Neutralization of a Conserved Amino Acid Residue in the Human Na+/Glucose Transporter (hSGLT1) Generates a Glucose-gated H+ Channel

The role of conserved Asp204 in the human high affinity Na+/glucose cotransporter (hSGLT1) was investigated by site-directed mutagenesis combined with functional assays exploiting the Xenopus oocyte expression system. Substitution of H+ for Na+ reduces the apparent affinity of hSGLT1 for glucose from 0.3 to 6 mm. The apparent affinity for H+ (7 μm) is about three orders of magnitude higher than for Na+ (6 mm). Cation/glucose cotransport exhibits a coupling ratio of 2 Na+ (or 2 H+):1. Pre-steady-state kinetics indicate that similar Na+- or H+-induced conformational changes are the basis for coupled transport. Replacing Asp204 with Glu increases the apparent affinity for H+ by >20-fold with little impact on the apparent Na+ affinity. This implies that the length of the carboxylate side chain is critical for cation selectivity. Neutralization of Asp204 (Asp → Asn or Cys) reveals glucose-evoked H+ currents that were one order of magnitude greater than Na+ currents. These phlorizin-sensitive H+ currents reverse and are enhanced by internal acidification of oocytes. Together with a H+ to sugar stoichiometry as high as 145:1, these results favor a glucose-gated H+ channel activity of the mutant. Our observations support the idea that cotransporters and channels share common features. The role of conserved Asp204 in the human high affinity Na+/glucose cotransporter (hSGLT1) was investigated by site-directed mutagenesis combined with functional assays exploiting the Xenopus oocyte expression system. Substitution of H+ for Na+ reduces the apparent affinity of hSGLT1 for glucose from 0.3 to 6 mm. The apparent affinity for H+ (7 μm) is about three orders of magnitude higher than for Na+ (6 mm). Cation/glucose cotransport exhibits a coupling ratio of 2 Na+ (or 2 H+):1. Pre-steady-state kinetics indicate that similar Na+- or H+-induced conformational changes are the basis for coupled transport. Replacing Asp204 with Glu increases the apparent affinity for H+ by >20-fold with little impact on the apparent Na+ affinity. This implies that the length of the carboxylate side chain is critical for cation selectivity. Neutralization of Asp204 (Asp → Asn or Cys) reveals glucose-evoked H+ currents that were one order of magnitude greater than Na+ currents. These phlorizin-sensitive H+ currents reverse and are enhanced by internal acidification of oocytes. Together with a H+ to sugar stoichiometry as high as 145:1, these results favor a glucose-gated H+ channel activity of the mutant. Our observations support the idea that cotransporters and channels share common features. human high affinity Na+/glucose transporter α-methyl-d-glucopyranoside d-glucose 3.2 μm H+ pH 5.5 100 mm Na+ hSGLT1-wild-type Na+/proline transporter 4-morpholinoethanesulfonic acid nanocoulomb(s) The human high affinity Na+/glucose cotransporter (hSGLT1)1 is a member of a family of secondary transporter proteins encompassing more than 55 homologues from archaea, bacteria, yeast, insects, and mammals (1Turk E. Wright E.M. J. Membr. Biol. 1997; 159: 1-20Crossref PubMed Scopus (191) Google Scholar, 2Reizer J. Reizer A. Saier Jr., M.H. Biochim. Biophys. Acta. 1994; 1197: 133-166Crossref PubMed Scopus (206) Google Scholar). This family uses electrochemical Na+ gradients to drive the coupled uphill transport of a variety of substrates (sugars, amino acids, vitamins, osmolytes, ions, myo-inositol, urea, and water). The expression of hSGLT1 in Xenopus laevisoocytes has resulted in a comprehensive study of both steady-state and pre-steady-state kinetics (3Hediger M.A. Coady M.J. Ikeda T.S. Wright E.M. Nature. 