A Superfamily of Voltage-gated Sodium Channels in Bacteria
2004; Elsevier BV; Volume: 279; Issue: 10 Linguagem: Inglês
10.1074/jbc.m313100200
ISSN1083-351X
AutoresRyuta Koishi, Haoxing Xu, Dejian Ren, Betsy Navarro, Benjamin W. Spiller, Qing Shi, David E. Clapham,
Tópico(s)Plant and Biological Electrophysiology Studies
ResumoNaChBac, a six-α-helical transmembrane-spanning protein cloned from Bacillus halodurans, is the first functionally characterized bacterial voltage-gated Na+-selective channel (Ren, D., Navarro, B., Xu, H., Yue, L., Shi, Q., and Clapham, D. E. (2001) Science 294, 2372-2375). As a highly expressing ion channel protein, NaChBac is an ideal candidate for high resolution structural determination and structure-function studies. The biological role of NaChBac, however, is still unknown. In this report, another 11 structurally related bacterial proteins are described. Two of these functionally expressed as voltage-dependent Na+ channels (NaVPZ from Paracoccus zeaxanthinifaciens and NaVSP from Silicibacter pomeroyi). NaVPZ and NaVSP share ∼40% amino acid sequence identity with NaChBac. When expressed in mammalian cell lines, both NaVPZ and NaVSP were Na+-selective and voltage-dependent. However, their kinetics and voltage dependence differ significantly. These single six-α-helical transmembrane-spanning subunits constitute a widely distributed superfamily (NaVBac) of channels in bacteria, implying a fundamental prokaryotic function. The degree of sequence homology (22-54%) is optimal for future comparisons of NaVBac structure and function of similarity and dissimilarity among NaVBac proteins. Thus, the NaVBac superfamily is fertile ground for crystallographic, electrophysiological, and microbiological studies. NaChBac, a six-α-helical transmembrane-spanning protein cloned from Bacillus halodurans, is the first functionally characterized bacterial voltage-gated Na+-selective channel (Ren, D., Navarro, B., Xu, H., Yue, L., Shi, Q., and Clapham, D. E. (2001) Science 294, 2372-2375). As a highly expressing ion channel protein, NaChBac is an ideal candidate for high resolution structural determination and structure-function studies. The biological role of NaChBac, however, is still unknown. In this report, another 11 structurally related bacterial proteins are described. Two of these functionally expressed as voltage-dependent Na+ channels (NaVPZ from Paracoccus zeaxanthinifaciens and NaVSP from Silicibacter pomeroyi). NaVPZ and NaVSP share ∼40% amino acid sequence identity with NaChBac. When expressed in mammalian cell lines, both NaVPZ and NaVSP were Na+-selective and voltage-dependent. However, their kinetics and voltage dependence differ significantly. These single six-α-helical transmembrane-spanning subunits constitute a widely distributed superfamily (NaVBac) of channels in bacteria, implying a fundamental prokaryotic function. The degree of sequence homology (22-54%) is optimal for future comparisons of NaVBac structure and function of similarity and dissimilarity among NaVBac proteins. Thus, the NaVBac superfamily is fertile ground for crystallographic, electrophysiological, and microbiological studies. Mammalian voltage-gated sodium (NaV) 1The abbreviations used are: NaV, voltage-gated sodium channel; CaV, voltage-gated calcium channel; 6TM, six-α-helical transmembrane-spanning; NaVBac, bacterial voltage-gated sodium channel; NaVPZ, NaVBac from P. zeaxanthinifaciens; NaVSP, NaVBac from S. pomeroyi; CHO, Chinese hamster ovary.1The abbreviations used are: NaV, voltage-gated sodium channel; CaV, voltage-gated calcium channel; 6TM, six-α-helical transmembrane-spanning; NaVBac, bacterial voltage-gated sodium channel; NaVPZ, NaVBac from P. zeaxanthinifaciens; NaVSP, NaVBac from S. pomeroyi; CHO, Chinese hamster ovary. and calcium (CaV) channels underlie membrane excitability, muscle contraction, and hormone secretion (1Hille B. Ion Channels of Excitable Membranes. Sinauer, Sunderland, MA2001Google Scholar). In contrast, the function of prokaryotic voltage-gated ion-selective channels is relatively unknown. Na+ channels may drive Na+-dependent flagellar motors in certain marine and alkaliphilic species (2Asai Y. Yakushi T. Kawagishi I. Homma M. J. Mol. Biol. 2003; 327: 453-463Crossref PubMed Scopus (98) Google Scholar, 3Kojima S. Yamamoto K. Kawagishi I. Homma M. J. Bacteriol. 