TMEM175 Is an Organelle K+ Channel Regulating Lysosomal Function

•Organelle patch clamping recorded a K+ channel on endosomes and lysosomes•Candidate gene screening found that a novel protein TMEM175 forms the channel•TMEM175 regulates lysosomal membrane potential and pH stability•Knocking out TMEM175 leads to abnormal autophagosome-lysosome fusion Potassium is the most abundant ion to face both plasma and organelle membranes. Extensive research over the past seven decades has characterized how K+ permeates the plasma membrane to control fundamental processes such as secretion, neuronal communication, and heartbeat. However, how K+ permeates organelles such as lysosomes and endosomes is unknown. Here, we directly recorded organelle K+ conductance and discovered a major K+-selective channel KEL on endosomes and lysosomes. KEL is formed by TMEM175, a protein with unknown function. Unlike any of the ∼80 plasma membrane K+ channels, TMEM175 has two repeats of 6-transmembrane-spanning segments and has no GYG K+ channel sequence signature-containing, pore-forming P loop. Lysosomes lacking TMEM175 exhibit no K+ conductance, have a markedly depolarized ΔΨ and little sensitivity to changes in [K+], and have compromised luminal pH stability and abnormal fusion with autophagosomes during autophagy. Thus, TMEM175 comprises a K+ channel that underlies the molecular mechanism of lysosomal K+ permeability. Potassium is the most abundant ion to face both plasma and organelle membranes. Extensive research over the past seven decades has characterized how K+ permeates the plasma membrane to control fundamental processes such as secretion, neuronal communication, and heartbeat. However, how K+ permeates organelles such as lysosomes and endosomes is unknown. Here, we directly recorded organelle K+ conductance and discovered a major K+-selective channel KEL on endosomes and lysosomes. KEL is formed by TMEM175, a protein with unknown function. Unlike any of the ∼80 plasma membrane K+ channels, TMEM175 has two repeats of 6-transmembrane-spanning segments and has no GYG K+ channel sequence signature-containing, pore-forming P loop. Lysosomes lacking TMEM175 exhibit no K+ conductance, have a markedly depolarized ΔΨ and little sensitivity to changes in [K+], and have compromised luminal pH stability and abnormal fusion with autophagosomes during autophagy. Thus, TMEM175 comprises a K+ channel that underlies the molecular mechanism of lysosomal K+ permeability. A hallmark of eukaryotic cells is the separation of cellular functions in membrane-bound organelles in the cytosol. Voltage gradients exist across both plasma and organelle membranes. The plasma membrane potential (Vm) regulates cellular processes fundamental to life, including fertilization, gene expression, secretion, neuronal communication, and the beating of cardiac cells. At rest, the plasma membrane is much more permeable to K+ than to Na+, leading to a resting Vm closer to the equilibrium potential of K+ (EK) (see Hille, 2001Hille B. Ion Channels of Excitable Membranes.Third Edition. Sinauer Associates, 2001Google Scholar for review). K+ channels represent the largest superfamily of ion-selective channels, with ∼80 pore-forming subunit-encoding genes and many more “auxiliary” subunit ones in humans (see Huang and Jan, 2014Huang X. Jan L.Y. Targeting potassium channels in cancer.J. Cell Biol. 2014; 206: 151-162Crossref PubMed Scopus (214) Google Scholar and Nichols and Lopatin, 1997Nichols C.G. Lopatin A.N. Inward rectifier potassium channels.Annu. Rev. Physiol. 