1987; 330: 379-381Crossref PubMed Scopus (787) Google Scholar, 4Parent L. Supplisson S. Loo D.D.F. Wright E.M. J. Membr. Biol. 1992; 125: 49-62Crossref PubMed Scopus (212) Google Scholar, 5Parent L. Supplisson S. Loo D.D.F. Wright E.M. J. Membr. Biol. 1992; 125: 63-79PubMed Google Scholar). A six-state ordered binding model has been proposed in which transport results from ligand-induced conformational changes (6Wright E.M. Loo D.D.F. Panayotova-Heiermann M. Lostao M.P. Hirayama B.A. Mackenzie B. Boorer K. Zampighi G. J. Exp. Biol. 1994; 196: 197-212Crossref PubMed Google Scholar, 7Wright E.M. Loo D.D.F. Turk E. Hirayama B.A. Curr. Opin. Cell Biol. 1996; 8: 468-473Crossref PubMed Scopus (57) Google Scholar). In this model Na+ binds before sugar, with a coupling ratio of 2 Na+:1 glucose, and voltage influences both Na+ binding and the conformational states of the unloaded transporter. Functional analysis of SGLT chimeras and truncated proteins strongly suggests that the sugar pathway is located in the C-terminal domain of the protein (8Panayotova-Heiermann M. Loo D.D.F. Kong C.T. Lever J.E. Wright E.M. J. Biol. Chem. 1996; 271: 10029-10034Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 9Panayotova-Heiermann M. Eskandari S. Turk E. Zampighi G.A. Wright E.M. J. Biol. Chem. 1997; 272: 20324-20327Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Site-directed thiol labeling of a residue in the proposed sugar pathway indicates that conformational changes are responsible for the coupling of Na+ and sugar transport (10Loo D.D.F. Hirayama B.A. Gallardo E.M. Lam J. Turk E. Wright E.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7789-7794Crossref PubMed Scopus (136) Google Scholar). We suggest that these conformational alterations are induced by cation binding in the N-terminal domain of the protein. Although the functional importance of the N terminus in cation binding/translocation was shown for another SGLT family member (the Na+/proline transporter (PutP) of Escherichia coli) (11Quick M. Jung H. Biochemistry. 1997; 36: 4631-4636Crossref PubMed Scopus (42) Google Scholar, 12Quick M. Jung H. Biochemistry. 1998; 37: 13800-13806Crossref PubMed Scopus (41) Google Scholar, 13Quick M. Stoelting S. Jung H. Biochemistry. 1999; 38: 13523-13529Crossref PubMed Scopus (25) Google Scholar), there is little information on the role of the N-terminal domain in hSGLT1. We have initiated a study to explore the role of N-terminal residues in hSGLT1 in cotransport. In the present study, we have targeted a conserved residue, Asp204, located in a short cytoplasmic loop of hSGLT1 connecting transmembrane domains V and VI, which has been implicated in cation selectivity in PutP (12Quick M. Jung H. Biochemistry. 1998; 37: 13800-13806Crossref PubMed Scopus (41) Google Scholar). Replacing Asp204 in hSGLT1 with Asn, Cys, or Glu dramatically modulated the steady-state and pre-steady-state kinetics of the transporter. Remarkably, although a transporter with a negative amino acid (Asp or Glu) exhibited cation/glucose cotransport with a stoichiometry of 2 (Na+ or H+) to 1, neutralization of Asp204 (by Asn or Cys) resulted in the activation of a glucose-activated H+ channel. A plasmid containing human SGLT1 (hSGLT1) cDNA was used as template for site-directed mutagenesis. Replacement of Asp204 with Asn, Cys, and Glu was performed using a two-step polymerase chain reaction protocol (14Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene. 1989; 77: 51-59Crossref PubMed Scopus (6768) Google Scholar). For each pair of mutagenic oligonucleotides the sequence of the sense primer is presented with the altered nucleotide(s) underlined: D204C, 5′-GATTTACACGTGCACCTTGC-3′; D204E, 5′-GATTTACACGGAAACCTTGC-3′; D204N, 5′-GATTTACACGAACACCTTGC-3′. Polymerase chain reaction products were digested with BglII and Eco47III, and the resulting 428-bp fragments were ligated into a similarly treated wild-type hSGLT1-containing plasmid. The fidelity of the inserted DNA fragments was confirmed by sequencing double-stranded DNA (Sequenase version 2.0, DNA sequencing kit, United States Biochemical, Cleveland, OH) after alkaline denaturation (15Hattori M. Sakaki Y. Anal. Biochem. 