1999; 181: 1927-1930Crossref PubMed Google Scholar, 4Krulwich T.A. Ito M. Guffanti A.A. Biochim. Biophys. Acta. 2001; 1505: 158-168Crossref PubMed Scopus (92) Google Scholar, 5McCarter L.L. Microbiol. Mol. Biol. Rev. 2001; 65: 445-462Crossref PubMed Scopus (262) Google Scholar, 6Yorimitsu T. Homma M. Biochim. Biophys. Acta. 2001; 1505: 82-93Crossref PubMed Scopus (120) Google Scholar). In marine vibrio, PomAB and MotXY have been proposed to form a functional Na+ channel (5McCarter L.L. Microbiol. Mol. Biol. Rev. 2001; 65: 445-462Crossref PubMed Scopus (262) Google Scholar, 6Yorimitsu T. Homma M. Biochim. Biophys. Acta. 2001; 1505: 82-93Crossref PubMed Scopus (120) Google Scholar), but the conductance has not been directly measured. In alkaliphilic bacteria, the prokaryotic ion channel responsible has not been identified. A bacterial 6-α-helical transmembrane (6TM) channel subunit NaChBac was expressed in CHO cells as a functional voltage-gated Na+ channel (7Ren D. Navarro B. Xu H. Yue L. Shi Q. Clapham D.E. Science. 2001; 294: 2372-2375Crossref PubMed Scopus (385) Google Scholar), but its role in bacteria is still being elucidated. The pore-forming subunits (α1) of mammalian NaV and CaV are composed of four similar repeats of 6TM domains (8Catterall W.A. Neuron. 2000; 26: 13-25Abstract Full Text Full Text PDF PubMed Scopus (1702) Google Scholar, 9Goldin A.L. Barchi R.L. Caldwell J.H. Hofmann F. Howe J.R. Hunter J.C. Kallen R.G. Mandel G. Meisler M.H. Netter Y.B. Noda M. Tamkun M.M. Waxman S.G. Wood J.N. Catterall W.A. Neuron. 2000; 28: 365-368Abstract Full Text Full Text PDF PubMed Scopus (653) Google Scholar), probably arising by gene duplication of a single 6TM gene (1Hille B. Ion Channels of Excitable Membranes. Sinauer, Sunderland, MA2001Google Scholar, 10Catterall W.A. Science. 2001; 294: 2306-2308Crossref PubMed Scopus (18) Google Scholar). The first bacterial voltage-gated Na+ channel (NaChBac) functionally expressed in mammalian cells was cloned from Bacillus halodurans (7Ren D. Navarro B. Xu H. Yue L. Shi Q. Clapham D.E. Science. 2001; 294: 2372-2375Crossref PubMed Scopus (385) Google Scholar). It contains a single 6TM domain of 274 amino acids but almost certainly forms a tetramer (7Ren D. Navarro B. Xu H. Yue L. Shi Q. Clapham D.E. Science. 2001; 294: 2372-2375Crossref PubMed Scopus (385) Google Scholar). NaChBac voltage-dependent activation and inactivation kinetics are 10-100 times slower than that of NaV (7Ren D. Navarro B. Xu H. Yue L. Shi Q. Clapham D.E. Science. 2001; 294: 2372-2375Crossref PubMed Scopus (385) Google Scholar). NaChBac inactivation may result from pore inactivation (C-type inactivation), since it does not contain an obvious cytoplasmic inactivation gate (7Ren D. Navarro B. Xu H. Yue L. Shi Q. Clapham D.E. Science. 2001; 294: 2372-2375Crossref PubMed Scopus (385) Google Scholar). Because single 6TM Na+-selective ion channels do not appear to be present in vertebrates, the 24 TM structure of NaVs may have arisen under evolutionary selective pressure. It has been proposed that highly Na+-selective NaV channels require pore asymmetry, which is achieved by the concatenated 4 × 6TM structure (11Sun Y.M. Favre I. Schild L. Moczydlowski E. J. Gen. Physiol. 1997; 110: 693-715Crossref PubMed Scopus (125) Google Scholar, 12Yamagishi T. Li R.A. Hsu K. Marban E. Tomaselli G.F. J. Gen. Physiol. 2001; 118: 171-182Crossref PubMed Scopus (32) Google Scholar). However, tetramers of identical 6TM NaChBac are equally Na+-selective as NaVs (7Ren D. Navarro B. Xu H. Yue L. Shi Q. Clapham D.E. Science. 