1997; 59: 171-191Crossref PubMed Scopus (660) Google Scholar for review). Despite the divergence in sequence and structure among the ∼80 K+ channels, their ion-selective filters are all formed by similar membrane re-entrant P loops containing the GYG/GFG K+ channel signature (Doyle et al., 1998Doyle D.A. Morais Cabral J. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. The structure of the potassium channel: molecular basis of K+ conduction and selectivity.Science. 1998; 280: 69-77Crossref PubMed Scopus (5732) Google Scholar, Heginbotham et al., 1994Heginbotham L. Lu Z. Abramson T. MacKinnon R. Mutations in the K+ channel signature sequence.Biophys. J. 1994; 66: 1061-1067Abstract Full Text PDF PubMed Scopus (688) Google Scholar, Jiang et al., 2003Jiang Y. Lee A. Chen J. Ruta V. Cadene M. Chait B.T. MacKinnon R. X-ray structure of a voltage-dependent K+ channel.Nature. 2003; 423: 33-41Crossref PubMed Scopus (1636) Google Scholar, MacKinnon and Miller, 1989MacKinnon R. Miller C. Mutant potassium channels with altered binding of charybdotoxin, a pore-blocking peptide inhibitor.Science. 1989; 245: 1382-1385Crossref PubMed Scopus (298) Google Scholar, Yellen et al., 1991Yellen G. Jurman M.E. Abramson T. MacKinnon R. Mutations affecting internal TEA blockade identify the probable pore-forming region of a K+ channel.Science. 1991; 251: 939-942Crossref PubMed Scopus (495) Google Scholar). In addition to voltage-dependent K+ channels, there is also a family of voltage- and time-independent “leak” (K2P) K+ channels (see Goldstein et al., 2001Goldstein S.A. Bockenhauer D. O’Kelly I. Zilberberg N. Potassium leak channels and the KCNK family of two-P-domain subunits.Nat. Rev. Neurosci. 2001; 2: 175-184Crossref PubMed Scopus (562) Google Scholar for review). K2Ps regulate resting background K+ conductance. Intriguingly, K2Ps are only found in eukaryotes. It is unknown whether the plasma membranes of bacteria and archaea also have “leak-like” K+ channels. In lysosomes and endosomes, electrical potential across the organelle membrane (ΔΨ, defined as Vcytosol − Vlumen; Bertl et al., 1992Bertl A. Blumwald E. Coronado R. Eisenberg R. Findlay G. Gradmann D. Hille B. Köhler K. Kolb H.-A. MacRobbie E. et al.Electrical measurements on endomembranes.Science. 1992; 258: 873-874Crossref PubMed Scopus (172) Google Scholar; Figure 1A) may vary among cells and individual organelles as the organelles mature from early to late endosomes. The ionic conductances determining ΔΨ have not been well studied. Earlier indirect studies using the measurement of each ion’s ability to influence lysosome luminal pH or to protect the organelles from osmotic lysis suggest that lysosomes are permeable to cations with an approximate permeability sequence of Cs+>K+>>Na+ (Casey et al., 1978Casey R.P. Hollemans M. Tager J.M. The permeability of the lysosomal membrane to small ions.Biochim. Biophys. Acta. 1978; 508: 15-26Crossref PubMed Scopus (30) Google Scholar, Henning, 1975Henning R. pH gradient across the lysosomal membrane generated by selective cation permeability and Donnan equilibrium.Biochim. Biophys. Acta. 1975; 401: 307-316Crossref PubMed Scopus (50) Google Scholar). Recent studies have uncovered several lysosomal ion channels/transporters conducting Cl−, H+, Ca2+, and Na+ (see Xu and Ren, 2015Xu H. Ren D. Lysosomal physiology.Annu. Rev. Physiol. 