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• W1964758210 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. InXenopus 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. 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. InXenopus 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. Potassium channels play key roles in the physiology of prokaryotic and eukaryotic organisms. In many cells, potassium channels are responsible for maintaining the resting membrane potential and for the modulation of firing properties in excitable cells (4Hille B. Ionic Channels of Excitable Membranes. 2nd Ed. Sinauer Associates Inc., Sunderland, MA1992: 115-139Google Scholar). The exceptionally diverse functional properties of potassium channels are matched by a large number of genes identified over the last few years (5Coetzee W.A. Amarillo Y. Chiu J. Chow A. Lau D. McCormack T. Moreno H. Nadal M.S. Ozaita A. Pountney D. Saganich M. Vega-Saenz de Miera E. Rudy B. Ann. N. Y. Acad. Sci. 1999; 868: 233-285Crossref PubMed Scopus (977) Google Scholar).Genome sequencing and molecular cloning has allowed the identification of a significant number of potassium channels possessing two and six transmembrane domains (TM) 1The abbreviations used are:TMtransmembrane domain(s)2Ptwo pore domains in tandem1Pone pore domainP1first poreP2second poreHOMOPIPEShomopiperazine-N,N′-bis-2-(ethanesulfonic acid)MES4-morpholineethanesulfonic acidCHOChinese hamster ovaryHEKhuman embryonic kidneypFpicofarads 1The abbreviations used are:TMtransmembrane domain(s)2Ptwo pore domains in tandem1Pone pore domainP1first poreP2second poreHOMOPIPEShomopiperazine-N,N′-bis-2-(ethanesulfonic acid)MES4-morpholineethanesulfonic acidCHOChinese hamster ovaryHEKhuman embryonic kidneypFpicofarads (5Coetzee W.A. Amarillo Y. Chiu J. Chow A. Lau D. McCormack T. Moreno H. Nadal M.S. Ozaita A. Pountney D. Saganich M. Vega-Saenz de Miera E. Rudy B. Ann. N. Y. Acad. Sci. 1999; 868: 233-285Crossref PubMed Scopus (977) Google Scholar). Despite the structural diversity, all these channels have a common feature consisting of a single pore-forming domain, which is essential for ion conduction and selectivity (1Biggin P.C. Roosild T. Choe S. Curr. Opin. Struct. Biol. 2000; 4: 456-461Crossref Scopus (46) Google Scholar, 2Doyle D.A. Morais Cabral J. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5684) Google Scholar). It is generally accepted that all these potassium channels aggregate as tetramers to form a functional channel, leaving the pore at the axis of a four-fold symmetry (1Biggin P.C. Roosild T. Choe S. Curr. Opin. Struct. Biol. 2000; 4: 456-461Crossref Scopus (46) Google Scholar, 6MacKinnon R. Nature. 1991; 350: 232-235Crossref PubMed Scopus (761) Google Scholar).Recently a new family of potassium channels characterized by the presence of two pore-forming domains in tandem (2P) has been identified (3Ketchum K.A. Joiner W.J. Sellers A.J. Kaczmarek L.K. Goldstein S.A. Nature. 1995; 376: 690-695Crossref PubMed Scopus (362) Google Scholar, 7Goldstein S.A. Bockenhauer D. O'Kelly I. Zilberberg N. Nat. Rev. Neurosci. 2001; 3: 175-184Crossref Scopus (555) Google Scholar, 8Lesage F. Lazdunski M. Am. J. Physiol. Renal Physiol. 2000; 279: F793-F801Crossref PubMed Google Scholar, 9Wang Z.W. Kunkel M.T. Wei A. Butler A. Salkoff L. Ann. N. Y. Acad. Sci. 1999; 868: 286-303Crossref PubMed Scopus (33) Google Scholar). These new potassium channels have either four (2P/4TM) or eight (2P/8TM) transmembrane segments and are highly conserved throughout evolution (7Goldstein S.A. Bockenhauer D. O'Kelly I. Zilberberg N. Nat. Rev. Neurosci. 2001; 3: 175-184Crossref Scopus (555) Google Scholar, 11Garcia E. Scanlon M. Naranjo D. J. Gen. Physiol. 1999; 4: 141-157Crossref Scopus (24) Google Scholar, 12Ko C.H. Gaber R.F. Mol. Cell. Biol. 1991; 8: 4266-4273Crossref Scopus (234) Google Scholar). Recent experimental evidence suggests that this family of potassium channels probably dimerizes to form functional channels (8Lesage F. Lazdunski M. Am. J. Physiol. Renal Physiol. 2000; 279: F793-F801Crossref PubMed Google Scholar). In general, the sequences of both selectivity filters of 2P channels are different, thus they are expected to make potassium-selective pores without the regular four-fold symmetry, but the functional significance of having two different pore-forming domains remains largely unknown.TOK1 was the first member identified from the 2P family of potassium channels (3Ketchum K.A. Joiner W.J. Sellers A.J. Kaczmarek L.K. Goldstein S.A. Nature. 