2001; 294: 2372-2375Crossref PubMed Scopus (385) Google Scholar). Therefore, it seems more likely that the concatenated 4 × 6TM structure of vertebrate NaVs provided an evolutionary advantage by increasing the speed of activation, inactivation, or recovery from inactivation. To begin to address these questions, we searched for other bacterial ion channel subunits that might reveal the range of the gating speeds provided by the simplest tetrameric Na+-selective channels. This information will also lay the groundwork for understanding their function in prokaryotes. Cloning of NaVPZ—NaChBac protein sequence (GenBank™ accession number NP_242367) was used for a BLASTP search. The nucleotide sequence of NaVPZ was obtained from the published genomic sequence of CcaA protein of Paracoccus zeaxanthinifaciens (CAD24429). We synthesized DNA of NaVPZ using the following oligonucleotides: 5′-TTACCATGGTAATGAGCCTGCGCGCGCGCCTCGACGCCCTTGTCCACGGGCGTCGTGCCCAGGGGGTGATCACCGGCGTCATCCTGTTCAA-3′ (1F), 5′-GTCCGCTGATCCTGCTGCTGGACGCGGCCTGCCTTGCCGTCTTCGTGGCCGAGATCGCGGCCAAGCTGATCGCGCGCGGCCCGCGCTTCT-3′ (2F), 5′-ATGCCGGCGGGGCAGGGCCTCTCGGTGCTGCGCGCGCTGCGCATCCTGCGTCTGCTGCGTCTGGTGTCGGTCACCCCGCGCCTGCGCCGC-3′ (3F), 5′-GCTGATGGGCGTGATCTTCTACATCTTCTCGGTCATGGCGACGAAGCTGTTCGGGGCGGGGTTCCCGGACTGGTTCGGCTCGCTTGGCAA-3′ (4F), 5′-GGATCGTGCGTCCGGTCATGCAGGAATATCCGCTGGCATGGCTGTTCTTCGTGCCGTTCATCCTGATCACGACCTTCGCGGTGATGAACC-3′ (5F), 5′-GAAAGCGCCGCCACCGACGCCTATCGCGACGAGGTGCTGATGCGCCTGCGCGCGATCGAGAAGCAGCTGGACGAAAGCGGCGGCCGTGGG-3′ (6F), 5′-CAGCAGCAGGATCAGCGGACCCGCGACCGCCATGACGCGGCCCGAGGTCTCCAGCCCCAGCAGGACGGCGTTGAACAGGATGACGCCGGT-3′ (1R), 5′-AGGCCCTGCCCCGCCGGCATCAGCGCGATGGCCACGACGCTGAAATCGAAGACGTTCCAGCCGTCGCGGAAGAAGCGCGGGCCGCGCGCG-3′ (2R), 5′-AGAAGATCACGCCCATCAGCAGGAAGACCGAGGCCATGCCCGGCATCGCGGCGAACAGCCCCTCGACCACGCGGCGCAGGCGCGGGGTGA-3′ (3R), 5′-CATGACCGGACGCACGATCCCCATCGACCAGCTTTCCAGCGTCATCACCTGGAACAGCGAATAGGCCGACTTGCCAAGCGAGCCGAACCA-3′ (4R), 5′-GCGTCGGTGGCGGCGCTTTCCTCGGCCTGGTGGGCATCCTGCATCGAGTTCACGATCAGACCGACGACAAGGTTCATCACCGCGAAGGTC-3′ (5R), and 5′-TTACTCGAGAGAACCGCGTGGCACCAGGACACGCCCACGGCCGCCGCTTTCGT-3′ (6R). After mixing 1 μm each of 2F, 3F, 1R, and 2R as a template, the first PCR was conducted using 1F and 3R as primers (10 μm each). Similarly, 4R, 5F, 5R, and 6F were mixed, and PCR was conducted using 4F and 6R as primers. These PCR products were excised from agarose gels. Purified fragments were mixed and used as templates for further PCR, using 1F and 6R as primers. The amplified fragment was digested with NcoI and XhoI and purified from an agarose gel. The PCR product was then cloned into pTrcHis2B (Invitrogen). This synthesized DNA contained additional methionine and valine codons prior to the first methionine as required by the cloning strategy. We also intentionally introduced 10 silent mutations (45G→ T, 321G→ T, 330G→ T, 564C→ G, 606C→ G, 624A→ C, 775A→ C, 777G→ T, and 783G→ T) into the synthetic DNA. An additional silent mutation (495T→ C) occurred during the PCR process. Finally, this plasmid DNA was digested with SalI and XhoI and then self-ligated after the linker sequence was deleted. The resultant expression clone was used to transform Escherichia coli BL-21 (Stratagene). NaVPZ was cloned into a modified pTracer-CMV2 vector (Invitrogen) containing enhanced green fluorescent protein for expression in mammalian cells. Briefly, DNA was amplified by PCR from the E. coli expression clone using the following primers: 5′-AATGGATCCATGAGCCTGCGCGCGCGC-3′ (containing a BamHI site) and 5′-ATTGAATTCTCAGACACGCCCACGGCCGCC-3′ (containing an EcoRI site). The PCR product was then cloned into the modified pTracer-CMV2 plasmid (Invitrogen) between the single restriction sites for BamHI and EcoRI. All clones were confirmed by DNA sequencing. Cloning of NaVSP—The NaChBac DNA sequence (NC_002570) was used for a TBLASTX search against the Microbial Genomic data base at NCBI. A sample of Silicibacter pomeroyi was obtained from the American Type Culture Collection (catalog no. 700808). S. pomeroyi genomic DNA was collected by standard procedures (13Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology.Vol. 1. John Wiley & Sons, Inc., New York1995: 24Google Scholar). The genomic sequence homologous to NaChBac was identified, and the preliminary sequence data was obtained from The Institute for Genomic Research (TIGR) site on the World Wide Web at www.tigr.org. NaVSP was cloned into a pTrcHis2A (Invitrogen) for