta3 a greater proportion of alphaIIA or SkM1 current was described by the fast time constant for both inactivation and recovery from inactivation. beta3 caused a hyperpolarizing shift in the voltage dependence of inactivation of alphaIIIA and reduced the slope factor. The voltage dependence of inactivation of SkM1 was described by a double Boltzmann equation. However, SkM1 co-expressed with beta3 was described by a single Boltzmann equation similar to one of the Boltzmann components for SkM1 expressed alone, with a small positive shift in V1/2 value and reduced slope factor. This is the first study demonstrating that beta3 is expressed in adult mammalian skeletal muscle and can functionally couple to the skeletal muscle alpha subunit, SkM1.