1995; 376: 690-695Crossref PubMed Scopus (362) Google Scholar). This is the only member from this family possessing eight TM (2P/8TM). 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 (10Bertl A. Bihler H. Reid J.D. Kettner C. Slayman C.L. J. Membr. Biol. 1998; 162: 67-80Crossref PubMed Scopus (48) Google Scholar).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 pore-forming 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.The results presented here indicate that each construct can form functional potassium channels in yeast and Xenopus laevisoocytes showing poor ionic selectivity and novel gating and pharmacological properties. Co-expression of both constructs partially restored wild type TOK1 channel properties. Given the proposed pore structure for 1P potassium channels based on the crystal structure of the KcsA potassium channel (2Doyle D.A. Morais Cabral J. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5684) Google Scholar), these results suggest a novel non-four-fold-symmetric architecture to attain potassium-selective pores.DISCUSSIONWe have divided a 2P potassium channel with eight 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.Expression of the two domains separately overcame the potassium auxotrophy in the potassium transport-deficient yeast double mutant Δtrk1,Δtrk2. Furthermore, experiments measuring potassium uptake with a potassium-selective electrode showed a clear phenotype in the Δtrk1,Δtrk2 double mutant, consisting in a reduced potassium uptake compared with that in wild type yeast. This phenotype was rescued by transforming the double mutant with plasmids containing wild type TOK1, TOK1A, or TOK1B separately.Electrophysiology experiments performed with X. laevisoocytes injected with TOK1A and TOK1B mRNA showed the appearance of inwardly rectifying, hyperpolarization-activated cationic currents not present in oocytes injected with mRNA from the human type II bradykinin receptor or in water-injected oocytes. Similar cationic currents were also observed in CHO and HEK293 cells transiently transfected with a plasmid containing the cDNA from TOK1B.Although it is possible that TOK1A and TOK1B (which share limited amino acid homology and are structurally very different) may be activators of cationic channels in yeast, oocytes, rodent, and human cells, we believe this hypothesis is less likely than that in which both constructs (which have the general features found in 1P potassium channels) may indeed produce functional cationic channels.Intersubunit interactions of the 2P from TOK1 may account for the differences in selectivity and single channel conductance observed between the wild type channel and both constructs (TOK1A and TOK1B). In this regard, it is worth mentioning that even when the amount of mRNA injected in the TOK1AB experiments was the same as that injected for TOK1A or TOK1B separately the amplitude of current produced by the co-injection was one-third of that obtained with the individual injection of the constructs.This observation is particularly important for two reasons. 1) If TOK1A and TOK1B are activators of endogenous channels, one would expect equivalent current amplitudes in the co-injection experiments and not less current as we have observed. This observation further supports the hypothesis that TOK1A and TOK1B may produce functional cationic channels; and 2) these observations suggest that some tetrameric combinations might result in nonfunctional or nonconducting channels. Homotetramers of TOK1A or TOK1B and the heterotetramer TOK1AB result in functional channels; however, it is possible that heterotetramerization of three TOK1A plus one TOK1B or vice versa may result in nonfunctional channels. Ongoing experiments with TOK1A and TOK1B constructs cloned in tandem may help to elucidate this point.If TOK1A and TOK1B are indeed producing cationic channels with similar properties, these results are particularly striking given the structural differences in both constructs (2TM versus 6TM), which may suggest that the pore domain (the only conserved sequence between them) might be the structure responsible for these functional properties. Considering that the individual expression of TOK1A and TOK1B results in channels with poor selectivity for potassium ions whereas TOK1AB and wild type TOK1 produced potassium-selective channels, one might speculate that the spatial rearrangement of the two pores from both constructs to form wild type TOK1 may provide a non-four-fold-symmetric potassium-selective architecture. This architecture contrasts with the proposed four-fold-symmetric architecture for 1P potassium-selective channels based on the crystal structure of the Streptomyces lividans KcsA potassium channel (2Doyle D.A. Morais Cabral J. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5684) Google Scholar).In this regard, recent experimental evidence obtained with tandem constructs (dimers) of the 1P potassium channel Drk1 suggests that mutations that alter the symmetry of the pore domain result in changes in selectivity, gating, and single channel conductance in the mutant dimers (27Chapman M.L. Krovetz H.S. VanDongen A.M. J. Physiol. 2001; 530: 21-33Crossref PubMed Scopus (38) Google Scholar).TOK1 is an outwardly rectifying potassium channel. Single channel openings are rarely observed in the hyperpolarized direction (3Ketchum K.A. Joiner W.J. Sellers A.J. Kaczmarek L.K. Goldstein S.A. Nature. 1995; 376: 690-695Crossref PubMed Scopus (362) Google Scholar). These properties are the opposite of what we have observed with the expression of TOK1A and TOK1B. The strong inward rectification observed in TOK1A and TOK1B may result from the voltage dependence properties of both channels since single channels open scarcely at voltages approaching the reversal potential and no discernible opening events were observed in the outward direction.In the family of inward rectifier potassium (Kir) channels, the unifying hypothesis to explain inward rectification proposes that the blockade by magnesium and/or polyamines produced by the depolarization is sufficient to account for the inward rectification in many members from this family (28Nichols C.G. Lopatin A.N. Annu. Rev. Physiol. 1997; 59: 171-191Crossref PubMed Scopus (657) Google Scholar). One cannot discard at the present time a similar mechanism involved in the strong rectification observed with TOK1A and TOK1B.Alternatively, the voltage dependence and gating differences observed between wild type TOK1 and the constructs (TOK1A and TOK1B) might be the result of a different spatial arrangement of the pore architecture. We do not have a definitive explanation at the present time to account for these differences.The structure of TOK1 resembles a six transmembrane domain Shaker-like channel attached to an inward rectifier-like channel, therefore the finding that separating the two pore-forming domains from TOK1 results in the appearance of potassium-permeable channels with novel selectivity, single channel conductance, and gating properties is very provocative.If indeed TOK1A and TOK1B produce functional channels (as suggested by the evidence in yeast, oocytes, and mammalian cells presented here) then these new channels may provide an excellent experimental tool to explore structural determinants of ionic selectivity, gating, and voltage dependence in 2P potassium channels and may help to understand the functional role of the sequence variations in the 2P found in this growing family of channels. Future experiments splitting the two pores from other 2P potassium channels may provide additional information about the non-four-fold-symmetric architecture of 2P potassium-selective channels. Potassium channels play key roles in the physiology of prokaryotic and eukaryotic organisms. In many cells, potassium channels are responsible for maintaining the resting membrane potential and for the modulation of firing properties in excitable cells (4Hille B. Ionic Channels of Excitable Membranes. 2nd Ed. Sinauer Associates Inc., Sunderland, MA1992: 115-139Google Scholar). The exceptionally diverse functional properties of potassium channels are matched by a large number of genes identified over the last few years (5Coetzee W.A. Amarillo Y. Chiu J. Chow A. Lau D. McCormack T. Moreno H. Nadal M.S. Ozaita A. Pountney D. Saganich M. Vega-Saenz de Miera E. Rudy B. Ann. N. Y. Acad. Sci. 1999; 868: 233-285Crossref PubMed Scopus (977) Google Scholar). Genome sequencing and molecular cloning has allowed the identification of a significant number of potassium channels possessing two and six transmembrane domains (TM) 1The abbreviations used are:TMtransmembrane domain(s)2Ptwo pore domains in tandem1Pone pore domainP1first poreP2second poreHOMOPIPEShomopiperazine-N,N′-bis-2-(ethanesulfonic acid)MES4-morpholineethanesulfonic acidCHOChinese hamster ovaryHEKhuman embryonic kidneypFpicofarads 1The abbreviations used are:TMtransmembrane domain(s)2Ptwo pore domains in tandem1Pone pore domainP1first poreP2second poreHOMOPIPEShomopiperazine-N,N′-bis-2-(ethanesulfonic acid)MES4-morpholineethanesulfonic acidCHOChinese hamster ovaryHEKhuman embryonic kidneypFpicofarads (5Coetzee W.A. Amarillo Y. Chiu J. Chow A. Lau D. McCormack T. Moreno H. Nadal M.S. Ozaita A. Pountney D. Saganich M. Vega-Saenz de Miera E. Rudy B. Ann. N. Y. Acad. Sci. 1999; 868: 233-285Crossref PubMed Scopus (977) Google Scholar). Despite the structural diversity, all these channels have a common feature consisting of a single pore-forming domain, which is essential for ion conduction and selectivity (1Biggin P.C. Roosild T. Choe S. Curr. Opin. Struct. Biol. 2000; 4: 456-461Crossref Scopus (46) Google Scholar, 2Doyle D.A. Morais Cabral J. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5684) Google Scholar). It is generally accepted that all these potassium channels aggregate as tetramers to form a functional channel, leaving the pore at the axis of a four-fold symmetry (1Biggin P.C. Roosild T. Choe S. Curr. Opin. Struct. Biol. 2000; 4: 456-461Crossref Scopus (46) Google Scholar, 6MacKinnon R. Nature. 1991; 350: 232-235Crossref PubMed Scopus (761) Google Scholar). transmembrane domain(s) two pore domains in tandem one pore domain first pore second pore homopiperazine-N,N′-bis-2-(ethanesulfonic acid) 4-morpholineethanesulfonic acid Chinese hamster ovary human embryonic kidney picofarads transmembrane domain(s) two pore domains in tandem one pore domain first pore second pore homopiperazine-N,N′-bis-2-(ethanesulfonic acid) 4-morpholineethanesulfonic acid Chinese hamster ovary human embryonic kidney picofarads Recently a new family of potassium channels characterized by the presence of two pore-forming domains in tandem (2P) has been identified (3Ketchum K.A. Joiner W.J. Sellers A.J. Kaczmarek L.K. Goldstein S.A. Nature. 1995; 376: 690-695Crossref PubMed Scopus (362) Google Scholar, 7Goldstein S.A. Bockenhauer D. O'Kelly I. Zilberberg N. Nat. Rev. Neurosci. 2001; 3: 175-184Crossref Scopus (555) Google Scholar, 8Lesage F. Lazdunski M. Am. J. Physiol. Renal Physiol. 2000; 279: F793-F801Crossref PubMed Google Scholar, 9Wang Z.W. Kunkel M.T. Wei A. Butler A. Salkoff L. Ann. N. Y. Acad. Sci. 1999; 868: 286-303Crossref PubMed Scopus (33) Google Scholar). These new potassium channels have either four (2P/4TM) or eight (2P/8TM) transmembrane segments and are highly conserved throughout evolution (7Goldstein S.A. Bockenhauer D. O'Kelly I. Zilberberg N. Nat. Rev. Neurosci. 2001; 3: 175-184Crossref Scopus (555) Google Scholar, 11Garcia E. Scanlon M. Naranjo D. J. Gen. Physiol. 1999; 4: 141-157Crossref Scopus (24) Google Scholar, 12Ko C.H. Gaber R.F. Mol. Cell. Biol. 1991; 8: 4266-4273Crossref Scopus (234) Google Scholar). Recent experimental evidence suggests that this family of potassium channels probably dimerizes to form functional channels (8Lesage F. Lazdunski M. Am. J. Physiol. Renal Physiol. 2000; 279: F793-F801Crossref PubMed Google Scholar). In general, the sequences of both selectivity filters of 2P channels are different, thus they are expected to make potassium-selective pores without the regular four-fold symmetry, but the functional significance of having two different pore-forming domains remains largely unknown. TOK1 was the first member identified from the 2P family of potassium channels (3Ketchum K.A. Joiner W.J. Sellers A.J. Kaczmarek L.K. Goldstein S.A. Nature. 1995; 376: 690-695Crossref PubMed Scopus (362) Google Scholar). This is the only member from this family possessing eight TM (2P/8TM). 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 (10Bertl A. Bihler H. Reid J.D. Kettner C. Slayman C.L. J. Membr. Biol. 1998; 162: 67-80Crossref PubMed Scopus (48) Google Scholar). 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 pore-forming 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. The results presented here indicate that each construct can form functional potassium channels in yeast and Xenopus laevisoocytes showing poor ionic selectivity and novel gating and pharmacological properties. Co-expression of both constructs partially restored wild type TOK1 channel properties. Given the proposed pore structure for 1P potassium channels based on the crystal structure of the KcsA potassium channel (2Doyle D.A. Morais Cabral J. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5684) Google Scholar), these results suggest a novel non-four-fold-symmetric architecture to attain potassium-selective pores. DISCUSSIONWe have divided a 2P potassium channel with eight 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.Expression of the two domains separately overcame the potassium auxotrophy in the potassium transport-deficient yeast double mutant Δtrk1,Δtrk2. Furthermore, experiments measuring potassium uptake with a potassium-selective electrode showed a clear phenotype in the Δtrk1,Δtrk2 double mutant, consisting in a reduced potassium uptake compared with that in wild type yeast. This phenotype was rescued by transforming the double mutant with plasmids containing wild type TOK1, TOK1A, or TOK1B separately.Electrophysiology experiments performed with X. laevisoocytes injected with TOK1A and TOK1B mRNA showed the appearance of inwardly rectifying, hyperpolarization-activated cationic currents not present in oocytes injected with mRNA from the human type II bradykinin receptor or in water-injected oocytes. Similar cationic currents were also observed in CHO and HEK293 cells transiently transfected with a plasmid containing the cDNA from TOK1B.Although it is possible that TOK1A and TOK1B (which share limited amino acid homology and are structurally very different) may be activators of cationic channels in yeast, oocytes, rodent, and human cells, we believe this hypothesis is less likely than that in which both constructs (which have the general features found in 1P potassium channels) may indeed produce functional cationic channels.Intersubunit interactions of the 2P from TOK1 may account for the differences in selectivity and single channel conductance observed between the wild type channel and both constructs (TOK1A and TOK1B). In this regard, it is worth mentioning that even when the amount of mRNA injected in the TOK1AB experiments was the same as that injected for TOK1A or TOK1B separately the amplitude of current produced by the co-injection was one-third of that obtained with the individual injection of the constructs.This observation is particularly important for two reasons. 1) If TOK1A and TOK1B are activators of endogenous channels, one would expect equivalent current amplitudes in the co-injection experiments and not less current as we have observed. This observation further supports the hypothesis that TOK1A and TOK1B may produce functional cationic channels; and 2) these observations suggest that some tetrameric combinations might result in nonfunctional or nonconducting channels. Homotetramers of TOK1A or TOK1B and the heterotetramer TOK1AB result in functional channels; however, it is possible that heterotetramerization of three TOK1A plus one TOK1B or vice versa may result in nonfunctional channels. Ongoing experiments with TOK1A and TOK1B constructs cloned in tandem may help to elucidate this point.If TOK1A and TOK1B are indeed producing cationic channels with similar properties, these results are particularly striking given the structural differences in both constructs (2TM versus 6TM), which may suggest that the pore domain (the only conserved sequence between them) might be the structure responsible for these functional properties. Considering that the individual expression of TOK1A and TOK1B results in channels with poor selectivity for potassium ions whereas TOK1AB and wild type TOK1 produced potassium-selective channels, one might speculate that the spatial rearrangement of the two pores from both constructs to form wild type TOK1 may provide a non-four-fold-symmetric potassium-selective architecture. This architecture contrasts with the proposed four-fold-symmetric architecture for 1P potassium-selective channels based on the crystal structure of the Streptomyces lividans KcsA potassium channel (2Doyle D.A. Morais Cabral J. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5684) Google Scholar).In this regard, recent experimental evidence obtained with tandem constructs (dimers) of the 1P potassium channel Drk1 suggests that mutations that alter the symmetry of the pore domain result in changes in selectivity, gating, and single channel conductance in the mutant dimers (27Chapman M.L. Krovetz H.S. VanDongen A.M. J. Physiol. 2001; 530: 21-33Crossref PubMed Scopus (38) Google Scholar).TOK1 is an outwardly rectifying potassium channel. Single channel openings are rarely observed in the hyperpolarized direction (3Ketchum K.A. Joiner W.J. Sellers A.J. Kaczmarek L.K. Goldstein S.A. Nature. 