VOLTAGE-GATED K+ CHANNELS CONTAIN MULTIPLE INTERSUBUNIT ASSOCIATION SITES

A domain in the cytoplasmic NH2 terminus of voltage-gated K+ channels supervises the proper assembly of specific tetrameric channels (Li, M. , Jan, J. M., and Jan, L. Y. (1992) Science 257, 1225-1230; Shen, N. V ., Chen X., Boyer, M. RI., and Pfaffinger, P. (1993) Neuron 11, 67-76) . It is referred to as a first tetramerization domain, or TI (Shen, N. V., Chen X., Boyer, RI. M., and Pfaffinger, P. (1993) Neuron 11, 67-7 6). However, a deletion mutant of Kv1.3 that lacks the first 141 amino acids, Kv1.3 (T1(-)) forms functional channels, suggesting that addit ional association sites in the central core of Kv1.3 mediate oligomeri zation. To characterize these sites, we have tested the abilities of c RNA Kv1.3 (T1(-)) fragments coinjected with Kv1.3 (T1(-)) to suppress current in Xenopus oocytes. The fragments include portions of the six putative transmembrane segments, S1 through S6, specifically: S1, S1-S 2, S1-S2-S3, S2-S3, S2-S3-S4, S3-S4, S3-S4-S5, S2 through COOH, S3 thr ough COOH, S4 through COOH, and S5-S6-COOH. Electrophysiologic experim ents show that the fragments S1-S2-S3, S3-S4-S5, S2 through COOH, and S3 through COOH strongly suppress Kv1.3 (T1(-)) current, while others do not. Suppression of expressed current is due to specific effects of the translated peptide Kv1.3 fragments, as validated by in vivo immun oprecipitation studies of a strong suppressor and a nonsuppressor. Pul se-chase experiments indicate that translation of truncated peptide fr agments neither prevents translation of Kv1.3 (T1(-)) nor increases it s rate of degradation. Co-immunoprecipitation experiments suggest that suppression involves direct association of a peptide fragment with Kv 1.3 (T1(-)). Fragments that strongly suppress Kv1.3 (T1(-)) also suppr ess an analogous NH2-terminal deletion mutant of Kv2.1 (Kv2.1 (Delta N 139)), an isoform belonging to a different subfamily. Our results indi cate that sites in the central core of Kv1.3 facilitate intersubunit a ssociation and that there are suppression sites in the central core, w hich are promiscuous across voltage-gated K+ channel subfamilies.