Voltage-gated potassium (Kv) channels sculpt neuronal excitability and play important developmental roles. Kv channels consist of pore-forming alpha- and auxiliary subunits. For many Kv alpha-subunits, existing mRNA probes and antibodies have allowed analysis of expression patterns, typically during adult stages. Here, we focus on the Kv2.2 alpha-subunit, for which the mRNA shows broad expression in the embryo and adult. A lack of suitable antibodies, however, has hindered detailed analysis of Kv2.2 protein localization, especially during development. We developed an antibody that specifically recognizes Kv2.2 protein in Xenopus laevis, a vertebrate well suited for study of early developmental stages. The Kv2.2 antibody recognized heterologously expressed Kv2.2 but not the closely related Kv2.1 protein. Immunodetection of the protein showed its presence at St 32 in ventrolateral regions of the hindbrain and spinal cord. At later stages, several sensory tissues (retina, otic, and olfactory epithelia) also expressed Kv2.2 protein. As development progressed in the central nervous system, Kv2.2 protein distribution expanded in close association with the cytoskeletal marker alpha-tubulin, consistent with growth of neuronal tracts. We analyzed the subcellular distribution of Kv2.2 protein within single cultured neurons. In addition to a surface membrane presence, Kv2.2 protein also resided intracellularly closely associated with alpha-tubulin, as in vivo. Furthermore, in contrast to Kv2.1, Kv2.2 protein localized to long, axonal-like processes, consistent with its in vivo location in tracts. Despite their primary sequence similarity, the contrasting localizations of Kv2.1 and Kv2.2 support different roles for the two during development and neuronal signaling.

Kv1 potassium channels consist of pore-forming alpha subunits as well as auxiliary beta subunits. In heterologous systems, Kv1alpha subunits suffice for induction of voltage-dependent potassium current (I(Kv)). Although Kv1 channels can be expressed without auxiliary subunits in heterologous systems, coexpression with Kvbeta subunits has dramatic effects on surface expression and kinetic properties. Much less is known about the functional roles of Kvbeta subunits in vivo, despite their presence in the majority of native Kv1 channel complexes. We used an antisense approach to probe the contribution of Kvbeta2 subunits to native Kv1 channel function in embryonic myocytes. We compared the effects of antisense Kvbeta2 treatment on the whole cell I(Kv) to those produced by overexpression of a dominant-negative Kv1alpha subunit. The reductions in the maximal potassium conductance produced by antisense Kvbeta2 treatment and elimination of Kv1alpha subunit function were not significantly different from each other. In addition, simultaneous elimination of Kv1alpha and Kvbeta2 subunit function resulted in no further reduction of the maximal conductance. The Kv channel complexes targeted by Kvbeta2 and/or Kv1alpha subunit elimination contributed to action potential repolarization because elimination of either or both subunits led to increases in the duration of the action potential. As for potassium conductance, the effects of elimination of both alpha and beta subunits on the duration of the action potential were not additive. Taken together, the results suggest that Kv1 potassium channel complexes in vivo have a strong requirement for both alpha and beta subunits.

Whereas Kvbeta2 subunits modulate potassium current properties carried by Kv1 channel complexes in heterologous systems, little is known about the contributions of Kvbeta2 subunits to native potassium channel function. Using antisense approaches and in situ recordings from Xenopus embryo spinal cord neurons, we tested the in vivo roles of Kvbeta2 subunits in modulation of voltage-dependent potassium current (IKv). We focused on 1) two different populations of dorsal spinal neurons that express both Kvbeta2 and Kv1 alpha-subunit genes and 2) the 24- and 48-h developmental period, during which IKv undergoes developmental regulation. At both 24 and 48 h, antisense methods produced efficient knock-down of both Kvbeta2 protein and IKv. At both times, dominant negative suppression of Kv1 channels also eliminated IKv, indicating that Kv1 channels require Kvbeta2 subunits to function in dorsal spinal neurons. Even though Kv1 channels determined the IKv values of both dorsal neuron types, comparisons of their IKv properties revealed important differences at both developmental stages. The latter results support the notion that different Kv1 alpha-subunits and/or posttranslational modifications underlie the IKv values of the two dorsal neuron types. Overall, the results demonstrate that Kvbeta2 subunits function in vivo as obligatory subunits of Kv1 channels in at least two neuron types and two different developmental stages.