2015; 77: 57-80Crossref PubMed Scopus (587) Google Scholar) for review). However, the molecular identity of the large K+ permeability, its contribution to ΔΨ, and ΔΨ’s roles in cellular functions are little understood. In this report, we discovered a “leak-like” K+ conductance KEL on endosomes and lysosomes. Using candidate gene screening, we found that TMEM175, a previously uncharacterized family of transmembrane proteins, form KEL. Bacteria and archaea also have TMEM175 homologs that form plasma membrane K+-permeable channels. Therefore, TMEM175 represents a new K+ channel family found in all the three domains of life. Unlike plasma membranes, the organelle membrane has little redundancy to K+ channel proteins; knocking out TMEM175 eliminates the K+ conductance and leads to compromised organelle lysosomal pH stability and abnormal organelle fusion during autophagy. To directly record the K+ permeability, we used whole-organelle patch clamp to measure the charge flow rates (currents) across lysosomal membranes with K+ as the major ion (Figure 1A). Similar to those we previously recorded from peritoneal macrophages (Cang et al., 2014Cang C. Bekele B. Ren D. The voltage-gated sodium channel TPC1 confers endolysosomal excitability.Nat. Chem. Biol. 2014; 10: 463-469Crossref PubMed Scopus (118) Google Scholar), lysosomes from primary cells cultured from mice, including glia (Figures 1B–1D), neurons, cardiac myocytes, and cardiac fibroblasts (Figure S1), all had high densities (∼50 pA/pF at +100 mV) of K+ currents. On plasma membranes, there is a large degree of heterogeneity in the kinetics of activation, inactivation, and deactivation in the K+ conductances (Hille, 2001Hille B. Ion Channels of Excitable Membranes.Third Edition. Sinauer Associates, 2001Google Scholar). Such heterogeneity appeared to be absent or minimal in lysosomes recorded from both excitable and non-excitable cells. In response to changes in voltage, the current amplitudes changed instantaneously, were proportional to the applied voltages, and had no obvious inactivation or rectification, properties of a voltage- and time-independent, leak-like K+ channel conductance (Figures 1C and S1). To test whether similar K+ conductance is present in other intracellular organelles, we patch clamped endosomes (Saito et al., 2007Saito M. Hanson P.I. Schlesinger P. Luminal chloride-dependent activation of endosome calcium channels: patch clamp study of enlarged endosomes.J. Biol. Chem. 2007; 282: 27327-27333Crossref PubMed Scopus (79) Google Scholar). To facilitate whole-organelle recording, we enlarged the endosomes with transfection of Rab5-Q79L, a Rab5 mutant that enlarges endosomes (Stenmark et al., 1994Stenmark H. Parton R.G. Steele-Mortimer O. Lütcke A. Gruenberg J. Zerial M. Inhibition of rab5 GTPase activity stimulates membrane fusion in endocytosis.EMBO J. 1994; 13: 1287-1296Crossref PubMed Scopus (771) Google Scholar). In endosomes tested in all the cell types cultured from mice, including neurons (Figures 1E and 1F), glia (Figure 1G) and cell lines, we detected K+ currents with properties similar to those found in lysosomes. These results indicate that both endosomes and lysosomes have a leak-like K+ conductance (KEL), which presumably underlies the K+ permeability. Consistent with the idea that KEL is constitutively open, the resting ΔΨ of lysosomes, like that of Vm, was highly dependent on [K+] (Figures 1H and 1I). We set out to identify the KEL-encoding gene. Among the ∼80 canonical K+ channels characterized on plasma membranes, only the K2P family has biophysical properties that resemble KEL (Goldstein et al., 2001Goldstein S.A. Bockenhauer D. O’Kelly I. Zilberberg N. Potassium leak channels and the KCNK family of two-P-domain subunits.Nat. Rev. Neurosci. 2001; 2: 175-184Crossref PubMed Scopus (562) Google Scholar). K2P1 protein has been detected in endosomes using immunostaining (Honoré, 2007Honoré E. The neuronal background K2P channels: focus on TREK1.Nat. Rev. Neurosci. 