expression in E. coli. Briefly, DNA was amplified by PCR from a S. pomeroyi genomic DNA using the following primers: 5′-AATCCATGGTAATGCAAAGAATGCAGGCCTTT-3′ (containing an NcoI site) and 5′-ATTCTCGAGAGAACCGCGTGGCACCAGCTTTTTGGTTTCACCAAG-3′ (containing a thrombin recognition site and an XhoI site). The PCR product was cloned into the pTrcHis2A plasmid using NcoI and XhoI sites. This cloned DNA contains additional methionine and valine codons prior to the first methionine as required by the cloning strategy. The resulting expression clone was used to transform E. coli BL-21. NaVSP was cloned into a modified pTracer-CMV2 vector containing enhanced green fluorescent protein for expression in mammalian cells. Briefly, DNA was amplified by PCR from a S. pomeroyi genomic DNA using the following primers: 5′-AATGGATCCATGCAAAGAATGCAGGCCTTT-3′ (containing a BamHI site) and 5′-ATTGAATTCTCACTTTTTGGTTTCACCAAG-3′ (containing an EcoRI site). The PCR product was cloned into the modified pTracer-CMV2 plasmid between the single restriction sites for BamHI and EcoRI. All clones were confirmed by DNA sequencing. Information related to the cloning of other NaChBac homologs is provided as supplementary data. Expression and Purification of Recombinant Proteins—100-300 ml of LB medium containing ampicillin (50 μg/ml) was inoculated from glycerol stocks and grown overnight at 30 °C. 40 ml of culture medium was inoculated into 2 liters of Terrific Broth medium (Invitrogen) containing ampicillin and grown at 37 °C to A600 = 1.2. Cells were induced with 1 mm 1-β-d-thiogalactopyranoside and grown at 37 °C for 3h. Cells were then suspended in PBS buffer (pH 8.0) containing protease inhibitors (Protease Inhibitor Mixture; Sigma) and lysed by sonication. The carboxyl-terminal histidine-tagged protein was extracted by homogenization and solubilization in 15 mmn-undecyl-β-d-thiomaltopyranoside (Anatrace). Following centrifugation, the supernatant was loaded onto a Talon Co2+ affinity column (Clontech). Resin was washed with 20 mm imidazole, and the protein was then eluted in the presence of 400 mm imidazole. Purified protein was resolved by 4-12% SDS-PAGE (Invitrogen) and stained with Coomassie Blue. Molecular weight marker was purchased from Invitrogen (BenchMark™ Prestained Protein Ladder). Mammalian Electrophysiology—NaVPZ and NaVSP as well as other NaChBac homologues were subcloned into an enhanced green fluorescence protein-containing pTracer-CMV2 vector (Invitrogen) for expression into CHO-K1 and HEK293T cells. CHO-K1 and HEK293T cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum at 37 °C under 5% CO2. DNA was transfected using LipofectAMINE™ 2000 (Invitrogen) and plated onto coverslips, and recordings were made after 12 h (NaVPZ) or 48h (NaVSP), respectively. Unless otherwise stated, the pipette solution contained 147 mm Cs+, 120 mm methane sulfonate, 8 mm NaCl, 10 mm EGTA, 2 mm Mg-ATP, and 20 mm HEPES (pH 7.4). Bath solution contained 140 mm NaCl, 2 mm CaCl2, 1 mm MgCl2, 5 mm KCl, 20 mm HEPES (pH 7.4), and 10 mm glucose. All experiments were conducted at 22 ± 2 °C. Unless otherwise indicated, all chemicals were dissolved in water. Nifedipine (dissolved in Me2SO) was purchased from Sigma. As reported previously (7Ren D. Navarro B. Xu H. Yue L. Shi Q. Clapham D.E. Science. 2001; 294: 2372-2375Crossref PubMed Scopus (385) Google Scholar), unknown agents, presumably leached from the perfusion tubing, caused fast inactivation, and these perfusion systems were subsequently avoided. Using the whole or partial sequence of NaChBac as the query, we performed standard BLASTP or TBLASTX searches on the GenBank™ data bases from various prokaryotic genomic sequencing projects. Several open reading frames with significant sequence homology (22-54%) to NaChBac were identified in the following species (Fig. 1A): Vibrio vulnificus (22%), Microbulbifer degradans (two genes, 32 and 33%, respectively), Colwellia psychrerythraea (two genes, 35 and 38%, respectively), Magnetococcus sp. (32%), S. pomeroyi (39%), P. zeaxanthinifaciens (39%), Hyphomonas neptunium (33%), Thermobifida fusca (30%), and Oceanobacillus iheyensis (54%). Among these species, V. vulnificus (14Strom M.S. Paranjpye R.N. Microbes Infect. 