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.
Highlights
Potassium channels play key roles in the physiology of prokaryotic and eukaryotic organisms
Dividing the Two Pore Domains from TOK1 Produces Novel Cationic Channels—TOK1 possesses eight transmembrane segments with two pores in tandem. This structure resembles a six transmembrane domain Shaker-like channel attached to an inward rectifier-like channel (Fig. 1A), dividing the two pore-forming domains from this channel would result in two structures that resemble single pore potassium channels
We have divided a 2P potassium channel with eight transmembrane domains (TM) to produce two constructs that resemble single pore potassium channels, one with two TM resembling a Kir-like inward rectifier potassium channel and the other with six TM resembling an outward rectifier of the Kv family of potassium channels
Summary
Potassium channels play key roles in the physiology of prokaryotic and eukaryotic organisms. A new family of potassium channels characterized by the presence of two pore-forming domains in tandem (2P) has been identified [3, 7,8,9]. The arrangement of the putative TM and the 2P in TOK1 results in a structure that resembles a six TM Shaker-like channel attached to an inward rectifier-like channel This potassium channel may be important for the maintenance of membrane voltage in yeast, which is essential for nutrient uptake and turgor regulation [10]. Despite over 60 genes identified so far encoding 2P potassium channels, very little is known about the functional significance of having two pore-forming domains in tandem. In an attempt to determine the role of each one of the two poreforming domains in this new family of potassium channels, we divided TOK1 at the intracellular linker between the sixth and seventh TM to produce two structures that resemble single pore domain channels, one with 6TM and the other with 2TM, each one with its respective pore-forming domain
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