2007; 8: 251-261Crossref PubMed Scopus (375) Google Scholar). Overexpressing K2P1 in HEK293T cells, however, led to no obvious increase in KEL-like currents recorded from endosomes (enlarged with Rab5-Q79L) or lysosomes (enlarged with vacuolin-1) (n = 15). We next used a whole-organelle patch-clamp-based candidate gene screening to test whether previously under-characterized proteins form KEL. We used the following candidate selection criteria: (1) the protein was found in our previous lysosomal proteomic analysis (Chapel et al., 2013Chapel A. Kieffer-Jaquinod S. Sagné C. Verdon Q. Ivaldi C. Mellal M. Thirion J. Jadot M. Bruley C. Garin J. et al.An extended proteome map of the lysosomal membrane reveals novel potential transporters.Mol. Cell. Proteomics. 2013; 12: 1572-1588Crossref PubMed Scopus (137) Google Scholar) or is known to lead to lysosome-related disease when mutated; (2) the protein is predicted to have at least one transmembrane spanning (TM) domain; and (3) the protein has not been functionally established as an ion channel/transporter. Because of the small size (∼−80 pA at −100 mV in HEK293T cell lysosomes) of the native KEL current (IKEL) and potential genetic redundancy, a loss-of-function approach with shRNAs to partially knockdown candidate proteins might not be sufficiently sensitive for the screening. We instead used a gain-of-function approach by testing whether overexpression of candidate proteins increased lysosomal currents. To screen proteins for channel current, we performed recordings under conditions that detected K+, Na+, and Cl− currents. We also included amino acids in the pipette solution to detect amino-acid-activated conductance. Out of the 16 candidates, 12 of which were detected in our previous proteomic analysis (Chapel et al., 2013Chapel A. Kieffer-Jaquinod S. Sagné C. Verdon Q. Ivaldi C. Mellal M. Thirion J. Jadot M. Bruley C. Garin J. et al.An extended proteome map of the lysosomal membrane reveals novel potential transporters.Mol. Cell. Proteomics. 2013; 12: 1572-1588Crossref PubMed Scopus (137) Google Scholar), only 1 (TMEM175) led to a large increase of IKEL-like currents (Figure 2). TMEM175 (transmembrane protein 175) has no significant sequence similarity to any other protein with known function. Homologs are found in archaea, bacteria, and eukaryotes and are grouped together into a superfamily by the presence of one (in archaea and bacteria) or two (in eukaryotes) copies of DUF1211 (domain-of-unknown-function). Within eukaryotes, TMEM175 is found in both unicellular organisms such as the choanoflagellate Salpingoeca rosetta (Genbank: XP_004995857) and marine microalgae Nannochloropsis gaditana (EWM26717) and multicellular organisms, including invertebrate and vertebrate animals. Among animals, TMEM175s are highly conserved (81% identity between human and mouse, 62% between human and zebrafish). Expressed-sequence-tag and RNA-sequencing databases suggest that human TMEM175 (hTMEM175) is expressed in all tissues/organs, including the heart, brain, testis, kidney, and liver, and in cultured cell lines. Using hydrophobicity analysis (Figure 3A) and a hidden Markov model method (Tusnády and Simon, 2001Tusnády G.E. Simon I. The HMMTOP transmembrane topology prediction server.Bioinformatics. 2001; 17: 849-850Crossref PubMed Scopus (1558) Google Scholar), hTMEM175 is predicted to have a two-repeat structure, with each repeat containing six TMs (S1–S6, Figure S2A). The two repeats have significant sequence similarity (Figure S2B), especially in the first TMs (IS1 and IIS1). This 2 × 6TM structure is similar to that of the two-repeat Na+ channels TPCs (Zhu et al., 2010Zhu M.X. Ma J. Parrington J. Calcraft P.J. Galione A. Evans A.M. Calcium signaling via two-pore channels: local or global, that is the question.Am. J. Physiol. Cell Physiol. 