2000; 2: 177-188Crossref PubMed Scopus (403) Google Scholar), M. degradans (15Gonzalez J.M. Weiner R.M. Int. J. Syst. Evol. Microbiol. 2000; 50: 831-834Crossref PubMed Scopus (44) Google Scholar), C. psychrerythraea (16Junge K. Eicken H. Deming J.W. Appl. Environ. Microbiol. 2003; 69: 4282-4284Crossref PubMed Scopus (55) Google Scholar), Magnetococcus sp. (17Meldrum F.C. Mann S. Heywood B.R. Frankel R.B. Bazylinski D.A. Proc. R. Soc. Lond. B. 1993; 251: 231-236Crossref Scopus (136) Google Scholar), S. pomeroyi (18Gonzalez J.M. Covert J.S. Whitman W.B. Henriksen J.R. Mayer F. Scharf B. Schmitt R. Buchan A. Fuhrman J.A. Kiene R.P. Moran M.A. Int. J. Syst. Evol. Microbiol. 2003; 53: 1261-1269Crossref PubMed Scopus (196) Google Scholar), P. zeaxanthinifaciens (19Berry A. Janssens D. Humbelin M. Jore J.P. Hoste B. Cleenwerck I. Vancanneyt M. Bretzel W. Mayer A.F. Lopez-Ulibarri R. Shanmugam B. Swings J. Pasamontes L. Int. J. Syst. Evol. Microbiol. 2003; 53: 231-238Crossref PubMed Scopus (83) Google Scholar), and H. neptunium (20Moore R.L. Weiner R.M. Gebers R. Int. J. Syst. Bacteriol. 1984; 34: 71-73Crossref Scopus (48) Google Scholar) were isolated from sea water or water. T. fusca (21McCarthy A.J. Cross T. J. Gen. Microbiol. 1984; 130: 5-25Google Scholar), a thermophilic Gram-positive bacteria, was isolated from soil but grows optimally in alkaliphilic conditions. O. iheyensis (22Lu J. Nogi Y. Takami H. FEMS Microbiol. Lett. 2001; 205: 291-297Crossref PubMed Google Scholar) and B. halodurans (23Takami H. Horikoshi K. Extremophiles. 2000; 4: 99-108Crossref PubMed Scopus (64) Google Scholar) are alkaliphilic Gram-positive bacteria isolated from deep sea water. Based on the degree of sequence homology, we consider these proteins to be NaChBac homologs (rather than orthologs). Hydrophobicity analysis of these proteins predicted that all have the 6TM architecture. Importantly, threonine, glutamate, and tryptophan residues are conserved in the pore region in all proteins (Fig. 1B). These residues have been shown to be critical for the cationic selectivity of NaChBac (24Yue L. Navarro B. Ren D. Ramos A. Clapham D.E. J. Gen. Physiol. 2002; 120: 845-853Crossref PubMed Scopus (127) Google Scholar). As is characteristic for voltage-gated channels (25Yellen G. Nature. 2002; 419: 35-42Crossref PubMed Scopus (533) Google Scholar), positively charged amino acids (Arg) are interspersed every 3 amino acids in the fourth putative transmembrane region (S4) (Fig. 1C). In V. vulnificus M06, however, the third arginine was not conserved. Based on the sequence homology and the structural similarity to NaChBac, it is likely that these proteins function as voltage-gated channels. We cloned all 11 sequences (see "Experimental Procedures") and studied them by expression in mammalian cell lines. As shown below, we were able to measure currents produced by two NaChBac homologs, NaVPZ (Fig. 2A) from P. zeaxanthinifaciens (a zeaxanthin-producing marine bacteria (19Berry A. Janssens D. Humbelin M. Jore J.P. Hoste B. Cleenwerck I. Vancanneyt M. Bretzel W. Mayer A.F. Lopez-Ulibarri R. Shanmugam B. Swings J. Pasamontes L. Int. J. Syst. Evol. Microbiol. 2003; 53: 231-238Crossref PubMed Scopus (83) Google Scholar)) and NaVSP (Fig. 2A) from S. pomeroyi (a dimethylsulfoniopropionate-degrading marine bacteria (18Gonzalez J.M. Covert J.S. Whitman W.B. Henriksen J.R. Mayer F. Scharf B. Schmitt R. Buchan A. Fuhrman J.A. Kiene R.P. Moran M.A. Int. J. Syst. Evol. Microbiol. 