1995; 376: 690-695Crossref PubMed Scopus (362) Google Scholar). These properties are the opposite of what we have observed with the expression of TOK1A and TOK1B. The strong inward rectification observed in TOK1A and TOK1B may result from the voltage dependence properties of both channels since single channels open scarcely at voltages approaching the reversal potential and no discernible opening events were observed in the outward direction.In the family of inward rectifier potassium (Kir) channels, the unifying hypothesis to explain inward rectification proposes that the blockade by magnesium and/or polyamines produced by the depolarization is sufficient to account for the inward rectification in many members from this family (28Nichols C.G. Lopatin A.N. Annu. Rev. Physiol. 1997; 59: 171-191Crossref PubMed Scopus (657) Google Scholar). One cannot discard at the present time a similar mechanism involved in the strong rectification observed with TOK1A and TOK1B.Alternatively, the voltage dependence and gating differences observed between wild type TOK1 and the constructs (TOK1A and TOK1B) might be the result of a different spatial arrangement of the pore architecture. We do not have a definitive explanation at the present time to account for these differences.The structure of TOK1 resembles a six transmembrane domain Shaker-like channel attached to an inward rectifier-like channel, therefore the finding that separating the two pore-forming domains from TOK1 results in the appearance of potassium-permeable channels with novel selectivity, single channel conductance, and gating properties is very provocative.If indeed TOK1A and TOK1B produce functional channels (as suggested by the evidence in yeast, oocytes, and mammalian cells presented here) then these new channels may provide an excellent experimental tool to explore structural determinants of ionic selectivity, gating, and voltage dependence in 2P potassium channels and may help to understand the functional role of the sequence variations in the 2P found in this growing family of channels. Future experiments splitting the two pores from other 2P potassium channels may provide additional information about the non-four-fold-symmetric architecture of 2P potassium-selective channels. We have divided a 2P potassium channel with eight 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. Expression of the two domains separately overcame the potassium auxotrophy in the potassium transport-deficient yeast double mutant Δtrk1,Δtrk2. Furthermore, experiments measuring potassium uptake with a potassium-selective electrode showed a clear phenotype in the Δtrk1,Δtrk2 double mutant, consisting in a reduced potassium uptake compared with that in wild type yeast. This phenotype was rescued by transforming the double mutant with plasmids containing wild type TOK1, TOK1A, or TOK1B separately. Electrophysiology experiments performed with X. laevisoocytes injected with TOK1A and TOK1B mRNA showed the appearance of inwardly rectifying, hyperpolarization-activated cationic currents not present in oocytes injected with mRNA from the human type II bradykinin receptor or in water-injected oocytes. Similar cationic currents were also observed in CHO and HEK293 cells transiently transfected with a plasmid containing the cDNA from TOK1B. Although it is possible that TOK1A and TOK1B (which share limited amino acid homology and are structurally very different) may be activators of cationic channels in yeast, oocytes, rodent, and human cells, we believe this hypothesis is less likely than that in which both constructs (which have the general features found in 1P potassium channels) may indeed produce functional cationic channels. Intersubunit interactions of the 2P from TOK1 may account for the differences in selectivity and single channel conductance observed between the wild type channel and both constructs (TOK1A and TOK1B). In this regard, it is worth mentioning that even when the amount of mRNA injected in the TOK1AB experiments was the same as that injected for TOK1A or TOK1B separately the amplitude of current produced by the co-injection was one-third of that obtained with the individual injection of the constructs. This observation is particularly important for two reasons. 