2010; 298: C430-C441Crossref PubMed Scopus (100) Google Scholar). TMEM175’s S4s have no obvious voltage-sensing domains characterized by the presence of charged residues, unlike in KVs, NaVs, CaVs, and TPCs. Intriguingly, the predicted S5–S6 linkers in both the repeats are short (4 aa). A GYG/GFG-signature-containing P loop (Figure S2A), a linker that reenters the membrane to form the selectivity filter of all the known K+ channels, is lacking (MacKinnon and Miller, 1989MacKinnon R. Miller C. Mutant potassium channels with altered binding of charybdotoxin, a pore-blocking peptide inhibitor.Science. 1989; 245: 1382-1385Crossref PubMed Scopus (298) Google Scholar, Miller, 1995Miller C. The charybdotoxin family of K+ channel-blocking peptides.Neuron. 1995; 15: 5-10Abstract Full Text PDF PubMed Scopus (318) Google Scholar). Using an accessible-cavity geometry-based set of criteria (Pore-Walker) (Nugent and Jones, 2012Nugent T. Jones D.T. Detecting pore-lining regions in transmembrane protein sequences.BMC Bioinformatics. 2012; 13: 169Crossref PubMed Scopus (47) Google Scholar), IS1, IIS1, and IIS2 are predicted to potentially contain channel pore-lining helices.Figure S2TMEM175 Has a Two-Repeat-of-Six-TM Predicted Structure, Related to Figure 3Show full caption(A) Human TMEM175 sequence with putative transmembrane helices in bold and boxed. The FSD signature in IS1 and IIS1 (highlighted in red) are conserved in all the TMEM175 homologs and are required for channel function. Transmembrane domains were predicted using a hidden Markov model-based topology prediction method (HMMTOP, http://www.enzim.hu/hmmtop/).(B) Alignment between the N-terminal half (aa 1-250) containing repeat I and the C-terminal half (aa 251- 504) containing repeat II of hTMEM175. Sequences were aligned using ClustalW2 program. “∗” and “”: indicate identical and conserved residues, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Human TMEM175 sequence with putative transmembrane helices in bold and boxed. The FSD signature in IS1 and IIS1 (highlighted in red) are conserved in all the TMEM175 homologs and are required for channel function. Transmembrane domains were predicted using a hidden Markov model-based topology prediction method (HMMTOP, http://www.enzim.hu/hmmtop/). (B) Alignment between the N-terminal half (aa 1-250) containing repeat I and the C-terminal half (aa 251- 504) containing repeat II of hTMEM175. Sequences were aligned using ClustalW2 program. “∗” and “”: indicate identical and conserved residues, respectively. TMEM175 was previously detected from lysosomal membrane protein preparations by mass spectrometry (Chapel et al., 2013Chapel A. Kieffer-Jaquinod S. Sagné C. Verdon Q. Ivaldi C. Mellal M. Thirion J. Jadot M. Bruley C. Garin J. et al.An extended proteome map of the lysosomal membrane reveals novel potential transporters.Mol. Cell. Proteomics. 2013; 12: 1572-1588Crossref PubMed Scopus (137) Google Scholar, Schröder et al., 2007Schröder B. Wrocklage C. Pan C. Jäger R. Kösters B. Schäfer H. Elsässer H.P. Mann M. Hasilik A. Integral and associated lysosomal membrane proteins.Traffic. 2007; 8: 1676-1686Crossref PubMed Scopus (145) Google Scholar). To elucidate the localization of TMEM175, we tagged hTMEM175 with fluorescence proteins at its N terminus (with EYFP) or C terminus (with EGFP) (Figure 3B). The tagged TMEM175 partially colocalized with endosomal (Rab5, Figures 3C and 3E) and lysosomal (Lamp1, Figures 3D and 3F; Chapel et al., 2013Chapel A. Kieffer-Jaquinod S. Sagné C. Verdon Q. Ivaldi C. Mellal M. Thirion J. Jadot M. Bruley C. Garin J. et al.An extended proteome map of the lysosomal membrane reveals novel potential transporters.Mol. Cell. Proteomics. 