2003; 53: 1261-1269Crossref PubMed Scopus (196) Google Scholar)). Isolation and sequencing of the gene encoding NaVPZ revealed an open reading frame of 262 amino acids with a predicted molecular size of 29 kDa. Similarly, the NaVSP gene encoded an open reading frame of 258 amino acids with a predicted molecular mass of 29 kDa. NaVPZ and NaVSP share 39% identity (60% similarity) and 39% identity (59% similarity) with NaChBac, respectively (Fig. 2B). Notably, NaVPZ is 65% identical (77% similar) to NaVSP. Upon electrophoresis, both NaVPZ and NaVSP proteins migrated as a single band (∼31 kDa; Fig. 2C), almost identical to the predicted molecular sizes of the His-tagged constructs. CHO-K1 or HEK293T cell lines were transfected with NaChBac homologs (in pTracer), and whole-cell currents were recorded 12-48 h after transfection (see "Experimental Procedures"). Among 11 NaChBac homologs, only two (NaVPZ and NaVSP) produced detectable currents. Similar current are not present in nontransfected or mock-transfected cells (data not shown). NaVPZ-transfected cells exhibited large (up to 10,000 pA; 10 nA) voltage-activated inward currents (Fig. 3, A-C). NaVPZ-mediated current (INaVPZ) activated with a time constant (τactivation) of 21.5 ± 1.3 ms at +10 mV (n = 19), significantly slower than both mammalian NaV channels (τactivation < 2 ms) and INaChBac (τactivation < 13 ms). Inactivation of INaVPZ was slow (τinactivation = 102 ± 4.2 ms at +10 mV, n = 19) compared with the typically fast inactivating NaV currents (τinactivation < 10 ms) but faster than INaChBac (τinactivation > 160 ms). Cation replacement by N-methyl-d-glucamine NMDG (bath) resulted in complete removal of voltage-dependent INaVPZ inward current (Fig. 3C). Similarly, no significant inward current was seen in isotonic [Ca2+]o (monovalent cations replaced with 105 mm Ca2+) (Fig. 3, B and C). INaVPZ reversed at +75 mV (Fig. 3C), close to the Nernst potential of Na+ under our recording conditions (ENa = +72 mV). These results, together with the large leftward shift of the reversal by external Na+ removal (N-methyl-d-glucamine+ and isotonic Ca2+ solution substitution), suggested that NaVPZ, like NaChBac, is a Na+-selective channel. We assumed that the outward currents in 0 mm [Na+]o (Fig. 3B) were carried by internal Na+ (8 mm [Na+]i). Due to the very negative Erev (Fig. 3C), sizable outward currents were observed at most voltages tested (Fig. 3, B and C). We evaluated the voltage-dependent activation of INaVPZ by measuring deactivation tail currents (Fig. 4A). A Boltzmann fit of the averaged activation curve yielded a V1/2 of -9.5 ± 0.8 mV (n = 9) and slope factor (κ) of 10.7 ± 0.7 mV per e-fold change in current (Fig. 4C). Steady-state inactivation of the channel was determined by sequential depolarization to test voltages followed by voltage clamp to the peak of activation at +10 mV (Fig. 4B). Steady-state inactivation was a steep function of voltage, with 50% inactivation at -35 ± 0.4 mV (n = 10) and slope factor (κ) of 6.3 ± 0.3 mV/e-fold (Fig. 4C). We investigated the time course of INaVPZ inactivation at -30 mV, where activation was minimal. The degree and speed of inactivation was strongly dependent on the duration of the inactivating prepulse (-30 mV; τ = 2123 ± 434 ms; n = 6; Fig. 4, D and E). INaVPZ recovered slowly with time constant, τ = 839 ± 90 ms (n = 7, HP = -90 mV, Fig. 4, F and G). NaVSP-transfected cells also yielded voltage-activated inward currents (Fig. 5A), peaking at ∼+30 mV. The Erev of NaVSP-mediated current (INaVSP) was +76 mV. Ion substitution (Ca2+ replacement) experiments confirmed that NaVSP, like NaVPZ, was also a Na+ -selective channel (data not shown). INaVSP activated and inactivated significantly faster than INaVPZ and INaChBac (τactivation = 3.4 ± 0.3 ms at +30 mV, n = 17; τinactivation = 35 ± 1.5 ms at +30 mV, n = 17) but still severalfold slower than NaV currents. The Boltzmann fit activation curve yielded a V of +21 ± 0.4 mV and κ of 11.8 ± 0.4 mV/e-fold change (n = 28; Fig. 5E). Steady state inactivation was strongly dependent on the voltage (κ = 10.3 ± 0.5 mV/e-fold), with half-inactivation at -22 ± 0.8 mV (n = 11; Fig. 5E). NaVPZ and NaVSP were sensitive to high concentrations of nifedipine (30 μm; data not shown). Expression of bacterial genes in systems where the protein can be studied (mammalian cells for patch clamp) is crucial to interpreting and extending static structural data through structure-function studies. Such functional expression is also important to understanding their native roles in bacteria. However, successful functional expression of bacterial proteins in