Voltage-gated K+ channels are protein complexes composed of ion-conducting integral membrane alpha subunits and cytoplasmic beta subunits. Here, we show that, in transfected mammalian cells, the predominant beta subunit isoform in brain, Kv beta 2, associates with the Kv1.2 alpha subunit early in channel biosynthesis and that Kv beta 2 exerts multiple chaperone-like effects on associated Kv1.2 including promotion of cotranslational N-linked glycosylation of the nascent Kv1.2 polypeptide, increased stability of Kv beta 2/Kv1.2 complexes, and increased efficiency of cell surface expression of Kv1.2. Taken together, these results indicate that while some cytoplasmic K+ channel beta subunits affect the inactivation kinetics of alpha subunits, a more general, and perhaps more fundamental, role is to mediate the biosynthetic maturation and surface expression of voltage-gated K+ channel complexes. These findings provide a molecular basis for recent genetic studies indicating that beta subunits are key determinants of neuronal excitability.

Recent cloning of K ϩ channel ␤ 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 ␣ subunits. Here, we examined the expression, distribution, and association of two of these ␤ subunits, Kv␤1 and Kv␤2, in adult rat brain. In situ hybridization using cRNA probes revealed that these ␤-subunit genes are heterogeneously expressed, with high densities of Kv␤1 mRNA in the striatum, CA1 subfield of the hippocampus, and cerebellar Purkinje cells, and high densities of Kv␤2 mRNA in the cerebral cortex, cerebellum, and brainstem. Immunohistochemical staining using subunit-specific monoclonal and affinity-purified polyclonal antibodies revealed that the Kv␤1 and Kv␤2 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 Kv␤2 is the major ␤ subunit present in rat brain membranes, and that most K ϩ channel complexes containing Kv␤1 also contain Kv␤2. Taken together, these data suggest that Kv␤2 is a component of almost all K ϩ channel complexes containing Kv1 ␣ subunits, and that individual channels may contain two or more biochemically and functionally distinct ␤-subunit polypeptides.

During neuronal differentiation and maturation, electrical excitability is essential for proper gene expression and the formation of synapses. The expression of ion channels is crucial for this process; in particular, voltage-gated K(+) channels function as the key determinants of membrane excitability. Previously, we reported that the A-type K(+) current (I(A)) and Kv4.2 K(+) channel subunit expression increased in cultured cerebellar granule cells with time. To examine the correlation between ion currents and the action potential, in the present study, we measured developmental changes of action potentials in cultured granule cells using the whole-cell patch-clamp method. In addition to an observed increment of I(A), we found that the Na(+) current also increased during development. The increase in both currents was accompanied by a change in the membrane excitability from the nonspiking type to the repetitive firing type. Next, to elucidate whether Kv4.2 is responsible for the I(A) and to assess the effect of Kv4 subunits on action potential waveform, we transfected a cDNA encoding a dominant-negative mutant Kv4.2 (Kv4.2dn) into cultured cells. Expression of Kv4.2dn resulted in the elimination of I(A) in the granule cells. This result demonstrates that members of the Kv4 subfamily are responsible for the I(A) in developing granule cells. Moreover, elimination of I(A) resulted in shortening of latency before the first spike generation. In contrast, expression of wild-type Kv4.2 resulted in a delay in latency. This indicates that appearance of I(A) is critically required for suppression of the excitability of granule cells during their maturation.