title: Voltage-gated K+ channels contain multiple intersubunit association sites.
abstract: 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. M., and Pfaffinger, P. (1993) Neuron 11, 67-76). It is referred to as a first tetramerization domain, or T1 (Shen, N. V., Chen X., Boyer, M. M., and Pfaffinger, P.(1993) Neuron 11, 67-76). However, a deletion mutant of Kv1.3 that lacks the first 141 amino acids, Kv1.3 (T1(-)) forms functional channels, suggesting that additional association sites in the central core of Kv1.3 mediate oligomerization. To characterize these sites, we have tested the abilities of cRNA Kv1.3 (T1(-)) fragments co-injected 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-S2, S1-S2-S3, S2-S3, S2-S3-S4, S3-S4, S3-S4-S5, S2 through COOH, S3 through COOH, S4 through COOH, and S5-S6-COOH. Electrophysiologic experiments 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 immunoprecipitation studies of a strong suppressor and a nonsuppressor. Pulse-chase experiments indicate that translation of truncated peptide fragments neither prevents translation of Kv1.3 (T1(-)) nor increases its rate of degradation. Co-immunoprecipitation experiments suggest that suppression involves direct association of a peptide fragment with Kv1.3 (T1(-)). Fragments that strongly suppress Kv1.3 (T1(-)) also suppress an analogous NH2-terminal deletion mutant of Kv2.1 (Kv2.1 (DeltaN139)), an isoform belonging to a different subfamily. Our results indicate that sites in the central core of Kv1.3 facilitate intersubunit association and that there are suppression sites in the central core, which are promiscuous across voltage-gated K+ channel subfamilies.
authors.affiliation: Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6085, USA.
authors.forename: L
authors.initials: L
authors.lastname: Tu
authors.forename: V
authors.initials: V
authors.lastname: Santarelli
authors.forename: Z
authors.initials: Z
authors.lastname: Sheng
authors.forename: W
authors.initials: W
authors.lastname: Skach
authors.forename: D
authors.initials: D
authors.lastname: Pain
authors.forename: C
authors.initials: C
authors.lastname: Deutsch
mesh_terms: D000818:Animals; D001665:Binding Sites; D005260:Female; D066298:In Vitro Techniques; D015640:Ion Channel Gating; D051664:Kv1.3 Potassium Channel; D015394:Molecular Structure; D009865:Oocytes / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated; D014176:Protein Biosynthesis; D018075:RNA, Complementary / Q000235:genetics; D017384:Sequence Deletion; D014982:Xenopus laevis
chemical_list: D051664:Kv1.3 Potassium Channel; D010446:Peptide Fragments; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D018075:RNA, Complementary