2013; 12: 1572-1588Crossref PubMed Scopus (137) Google Scholar) markers. Because GFP and YFP lose fluorescence at acidic pH found in the lumen of lysosomes, the detection of bright YFP and GFP signals overlapping with Lamp1 suggests that the N and C termini of TMEM175 are localized in the cytosol (Figure 3A), a topology similar to that of TPCs (Zhu et al., 2010Zhu M.X. Ma J. Parrington J. Calcraft P.J. Galione A. Evans A.M. Calcium signaling via two-pore channels: local or global, that is the question.Am. J. Physiol. Cell Physiol. 2010; 298: C430-C441Crossref PubMed Scopus (100) Google Scholar). We transfected HEK293T cells with YFP-tagged hTMEM175 to identify TMEM175-expressing organelles to use for patch clamping. Overexpressing hTMEM175 led to large K+ currents in both endosomes (438.5 ± 133.5 pA/pF at +100 mV; n = 13; Figures 4A–4F) and lysosomes (374.3 ± 96.8 pA/pF, n = 6; Figures 4G–4I). Like the native IKEL, TMEM175 currents (ITMEM175) rose and fell instantaneously upon voltage changes without rectification (Figures 4E and 4H). Unlike KVs, TMEM175 did not inactivate even when tested with pulses of 10 s duration (Figure S3A).Figure S3Properties of TMEM175, Related to Figure 4Show full caption(A) Current traces elicited by voltage pulses of 10 s duration from an hTMEM175 expressing lysosome demonstrating that TMEM175 does not inactivate. Removal of K+ in the bath eliminated the outward current (not shown). Representative of 3.(B) Representative current traces (elicited by voltage ramps) recorded from hTMEM175 mutant with amino acid 39 (F39) within the conserved FSD signature in IS1 transmembrane domain mutated to valine. Recordings were done with 150 mM K+ containing pipette.(C) Averaged current amplitudes (at +100 mV) of mutants R35A, F39V, S40A, D41A, D41N and D41E. Data for the wild-type were taken from Figure 4F and was replotted for comparison.(D–G) Pharmacology of TMEM175. Outward currents were recorded with 150 mM K+ in the bath (cytosol) and 150 mM NMDG in pipette (lumen). Representative currents in the absence or presence of drugs are in (D).(E and F) Dose responses (n = 5 to 7). (G) Current amplitudes (at +100 mV) normalized to those in the absence of drugs. Numbers of organelles recorded are in parentheses. Data are presented as mean ± SEM.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Current traces elicited by voltage pulses of 10 s duration from an hTMEM175 expressing lysosome demonstrating that TMEM175 does not inactivate. Removal of K+ in the bath eliminated the outward current (not shown). Representative of 3. (B) Representative current traces (elicited by voltage ramps) recorded from hTMEM175 mutant with amino acid 39 (F39) within the conserved FSD signature in IS1 transmembrane domain mutated to valine. Recordings were done with 150 mM K+ containing pipette. (C) Averaged current amplitudes (at +100 mV) of mutants R35A, F39V, S40A, D41A, D41N and D41E. Data for the wild-type were taken from Figure 4F and was replotted for comparison. (D–G) Pharmacology of TMEM175. Outward currents were recorded with 150 mM K+ in the bath (cytosol) and 150 mM NMDG in pipette (lumen). Representative currents in the absence or presence of drugs are in (D). (E and F) Dose responses (n = 5 to 7). (G) Current amplitudes (at +100 mV) normalized to those in the absence of drugs. Numbers of organelles recorded are in parentheses. Data are presented as mean ± SEM. Removing K+ from the bath containing Cl− abolished the current, indicating that hTMEM175 is not permeable to anions (Figure 5A). The