mammalian cells is rare. Here, we identified 11 putative 6TM Na+ prokaryotic channel subunit genes and were able to functionally express 2 of the 11 in mammalian cells, where their electrophysiological properties could be studied. Both channels were Na+-selective and activated by voltage. One conclusion based on comparison of bacterial channels and NaVs is that the major evolutionary pressure for gene duplication and concatenation of subunits was to increase the speed of channel gating. Given the high selectivity of presumed homomeric bacterial Na+ channels, the case for pore asymmetry as a means to increase Na+ selectivity seems a less likely scenario. Little is known about the molecular determinants that control mammalian NaV activation rates. Mammalian NaV channels activate and inactivate within a few milliseconds (<10 ms), roughly 10-100 times faster than NaChBac, the only bacterial voltage-gated channel functionally expressed up to now. Interestingly, NaVSP activation is ∼4-fold faster than NaChBac, whereas NaVPZ activation is ∼2 times slower than NaChBac, despite 77% sequence homology between NaVSP and NaVPZ. In NaVSP, NaVPZ, and NaChBac, the S4 domain and short S3-S4 linker are highly conserved, suggesting that the structural determinants for the kinetics differences are located elsewhere. Notably, there are several NaVSP-specific residues in the putative pore-forming domains (Gly143 in S5, Ile172 in the pore loop, Val189 in the linker between the P loop and S6, and Met202 in S6). These residues may contribute to the relatively fast activation kinetics of NaVSP. Na+ channel inactivation mechanisms are better understood than those of activation. Interdomain linkers mediate fast inactivation in NaVs by "ball and chain" or N type inactivation (26Vassilev P.M. Scheuer T. Catterall W.A. Science. 1988; 241: 1658-1661Crossref PubMed Scopus (314) Google Scholar), but these domains are obviously missing in tetramers of 6TM bacterial channels. Additionally, the removal of segments within the N and C cytoplasmic domains of NaChBac (24Yue L. Navarro B. Ren D. Ramos A. Clapham D.E. J. Gen. Physiol. 2002; 120: 845-853Crossref PubMed Scopus (127) Google Scholar) (unpublished data) does not substantially alter its inactivation rate. If the cytoplasmic domains do not participate in inactivation, we can then begin to look at other domains. Studies on 6TM HERG K+ channels indicated that the S5-P linker was crucial for its C-type inactivation, probably by providing allosteric coupling between its outer mouth and the voltage sensor (27Liu J. Zhang M. Jiang M. Tseng G.N. J. Gen. Physiol. 2002; 120: 723-737Crossref PubMed Scopus (102) Google Scholar). NaChBac inactivates with a time constant similar to NaVPZ, but the NaChBac S5-P linker (from Gln167 to Ser180) has low homology to NaVPZ. NaChBac and NaVPZ inactivates 5-fold more slowly than NaVSP. In the pore-S6 linker, NaVSP lacks the negatively charged glutamate present in both NaChBac and NaVPZ. Future studies will focus on this and other sequence differences. However, the difficulty of obtaining functional expression of many mutants highlights the need for structural data. By exclusion of alternative mechanisms, we hypothesize that C-type inactivation, in which the Na+ pore is shut, is the more likely mechanism for NaVBac channel inactivation. Na+ channels have been proposed to play a central role in Na+-dependent flagellar mobility in some prokaryotes. Marine Vibrio species utilize their Na+-driven polar flagella for swimming (5McCarter L.L. Microbiol. Mol. Biol. Rev. 2001; 65: 445-462Crossref PubMed Scopus (262) Google Scholar, 6Yorimitsu T. Homma M. Biochim. Biophys. Acta. 2001; 1505: 82-93Crossref PubMed Scopus (120) Google Scholar), and in the alkaliphilic Bacillus species, [Na+]o determines the activity of the flagellar motor (4Krulwich T.A. Ito M. Guffanti A.A. Biochim. Biophys. Acta. 2001; 1505: 158-168Crossref PubMed Scopus (92) Google Scholar, 28Imae Y. Atsumi T. J. Bioenerg. Biomembr. 