1) If TOK1A and TOK1B are activators of endogenous channels, one would expect equivalent current amplitudes in the co-injection experiments and not less current as we have observed. This observation further supports the hypothesis that TOK1A and TOK1B may produce functional cationic channels; and 2) these observations suggest that some tetrameric combinations might result in nonfunctional or nonconducting channels. Homotetramers of TOK1A or TOK1B and the heterotetramer TOK1AB result in functional channels; however, it is possible that heterotetramerization of three TOK1A plus one TOK1B or vice versa may result in nonfunctional channels. Ongoing experiments with TOK1A and TOK1B constructs cloned in tandem may help to elucidate this point. If TOK1A and TOK1B are indeed producing cationic channels with similar properties, these results are particularly striking given the structural differences in both constructs (2TM versus 6TM), which may suggest that the pore domain (the only conserved sequence between them) might be the structure responsible for these functional properties. Considering that the individual expression of TOK1A and TOK1B results in channels with poor selectivity for potassium ions whereas TOK1AB and wild type TOK1 produced potassium-selective channels, one might speculate that the spatial rearrangement of the two pores from both constructs to form wild type TOK1 may provide a non-four-fold-symmetric potassium-selective architecture. This architecture contrasts with the proposed four-fold-symmetric architecture for 1P potassium-selective channels based on the crystal structure of the Streptomyces lividans KcsA potassium channel (2Doyle D.A. Morais Cabral J. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5684) Google Scholar). In this regard, recent experimental evidence obtained with tandem constructs (dimers) of the 1P potassium channel Drk1 suggests that mutations that alter the symmetry of the pore domain result in changes in selectivity, gating, and single channel conductance in the mutant dimers (27Chapman M.L. Krovetz H.S. VanDongen A.M. J. Physiol. 2001; 530: 21-33Crossref PubMed Scopus (38) Google Scholar). TOK1 is an outwardly rectifying potassium channel. Single channel openings are rarely observed in the hyperpolarized direction (3Ketchum K.A. Joiner W.J. Sellers A.J. Kaczmarek L.K. Goldstein S.A. Nature. 1995; 376: 690-695Crossref PubMed Scopus (362) Google Scholar). These properties are the opposite of what we have observed with the expression of TOK1A and TOK1B. The strong inward rectification observed in TOK1A and TOK1B may result from the voltage dependence properties of both channels since single channels open scarcely at voltages approaching the reversal potential and no discernible opening events were observed in the outward direction. In the family of inward rectifier potassium (Kir) channels, the unifying hypothesis to explain inward rectification proposes that the blockade by magnesium and/or polyamines produced by the depolarization is sufficient to account for the inward rectification in many members from this family (28Nichols C.G. Lopatin A.N. Annu. Rev. Physiol. 1997; 59: 171-191Crossref PubMed Scopus (657) Google Scholar). One cannot discard at the present time a similar mechanism involved in the strong rectification observed with TOK1A and TOK1B. Alternatively, the voltage dependence and gating differences observed between wild type TOK1 and the constructs (TOK1A and TOK1B) might be the result of a different spatial arrangement of the pore architecture. We do not have a definitive explanation at the present time to account for these differences. The structure of TOK1 resembles a six transmembrane domain Shaker-like channel attached to an inward rectifier-like channel, therefore the finding that separating the two pore-forming domains from TOK1 results in the appearance of potassium-permeable channels with novel selectivity, single channel conductance, and gating properties is very provocative. If indeed TOK1A and TOK1B produce functional channels (as suggested by the evidence in yeast, oocytes, and mammalian cells presented here) then these new channels may provide an excellent experimental tool to explore structural determinants of ionic selectivity, gating, and voltage dependence in 2P potassium channels and may help to understand the functional role of the sequence variations in the 2P found in this growing family of channels. Future experiments splitting the two pores from other 2P potassium channels may provide additional information about the non-four-fold-symmetric architecture of 2P potassium-selective channels. pGEMA-TOK1 was a kind donation from Dr. Karen Ketchum (Institute for Genomic Research, Rockville, MD). We thank Velia Cardin, Alicia Sampieri, and Veronica Morales-Tlalpan for initial experiments with TOK1B-transfected mammalian cells. The services from the Molecular Biology unit, library, and computer facility at the Instituto de Fisiologia Celular and the excellent technical assistance from Beatriz Aguirre, Miguel Angel Hernandez, and Gloria Salgado are greatly appreciated." @default.
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