title: Splitting the two pore domains from TOK1 results in two cationic channels with novel functional properties.
abstract: Potassium channels are membrane-spanning proteins with several transmembrane segments and a single pore region where ion conduction takes place (Biggin, P. C., Roosild, T., and Choe, S. (2000) Curr. Opin. Struct. Biol. 4, 456-461; Doyle, D. A., Morais Cabral, J., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T., and MacKinnon, R. (1998) Science 280, 69-77). TOK1, a potassium channel identified in the yeast Saccharomyces cerevisiae, was the first described member from a growing new family of potassium channels with two pore domains in tandem (2P) (Ketchum, K. A., Joiner, W. J., Sellers, A. J., Kaczmarek, L. K., and Goldstein, S. A. (1995) Nature 376, 690-695). In an attempt to understand the relative contribution of each one of the 2P from TOK1 to the functional properties of this channel, we split and expressed the pore domains separately or in combination. Expression of the two domains separately rescued a potassium transport-deficient yeast mutant, suggesting that each domain forms functional potassium-permeable channels in yeast. In Xenopus laevis oocytes expression of each pore domain resulted in the appearance of unique inwardly rectifying cationic channels with novel gating and pharmacological properties. Both pore domains were poorly selective to potassium; however, upon co-expression they partially restored TOK1 channel selectivity. The single channel conductance was different in both pore domains with 7 +/- 1 (n = 12) and 15 +/- 2 (n = 12) picosiemens for the first and second domain, respectively. In light of the known structure of the Streptomyces lividans KcsA potassium channel pore (see Doyle et al. above), these results suggest a novel non-four-fold-symmetric architecture for 2P potassium-selective channels.
authors.affiliation: Departamento de Genética Molecular, Biofisica y Biologia Celular, Instituto de Fisiologia Celular, Universidad Nacional Autónoma de México, Ciudad Universitaria, México D.F. 04510, México.
authors.forename: Carlos
authors.initials: C
authors.lastname: Saldaña
authors.forename: David
authors.initials: D
authors.lastname: Naranjo
authors.forename: Roberto
authors.initials: R
authors.lastname: Coria
authors.forename: Antonio
authors.initials: A
authors.lastname: Peña
authors.forename: Luis
authors.initials: L
authors.lastname: Vaca
mesh_terms: D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D002412:Cations; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004594:Electrophysiology; D005816:Genetic Complementation Test; D006801:Humans; D007473:Ion Channels / Q000737:chemistry*; D007700:Kinetics; D008954:Models, Biological; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D018075:RNA, Complementary / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013997:Time Factors; D014162:Transfection; D014981:Xenopus
chemical_list: D002412:Cations; D018076:DNA, Complementary; D007473:Ion Channels; D015221:Potassium Channels; D020033:Protein Isoforms; D018075:RNA, Complementary; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; C095220:TOK1 protein, S cerevisiae