1986; 152: 232-238Crossref PubMed Scopus (1053) Google Scholar). Each mutagenized DNA template was linearized with XbaI, transcribed, and cappedin vitro using the T3 RNA promoter (MEGAscript kit, Ambion, Austin, TX). X. laevis oocytes were injected with 50 ng of mRNA and were incubated in Barth medium containing gentamicin (5 mg/ml) at 18 °C for 3–7 days (4Parent L. Supplisson S. Loo D.D.F. Wright E.M. J. Membr. Biol. 1992; 125: 49-62Crossref PubMed Scopus (212) Google Scholar). For transport and electrophysiological experiments, oocytes were bathed in an assay buffer composed of 2 mm KCl, 1 mmMgCl2, 1 mm CaCl2, 10 mm HEPES-Tris, pH 7.5, and a combination of Na+or choline+ chloride salts to give a final concentration of 100 mm. For H+ activation experiments the pH of 100 mm choline buffer was varied between 8.0 and 4.5 by titration with Tris or Mes. Uptake of methyl-α-d-[U-14C]glucopyranoside (293 Ci/mol, Amersham Pharmacia Biotech) and electrophysiological measurements using the two-microelectrode voltage clamp technique were performed as described (16Ikeda T.S. Hwang E.S. Coady M.J. Hirayama B. Hediger M.A. Wright E.M. J. Membr. Biol. 1989; 110: 87-95Crossref PubMed Scopus (141) Google Scholar, 17Loo D.D.F. Hazama A. Supplisson S. Turk E. Wright E.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5767-5771Crossref PubMed Scopus (199) Google Scholar). The stoichiometry of cation-coupledd-[U-14C]glucose (316 Ci/mol, ICN Radiochemicals) uptake was determined under voltage clamp conditions (18Mackenzie B. Loo D.D.F. Wright E.M. J. Membr. Biol. 1998; 162: 101-106Crossref PubMed Scopus (106) Google Scholar). Sugar-evoked steady-state currents were fitted to Eq. 1, IGlc=ImaxGlc×[Glc]K0.5Glc+[Glc],Equation 1 where I Glc and ImaxGlc representd-glucose-induced current and maximald-glucose-induced current, respectively, at saturating [cation], [Glc] is the concentration of d-glucose, and K0.5Glc is [Glc] at 0.5 ImaxGlc. Kinetic parameters for the phlorizin-inhibited cation leak or cation-activated glucose transport were determined by using Eq. 2,Ication=Imaxcation×[C]n(K0.5cation)n+[C]n,Equation 2 where I cation and Imaxcation is the cation-evoked (leak) current and the maximal cation (leak) current at saturating cation concentration [C], respectively, K0.5cation is [C] at 0.5 and the for each voltage were by pre-steady-state current the and the steady-state currents from the with and were fitted to Eq. D.D.F. Hazama A. Supplisson S. Turk E. Wright E.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5767-5771Crossref PubMed Scopus (199) Google Scholar, A. Loo D.D.F. Wright E.M. J. Membr. Biol. 1997; PubMed Scopus (92) Google with and at and V 0.5 at which of the in the has is the apparent of the and have experiments were at three with oocytes from were performed using the in are on from a on a and of the In these between Na+ and H+ kinetics were performed in the oocyte D204C, D204E, or For of kinetics and the kinetics of a transporter with a at oocytes of the were on the 10 The Asp204 → Glu resulted in a of uptake by the uptake of and were by In the of the of uptake in oocytes was about 1 a to oocytes with the transporter Asp204 with or Asn the uptake by and This the enhanced apparent affinity for sugar of parameters in of Na+ and the maximal by the for saturating For at the the maximal is greater than the at and for sugar transport are shown I and were determined at 100 mm Na+ and 3.2 the maximal by the for saturating For at the the maximal is greater than the at in a and for sugar transport are shown I and were determined at 100 mm Na+ and 3.2 steady-state for D204C, and in the of and at saturating For both were and at A similar was for in in the currents exhibited a with and exhibited with of the currents at the negative in or in The glucose-evoked currents in for these were greater than the Na+ currents In for the glucose currents at more than at A of the currents at V similar in the of for and and a of about for In and currents by 100 at and the and exhibited than of the current for We the sugar kinetics of each transporter by [Glc] from to 100 mm in the of The apparent affinity at V are shown in described B.A. Lostao M.P. Panayotova-Heiermann M. Loo D.D.F. Turk E. Wright E.M. J. 1996; Google Scholar), hSGLT1 