Here we investigate differences in the temporal and spatial patterning, and subunit interactions of two of the major Na(+) channel alpha-subunit isoforms in mammalian brain, the type I and type II Na(+) channels. By using subtype-specific antibodies, we find that both isoforms are abundant in adult rat brain, where both interact with the covalently bound beta2 auxiliary subunit. Immunoblot analysis reveals complementary levels of type I and type II in different brain regions, with the highest levels of type I in brainstem, cortex, substantia nigra, and caudate, where it is found predominantly on the soma of neurons, and the highest levels of type II in globus pallidus, hippocampus and thalamus, where it is preferentially localized to axons. Developmentally, type I Na(+) channel polypeptide expression in brain increases dramatically during the third postnatal week, peaks at the end of the first postnatal month, and then decreases such that adult levels are approximately 50% of those at peak. Type II Na(+) channel polypeptide expression in brain also undergoes large increases in the third postnatal week, but levels continue to increase such that peak expression levels are maintained in adult animals. Type I Na(+) channels are found associated with the auxiliary beta2 subunit at all ages, whereas free type II Na(+) channels exist during the first two postnatal weeks. Thus, although expression of these two Na(+) channel alpha subunits in heterologous systems yields currents with very similar electrophysiological and pharmacological properties, their distinct spatial and temporal patterning, and association with auxiliary subunits in brain, suggest that they perform distinct, nonoverlapping functions in situ.

The differential expression and association of cytoplasmic beta-subunits with pore-forming alpha-subunits may contribute significantly to the complexity and heterogeneity of voltage-gated K+ channels in excitable cells. Here we examined the association and colocalization of two mammalian beta-subunits, Kvbeta1 and Kvbeta2, with the K+ channel alpha-subunits Kv1.1, Kv1.2, Kv1.4, Kv1.6, and Kv2.1 in adult rat brain. Reciprocal coimmunoprecipitation experiments using subunit-specific antibodies indicated that Kvbeta1 and Kvbeta2 associate with all the Kv1 alpha-subunits examined, and with each other, but not with Kv2.1. A much larger portion of the total brain pool of Kv1-containing channel complexes was found associated with Kvbeta2 than with Kvbeta1. Single- and multiple-label immunohistochemical staining indicated that Kvbeta1 codistributes extensively with Kv1.1 and Kv1.4 in cortical interneurons, in the hippocampal perforant path and mossy fiber pathways, and in the globus pallidus and substantia nigra. Kvbeta2 codistributes extensively with Kv1.1 and Kv1.2 in all brain regions examined and was strikingly colocalized with these alpha-subunits in the juxtaparanodal region of nodes of Ranvier as well as in the axons and terminals of cerebellar basket cells. Taken together, these data provide a direct demonstration that Kvbeta1 and Kvbeta2 associate and colocalize with Kv1 alpha-subunits in native tissues and provide a biochemical and neuroanatomical basis for the differential contribution of Kv1 alpha- and beta-subunits to electrophysiologically diverse neuronal K+ currents.

Molecular characterization of mammalian voltage-sensitive K+ channel genes and their expression became possible with the cloning of the Shaker locus of Drosophila. However, analysis of the expression patterns and subunit composition of native K+ channel protein complexes requires immunological probes specific for the individual K+ channel gene products expressed in excitable tissue. Here, we describe the generation and characterization of monoclonal antibodies (mAbs) against eight distinct mammalian K+ channel polypeptides; the Kv1.1, Kv1.2, Kv1.4, Kv1.5 and Kv1.6 Shaker-related α-subunits, the Kv2.1 Shab-related α-subunit, and the kvβ1 and Kvβ2 β-subunits. We characterized the subtype-specificity of these mAbs against native K+ channels in mammalian brain and against recombinant K+ channels expressed in transfected mammalian cells. In addition, we used these mAbs to investigate the cellular and subcellular distribution of the corresponding polypeptides in rat cerebral cortex, as well as their expression levels across brain regions. Copyright © 1996 Elsevier Science Ltd

Recent cloning of K+channel β subunits revealed that these cytoplasmic polypeptides can dramatically alter the kinetics of current inactivation and promote efficient glycosylation and surface expression of the channel-forming α subunits. Here, we examined the expression, distribution, and association of two of these β subunits, Kvβ1 and Kvβ2, in adult rat brain.In situhybridization using cRNA probes revealed that these β-subunit genes are heterogeneously expressed, with high densities of Kvβ1 mRNA in the striatum, CA1 subfield of the hippocampus, and cerebellar Purkinje cells, and high densities of Kvβ2 mRNA in the cerebral cortex, cerebellum, and brainstem. Immunohistochemical staining using subunit-specific monoclonal and affinity-purified polyclonal antibodies revealed that the Kvβ1 and Kvβ2 polypeptides frequently co-localize and are concentrated in neuronal perikarya, dendrites, and terminal fields, and in the juxtaparanodal region of myelinated axons. Immunoblot and reciprocal co-immunoprecipitation analyses indicated that Kvβ2 is the major β subunit present in rat brain membranes, and that most K+channel complexes containing Kvβ1 also contain Kvβ2. Taken together, these data suggest that Kvβ2 is a component of almost all K+channel complexes containing Kv1 α subunits, and that individual channels may contain two or more biochemically and functionally distinct β-subunit polypeptides.