NBCe1-A Transmembrane Segment 1 Lines the Ion Translocation Pathway

The electrogenic Na+/ HCO3- cotransporter (NBCe1-A) transports sodium and bicarbonate across the basolateral membrane of the renal proximal tubule. In this study the structural requirement of transmembrane segment 1 (TM1) residues in mediating NBCe1-A transport was investigated. Twenty-five introduced cysteine mutants at positions Gln-424 to Gly-448 were tested for their sensitivity to the methanethiosulfonate reagents (2-sulfonatoethyl) methanethiosulfonate (MTSES), [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET), and (2-aminoethyl) methanethiosulfonate (MTSEA). Two mutants, T442C and A435C, showed 100 and 70% sensitivity, respectively, to inhibition by all the three methanethiosulfonate (MTS) reagents, I441C had >50% sensitivity to MTSET and MTSEA, and A428C had 50% sensitivity to MTSEA inhibition. A helical wheel plot showed that mutants T442C, A435C, and A428C are clustered on one face of TM1 within a 100° arc. Topology analysis of TM1 with biotin maleimide and 2-((5(6)-tetramethylrhodamine)carboxylamino) ethyl methanethiosulfonate (MTS-TAMRA) revealed Thr-442 marks the C-terminal end of TM1 and that extracellular FGGLLG stretch is in a small aqueous-accessible cavity. Functional studies indicated that Thr-442 resides in a narrow region of the ion translocation pore with strong δ- helical dipole influence. Analysis of the corresponding residue of NBCe1-A-Thr-442 in AE1 (Thr-422) shows it is functionally insensitive to MTSES and unlabeled with MTS-TAMRA, indicating that AE1-TM1 is oriented differently from NBCe1-A. In summary, we have identified residues Thr-442, Ala-435, and Ala-428 in TM1 lining the ion translocation pore of NBCe1-A. Our findings are suggestive of a δ- helical dipole at the C-terminal end of TM1 involving Thr-442 that plays a critical role in the function of the cotransporter. The electrogenic Na+/ HCO3- cotransporter (NBCe1-A) transports sodium and bicarbonate across the basolateral membrane of the renal proximal tubule. In this study the structural requirement of transmembrane segment 1 (TM1) residues in mediating NBCe1-A transport was investigated. Twenty-five introduced cysteine mutants at positions Gln-424 to Gly-448 were tested for their sensitivity to the methanethiosulfonate reagents (2-sulfonatoethyl) methanethiosulfonate (MTSES), [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET), and (2-aminoethyl) methanethiosulfonate (MTSEA). Two mutants, T442C and A435C, showed 100 and 70% sensitivity, respectively, to inhibition by all the three methanethiosulfonate (MTS) reagents, I441C had >50% sensitivity to MTSET and MTSEA, and A428C had 50% sensitivity to MTSEA inhibition. A helical wheel plot showed that mutants T442C, A435C, and A428C are clustered on one face of TM1 within a 100° arc. Topology analysis of TM1 with biotin maleimide and 2-((5(6)-tetramethylrhodamine)carboxylamino) ethyl methanethiosulfonate (MTS-TAMRA) revealed Thr-442 marks the C-terminal end of TM1 and that extracellular FGGLLG stretch is in a small aqueous-accessible cavity. Functional studies indicated that Thr-442 resides in a narrow region of the ion translocation pore with strong δ- helical dipole influence. Analysis of the corresponding residue of NBCe1-A-Thr-442 in AE1 (Thr-422) shows it is functionally insensitive to MTSES and unlabeled with MTS-TAMRA, indicating that AE1-TM1 is oriented differently from NBCe1-A. In summary, we have identified residues Thr-442, Ala-435, and Ala-428 in TM1 lining the ion translocation pore of NBCe1-A. Our findings are suggestive of a δ- helical dipole at the C-terminal end of TM1 involving Thr-442 that plays a critical role in the function of the cotransporter. Bicarbonate transport processes play an essential role in systemic acid-base balance and intracellular pH regulation (1Pushkin A. Kurtz I. Am. J. Physiol. Renal Physiol. 