exhibited a K0.5Glc of 0.3 mm in for the apparent affinity for Glc was by K0.5Glc 6 mm). A similar of the apparent Glc affinity in was for each transporter with a of A of of Glu in of Asp204 the apparent affinity for Glc in or about The with a side chain at → Asn or which apparent for Although the K0.5Glc of in a of K0.5Glc was in mm). exhibited a more of the apparent affinity for Glc with a and of K0.5Glc in and in a from to the K0.5Glc determined in for to exhibited an K0.5Glc in voltage of the for the The ImaxGlc for each transporter with and were to the currents by 100 The cation impact on the magnitude of ImaxGlc for and exhibited a ImaxGlc in and the similar ImaxGlc of these shown at were by more the of of the ImaxGlc was for each transporter at in the of Asp204 in hSGLT1 the sugar of the oocytes were at and currents by 10 mm sugar were in the of or the selectivity for each transporter was in the order Glc with the of the cation on the apparent Glc affinity for and D204E, each sugar induced currents in the of than in The on the apparent Glc affinity of was by currents in and than with In and exhibited currents similar to the currents by each sugar in were than the currents for We determined the apparent cation concentration for Na+- and glucose transport K0.5cation for at V For the (7 pH was about three orders of magnitude than the (6 mm). the of the of Asp204 with a amino acid side chain → Asn or Cys) in one These exhibited a the was the Glu in of Asp204 by about mm). this of this transporter by a of the pH of the apparent concentration for glucose transport from pH to In for each transporter was and about from to the voltage by a of about The of for cation-activated glucose transport was than for both Na+ and H+ and was by voltage to Na+ of was for H+ was on the and exhibited an Na+ H+ of at for each transporter was from ImaxGlc in this in was for and that the of and and the of at the at V a transporter with a amino acid side chain at exhibited a D204N, than ImaxGlc in D204N, and with more negative the Na+ and H+ each or was varied between and 100 mm Na+ or between and μm H+ and phlorizin-sensitive currents and choline currents at pH 8.0 were from the currents. The kinetics of the cation leak were by the to Eq. In for each transporter for glucose cotransport and leak were the for the leak was than exhibited a leak of and of Asp204 with Asn 2 or Glu the leak to The leak for was to than for and there was H+ leak Although the for the Na+ or the H+ leak was for the of for each transporter was A current of oocytes injected with or in the of or after the from the to the to in the 1 each transporter exhibited currents that to a steady-state with a These were after of saturating The of a negative amino acid side chain at a of the current in of and were determined from the in and shown in A. of and was as a of a to for both was and shown as the the to for with in with in and with in with in The was by of the each V a of the the voltage a were fitted to Eq. to the 0.5 at which of the has in the and apparent of the of and were in and and of and were about The of by V 0.5 of from 1 to Glu in of Asp204 exhibited a V 0.5 in of 2 0.5 of this transporter in was to more In the of on the is shown for and or 3.2 respectively, the for at negative exhibited a the higher than 3.2 the of the transporter at V 0.5 as a of from 100 to V 0.5 of from 1 to 2 V 0.5 as a of a with a of in 0.5 for was on of In H+ and exhibited a similar of V 0.5 of was by about the concentration shown in and exhibited a of of and steady-state currents from the currents a of the pre-steady-state currents. was in on voltage in In the of the for the were and described a The for and was and at In in the of the to the of was dramatically to the of at and exhibited pre-steady-state currents that were at the of and from the voltage The stoichiometry of cotransport was determined by the into oocytes or and the of the current with the that the A of the glucose uptake by oocytes a in both with a of glucose in 2 0.3 in In the of the of the of was Replacing