current increased when K+ was replaced with Rb+, suggesting that, like many canonical K+ channels, hTMEM175 is Rb+ permeable (Figure 5A). To determine hTMEM175’s cation selectivity, we recorded ITMEM175 under various ionic conditions. Replacing bath K+ with NMDG, Na+, or Ca2+ largely abolished the outward currents (moving into lysosomes) (Figures 5A–5C), suggesting that hTMEM175 is minimally permeable to NMDG, Na+ and Ca2+. We recorded IhTMEM175 under bi-ionic conditions and used the resulting reversal potentials to quantify hTMEM175’s relative permeability (Figures 5D and 5E). hTMEM175 was selective for K+ over Na+ and Ca2+ (PK/PNa = 36.0 ± 4.4, n = 11; PK/PCa = 141.6 ± 27.7, n = 9), with a PK/PNa within the range of those of KV and K2P channels (Heginbotham et al., 1994Heginbotham L. Lu Z. Abramson T. MacKinnon R. Mutations in the K+ channel signature sequence.Biophys. J. 1994; 66: 1061-1067Abstract Full Text PDF PubMed Scopus (688) Google Scholar, Honoré, 2007Honoré E. The neuronal background K2P channels: focus on TREK1.Nat. Rev. Neurosci. 2007; 8: 251-261Crossref PubMed Scopus (375) Google Scholar). Unlike the canonical K+ channels but similar to the behavior of lysosomal membranes (Casey et al., 1978Casey R.P. Hollemans M. Tager J.M. The permeability of the lysosomal membrane to small ions.Biochim. Biophys. Acta. 1978; 508: 15-26Crossref PubMed Scopus (30) Google Scholar, Henning, 1975Henning R. pH gradient across the lysosomal membrane generated by selective cation permeability and Donnan equilibrium.Biochim. Biophys. Acta. 1975; 401: 307-316Crossref PubMed Scopus (50) Google Scholar), hTMEM175 permeated Cs+ better than K+ (Figure 5B; PK/PCs = 0.51 ± 0.03, n = 9). We tested whether K+ conduction through hTMEM175 required a co-movement of H+, a property of H+ cotransporter/exchangers. Under conditions where there was no H+ gradient (pHlumen = pHcytosol = 7.2) and K+ was the only other permeable cation, the reversal potential follows the changes in [K+]cyt with a slope of 56.8 mV/decade (Figure 5F), close to that of a pure K+ electrode (58.8 mV/decade at 23°C), suggesting that H+ co-movement is not required (Accardi and Miller, 2004Accardi A. Miller C. Secondary active transport mediated by a prokaryotic homologue of ClC Cl- channels.Nature. 2004; 427: 803-807Crossref PubMed Scopus (517) Google Scholar). The highest sequence similarity among TMEM175s is found in IS1 and IIS1. These two regions contain an FSD signature conserved in TMEM175s of eukaryotes (Figure S2A), bacteria, and archaea. TMEM175 with the FSD signature mutated generated proteins comparable to that of the wild-type (WT; as judged by the intensity of the attached YFP protein, data not shown). However, the mutants exhibited no active channel activity (Figures S3B and S3C). ITMEM175 was not inhibited by Cs+, Ba2+, tetraethylammonium, or quinine at concentrations commonly used to block canonical K+ channels but was sensitive to Zn2+ (IC50, 38.4 μM) and 4-aminopyridine (4-AP; IC50, 35.0 μM) (Figures S3D–S3G). The lack of voltage dependence and the distinct pharmacological properties suggest that ITMEM175 was not the result of a potential upregulation of canonical K+ channel proteins caused by overexpressing TMEM175. The predicted 2 × 6TM structure of hTMEM175 is similar to that of the TPC channels but deviates from those of most other ion channels whose structures are 2TMs, 6TMs, or 4 × 6TM. To test whether proteins with only one of the two repeats can form ion channels, we expressed the bacterial TMEM175 homologs (bacTMEM175s) from gram-positive (Chryseobacterium; Genbank: KFF73457; cbTMEM175) and gram-negative (Streptomyces collinus, Genbank: AGS72644; scTMEM175) bacteria in HEK293T cells. The prokaryotic TMEM175s are small (192 aa in cbTMEM175 and 206 aa in scTMEM175) and are predicted to have only one 6TM repeat (Figure 6A). Bacterial and human TMEM175s are conserved, especially in the first two TMs (cbTMEM175 versus hTMEM175 repeat II: 39% identity, 56% similarity; Figures S4A–S4D). As a reference, the mammalian protein with the highest similarity to the bacterial Na+ channel NaChBac is the 4 × 6TM T-type Ca2+ channel CaV3.1. The identity and similarity between CaV3.1 and NaChBac are 27% and 51%, respectively.Figure S4Bacterial TMEM175 Homologs, Related to Figure 6Show full caption(A–E) Partial sequences of cbTMEM175 (from gram-positive bacterium Chryseobacterium; full-length, 192 aa) and scTMEM175 (from gram-negative bacterium Streptomyces collinus; full-length, 206 aa) are aligned against repeats I and II of hTMEM175 (A-D) and against each other (E). Putative S1 and S2 transmembrane segments are highlighted.(F–H) Functional expression of scTMEM175. Whole-cell recordings were done with scTMEM175-transfected HEK293T cells. (F) Representative whole-cell currents recorded with bath containing 150 mM K+ or 150 mM NMDG using a ramp protocol (−100 mV to +100 mV in 1 s, Vh = 0 mV). (G) Averaged current sizes at −100 mV. (H) Drug sensitivity presented as the current amplitudes (at −100 mV) after drug application normalized to those before. See Figure 6 for control recordings from mock-transfected cells. Data are presented as mean ± SEM.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A–E) Partial sequences of cbTMEM175 (from gram-positive bacterium Chryseobacterium; full-length, 192 aa) and scTMEM175 (from gram-negative bacterium Streptomyces collinus; full-length, 206 aa) are aligned against repeats I and II of hTMEM175 (A-D) and against each other (E). Putative S1 and S2 transmembrane segments are highlighted. (F–H) Functional expression of scTMEM175. Whole-cell recordings were done with scTMEM175-transfected HEK293T cells. (F) Representative whole-cell currents recorded with bath containing 150 mM K+ or 150 mM NMDG using a ramp protocol (−100 mV to +100 mV in 1 s, Vh = 0 mV). (G) Averaged current sizes at −100 mV. (H) Drug sensitivity presented as the current amplitudes (at −100 mV) after drug application normalized to those before. See Figure 6 for control recordings from mock-transfected cells. Data are presented as mean ± SEM. Unlike most other bacterial ion channel proteins, bacTMEM175s were readily expressed in mammalian cells. HEK293T transfected with bacTMEM175s had large K+ currents when recorded under symmetric [K+] conditions (Figures 6, S4F, and S4G). Thus, prokaryotes use TMEM175 homologs, instead of K2Ps, to form K+-permeable leak-like channels. As bacteria and archaea have no intracellular organelles, the function of TMEM175 is perhaps to set the membrane potentials of the cells and/or to regulate cellular osmolarity. The sequence similarity between the two bacTMEM175s (35% identity, 59% similarity) is only as large as the similarity between each and hTMEM175 (Figure S4). The two, however, generated currents with similar properties. Like hTMEM175, bacTMEM175s also had linear current (I)-voltage (V) relationships and the currents did not inactivate (Figures 6D, 6E, and S4F). Compared to hTMEM175, bacTMEM175s were less permeable to Cs (PK/PCs: 2.1 ± 0.7, n = 4 for cbTMEM175; 2.3 ± 0.3, n = 5 for scTMEM175) and were noticeably permeable to Na+ (PK/PNa: 2.4 ± 0.5, n = 4 for cbTMEM175; 4.4 ± 1.4, n = 5 for scTMEM175). Unlike hTMEM175, bacTMEM175s were not inhibited by 4-AP when applied either in the bath or in the pipette solutions (Figures