1989; 21: 705-716Crossref PubMed Scopus (84) Google Scholar). Under alkaliphilic conditions, the H+-motive force is weak due to the high intracellular pH (pH 8-9) of these bacteria (4Krulwich T.A. Ito M. Guffanti A.A. Biochim. Biophys. Acta. 2001; 1505: 158-168Crossref PubMed Scopus (92) Google Scholar, 29Krulwich T.A. Ito M. Gilmour R. Sturr M.G. Guffanti A.A. Hicks D.B. Biochim. Biophys. Acta. 1996; 1275: 21-26Crossref PubMed Scopus (49) Google Scholar). Therefore, a Na+ cycle driven by the Na+ channel may have evolved to power the flagellar motor (4Krulwich T.A. Ito M. Guffanti A.A. Biochim. Biophys. Acta. 2001; 1505: 158-168Crossref PubMed Scopus (92) Google Scholar, 28Imae Y. Atsumi T. J. Bioenerg. Biomembr. 1989; 21: 705-716Crossref PubMed Scopus (84) Google Scholar, 30Hirota N. Imae Y. J. Biol. Chem. 1983; 258: 10577-10581Abstract Full Text PDF PubMed Google Scholar, 31Sugiyama S. Matsukura H. Imae Y. FEBS Lett. 1985; 182: 265-268Crossref PubMed Scopus (28) Google Scholar, 32Imae Y. Matsukura H. Kobayashi S. Methods Enzymol. 1986; 125: 582-592Crossref PubMed Scopus (21) Google Scholar). Interestingly, bacteria whose flagellar motors are powered by Na+ (as opposed to H+) express NaChBac homologs. In bacteria, the Na+/H+ exchanger prevents cytotoxic Na+ accumulation and also supports pH homeostasis at elevated pH (4Krulwich T.A. Ito M. Guffanti A.A. Biochim. Biophys. Acta. 2001; 1505: 158-168Crossref PubMed Scopus (92) Google Scholar, 31Sugiyama S. Matsukura H. Imae Y. FEBS Lett. 1985; 182: 265-268Crossref PubMed Scopus (28) Google Scholar, 33Booth I.R. Edwards M.D. Miller S. Biochemistry. 2003; 42: 10045-10053Crossref PubMed Scopus (37) Google Scholar). In low [Na+]o environments or in the absence of solutes to support Na+ uptake through Na+-coupled solute transporters, the pH homeostasis function may rely on a Na+ channel (4Krulwich T.A. Ito M. Guffanti A.A. Biochim. Biophys. Acta. 2001; 1505: 158-168Crossref PubMed Scopus (92) Google Scholar, 31Sugiyama S. Matsukura H. Imae Y. FEBS Lett. 1985; 182: 265-268Crossref PubMed Scopus (28) Google Scholar, 33Booth I.R. Edwards M.D. Miller S. Biochemistry. 2003; 42: 10045-10053Crossref PubMed Scopus (37) Google Scholar). We propose that sustained voltage-gated Na+ channel opening is primarily responsible for this Na+ entry (33Booth I.R. Edwards M.D. Miller S. Biochemistry. 2003; 42: 10045-10053Crossref PubMed Scopus (37) Google Scholar). It is possible that some mammalian Na+ channels play a role in Na+ or H+ homeostasis. Interestingly, the mammalian persistent and resurgent Na+ currents have similar kinetics to NaVBac (34Akopian A.N. Sivilotti L. Wood J.N. Nature. 1996; 379: 257-262Crossref PubMed Scopus (911) Google Scholar, 35Do M.T. Bean B.P. Neuron. 2003; 39: 109-120Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). These persistent Na+ currents may be mediated by subthreshold gating of fast NaV channels (36Taddese A. Bean B.P. Neuron. 2002; 33: 587-600Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar) or perhaps by NaV1.8 and NaV1.9. NaChBac selectivity is converted from Na+ to Ca2+ by replacing an amino acid adjacent to glutamatic acid in the putative pore domain by a negatively charged aspartate (from TLESWAS to TLEDWAS or TLDDWAD) (24Yue L. Navarro B. Ren D. Ramos A. Clapham D.E. J. Gen. Physiol. 2002; 120: 845-853Crossref PubMed Scopus (127) Google Scholar). Interestingly, two bacterial strains (C. psychrerythraea and M. degradans) have a putative pore sequence (TFEDWTD) similar to that of the Ca2+-selective NaChBac mutant. We have not been able to functionally express these channel subunits in mammalian cells, but one possibility is that these proteins form heteromeric channels with other related subunits in the same species. We are grateful for bacteria provided by Drs. Hideto Takami, Arthur. A. Guffanti, Terry. A. Krulwich (O. iheyensis HTE831), and James D. Oliver (V. vulnificus M06) and genomic DNA from Dr. Barbara Methe (C. psychrerythraea 34H). We also thank the members of the Clapham laboratory, Nat Blair, and Dr. Terry Krulwich (Mt. Sinai School of Medicine) for valuable comments and encouragement. Download .pdf (.01 MB) Help with pdf files
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