with the for to the concentration in to the for was on the stoichiometry was for or glucose uptake or current was in oocytes. We the of acidification on H+ currents. of the was by of choline chloride with G.A. Loo D.D.F. M. Eskandari S. Wright E.M. J. 1999; PubMed Scopus Google Scholar). on the of internal acidification of oocytes or in the of μm H+ In the of the currents for with and at acidification the magnitude of currents with at on currents at more negative than currents were after internal acidification of oocytes under 6 the that the of in the was about that of for for of mm the and currents for 6 The present study and on the kinetics of SGLT1 (4Parent L. Supplisson S. Loo D.D.F. Wright E.M. J. Membr. Biol. 1992; 125: 49-62Crossref PubMed Scopus (212) Google Scholar, 5Parent L. Supplisson S. Loo D.D.F. Wright E.M. J. Membr. Biol. 1992; 125: 63-79PubMed Google the steady-state and pre-steady-state kinetics of the transporter in Na+ are in with observations on hSGLT1 (10Loo D.D.F. Hirayama B.A. Gallardo E.M. Lam J. Turk E. Wright E.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7789-7794Crossref PubMed Scopus (136) Google Scholar, 17Loo D.D.F. Hazama A. Supplisson S. Turk E. Wright E.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5767-5771Crossref PubMed Scopus (199) Google Scholar, B.A. Lostao M.P. Panayotova-Heiermann M. Loo D.D.F. Turk E. Wright E.M. J. 1996; Google Scholar, Coady M.J. A. Biophys. J. Full Text PDF PubMed Scopus Google Scholar, Coady M.J. Biophys. J. 1996; Full Text PDF PubMed Scopus Google Scholar, Coady M.J. B. Biophys. J. 1997; Full Text PDF PubMed Scopus Google Scholar). we and on SGLT1 B.A. Loo D.D.F. Wright E.M. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, B.A. Loo D.D.F. Wright E.M. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google that H+ drive sugar cotransport hSGLT1. The kinetics of H+ and Na+ sugar cotransport are the apparent affinity of hSGLT1 for H+ is greater than for and the apparent affinity for sugar is about in than in In the of the stoichiometry the and voltage of transport are for Na+ and H+ cotransport in the the pre-steady-state parameters V and are in and The are that the for the leak pathway D.D.F. Hirayama B.A. G. Wright E.M. J. 1999; PubMed Scopus Google is about higher in than in and that is about one order of magnitude for the leak than for the cotransport. The is with the results for hSGLT1 by Coady M.J. B. Biophys. J. 1997; Full Text PDF PubMed Scopus Google Scholar). In to a for Na+ 1 and for the present study that the for both Na+ and H+ are Together with the concentration 0.5 in or this that Na+ or to SGLT1 before This is in to the Na+ (or binding sequence model proposed by Coady M.J. B. Biophys. J. 1997; Full Text PDF PubMed Scopus Google on model is with into that the for the H+ leak current was and that the currents for the Na+ leak were greater than with 100 in the present that Na+ is the to the transporter for the steady-state the of Na+/glucose cotransport is of (4Parent L. Supplisson S. Loo D.D.F. Wright E.M. J. Membr. Biol. 1992; 125: 49-62Crossref PubMed Scopus (212) Google Scholar, 5Parent L. Supplisson S. Loo D.D.F. Wright E.M. J. Membr. Biol. 1992; 125: 63-79PubMed Google Scholar). explore the role of the N terminus of hSGLT1 in we have the of a conserved amino acid residue, This residue is with in the Na+/proline cotransporter which is for cation selectivity and binding and transport (12Quick M. Jung H. Biochemistry. 1998; 37: 13800-13806Crossref PubMed Scopus (41) Google Scholar). Replacing Asp204 functional of on the or of a amino acid side chain at This in to where a than a residue at is for The of of a transporter with a Glu in of of the coupling the apparent affinity for glucose is by a cation on the glucose The apparent affinity of the transporter for Na+ is by the and ImaxGlc in and the leak are to These are by similar by these the exhibits an apparent H+ affinity. This implies that the side chain at by one dramatically or the of the cation A similar was for the transporter or M.P. A. Y. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). The apparent H+ affinity of is by the alkaline in the at binding of H+ conformational changes that are the basis for coupled transport (10Loo D.D.F. Hirayama B.A. Gallardo E.M. Lam J. Turk E. Wright E.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7789-7794Crossref PubMed Scopus (136) Google Scholar). This is the for the apparent of the of on H+ is the cation than μm at is in this between and μm is similar to from 1 μm to μm The in apparent affinity is by a of V 0.5 and a of at is for in from to to of the model for SGLT1 L. Supplisson S. Loo D.D.F. Wright E.M. J. Membr. Biol. 1992; 125: 63-79PubMed Google Scholar), this of and the higher are to higher binding and for the This is with the that H+ leak is and the glucose-evoked H+ currents were about for than for the of in H+ at the for cotransport for at greater than The of with a side chain at (Asp → Asn or using thiol the cytoplasmic of Asp204 (1Turk E. Wright E.M. J. Membr. Biol. 1997; 159: 1-20Crossref PubMed Scopus (191) Google E. Lostao M.P. Wright E.M. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google is is from the of a (by have a on the kinetics of the transporter. on this we a role of Asp204 in with a amino acid as proposed for residues in the M. Biochemistry. 1993; PubMed Scopus Google Scholar, M. Biochemistry. 1993; PubMed Scopus Google Scholar, Hwang J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). as by the of for and D204N, of the negative at reduces the of in the These a of the protein to the a common with the expression of a protein in a expression Turk E. Hirayama B.A. B. Wright E.M. Biochim. Biophys. Acta. 1999; PubMed Scopus Google Scholar, M.P. Hirayama B.A. Panayotova-Heiermann M. Wright E.M. PubMed Scopus Google Scholar). This from a analysis of the pre-steady-state kinetics of or of steady-state kinetics of and a of K0.5Glc in and The that this a in the idea that cation and sugar are in or A similar is proposed for the of E. where an N-terminal domain of the transporter a role in connecting and in of coupling Y. G. B. Biochemistry. PubMed Scopus Google Scholar, E. G. Biochemistry. PubMed Scopus Google Scholar). Although the apparent affinity parameters of the for and were altered and the steady-state activation of the currents a 1, the coupling ratio of Na+ to glucose transport the for Na+/glucose cotransport was indicates that neutralization of Asp204 and in hSGLT1 results in the activation of a glucose-activated H+ channel. This is on the observations The glucose-evoked are an order of magnitude greater than the the H+ currents the voltage this that the increases by more than one order of magnitude that in The H+ currents reverse at the Na+ currents for or or the H+ and Na+ currents for or These H+ currents are enhanced by internal acidification of the oocyte 6 and are by the of 6 The H+ currents for the coupling of cotransport. The stoichiometry increases from the of 2 to as high as The Na+/glucose stoichiometry for both and was is in the H+ leak currents for these The for the H+ leak currents for and are to that for the wild-type protein These results that changes in a side chain with an or are to a glucose-activated This channel activity is with sugar the is to the H+ channel is to the of on results we neutralization of this conserved residue a H+ or with the coupling cation of the the H+ Asp204 is located in a cytoplasmic that this residue is of the H+ with the of high of this this an Our observations support to the that cotransporters and channels share in For the cotransporters as ligand-induced chloride channels Curr. Opin. 1996; PubMed Scopus Google for this that hSGLT1 to in channel activity is in the mutagenized the a channel for the cation for and SGLT1 as a S. Wright E.M. M. Zampighi G.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar). The cotransporter as a in oocyte and has been that the chloride channel is by the S. M. M.P. Wright E.M. Zampighi G.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). We are to and for with the and for critical