2006; 290: 580-599Crossref PubMed Scopus (145) Google Scholar). NBCe1-A belongs to the SLC4 bicarbonate transporter family containing three functionally distinct groups of transporters: Na+-independent Cl-/ HCO3- exchangers, Na+- HCO3- cotransporters, and Na+-driven Cl-/ HCO3- exchangers, of which only NBCe1 and NBCe2 are electrogenic. Three NBCe1 variants have been reported; they are NBCe1-A, which is found in the kidney and eye, NBCe1-B, which is expressed in pancreas, duodenum, colon, and several other tissues, and NBCe1-C, which is predominantly expressed in the brain. NBCe1-A differs from NBCe1-B/NBCe1-C in the N-terminal initial 85 amino acids, and NBCe1-C differs from NBCe1-A/NBCe1-B in the last 61 amino acids in the C-terminal tail (1Pushkin A. Kurtz I. Am. J. Physiol. Renal Physiol. 2006; 290: 580-599Crossref PubMed Scopus (145) Google Scholar). NBCe1 mediates the electrogenic cotransport of Na+ and HCO3- across plasma membranes in specific cell types. In kidney, NBCe1-A in proximal tubule cells mediates the basolateral efflux of HCO3- from cell to the blood, thereby reabsorbing ∼80% of the filtered HCO3- load. In pancreas, NBCe1-B expressed in duct epithelial cells contributes to the basolateral influx of HCO3- from blood to cell, during the process of secretin-evoked pancreatic fluid secretion. The ion transport stoichiometry of NBCe1-A is 1 Na+/3 HCO3- but can be altered to 1Na+/2 HCO3- upon phosphorylation of a serine residue near the C terminus (2Gross E. Hawkins K. Pushkin A. Sassani P. Dukkipati R. Abuladze N. Hopfer U. Kurtz I. J. Physiol. (Lond.). 2001; 537: 659-665Crossref Scopus (81) Google Scholar). In addition, the cellular environment appears to play a role in modulating the transport stoichiometry of NBCe1 proteins (3Gross E. Abuladze N. Pushkin A. Kurtz I. Cotton C.U. J. Physiol. (Lond.). 2001; 531: 375-382Crossref Scopus (55) Google Scholar). Ten mutations in the SLC4 gene have been reported that cause autosomal recessive proximal renal tubular acidosis with ocular and intracerebral abnormalities (1Pushkin A. Kurtz I. Am. J. Physiol. Renal Physiol. 2006; 290: 580-599Crossref PubMed Scopus (145) Google Scholar). NBCe1-A is a 140-kDa glycoprotein containing 1035 amino acids. Glycosylation studies indicate that it transverses the lipid bilayer a minimum of 10 times (4Tatishchev S. Abuladze N. Pushkin A. Newman D. Liu W. Weeks D. Sachs G. Kurtz I. Biochemistry. 2003; 42: 755-765Crossref PubMed Scopus (32) Google Scholar), and hydropathy analyses predict up to 14 transmembrane regions. Based on anion exchanger 1 (AE1) topology (5Zhu Q. Lee D.W. Casey J.R. J. Biol. Chem. 2003; 278: 3112-3120Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), NBCe1-A is predicted to transverse the lipid bilayer 13 times with 2 reentrant loops (6Boron W.F. J. Am. Soc. Nephrol. 2006; 17: 2368-2382Crossref PubMed Scopus (125) Google Scholar). Both N and C termini of NBCe1-A are located in cytoplasm, with a large extracellular loop proposed between transmembrane segment (TM) 2The abbreviations used are: TM, transmembrane; BM, biotin maleimide, 3-(N-maleimidylpropionyl) biocytin; MTS, methanethiosulfonate; MTSEA, (2-aminoethyl) methanethiosulfonate; MTSET, [2-(trimethylammonium-)ethyl] methanethiosulfonate; MTSES, (2-sulfonatoethyl) methanethiosulfonate; MTS-TAMRA, 2-((5(6)-tetramethylrhodamine)carboxylamino) ethyl methanethiosulfonate; BCECF-AM, 2′,7′-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein tetrakis(acetoxymethyl) ester; LeuT, leucine transporter; AE1, anion exchanger 1; PBS, phosphate-buffered saline. 5 and 6 containing two glycosylation sites (Fig. 1A). NBCe1-A is predominantly a homodimer that is composed of functionally active monomers and whose oligomeric state is dependent on S-S bond formation (7Kao L. Sassani P. Azimov R. Pushkin A. Abuladze N. Peti-Peterdi J. Liu W. Newman D. Kurtz I. J. Biol. Chem. 2008; 283: 26782-26794Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). We previously performed a large scale mutagenesis study on NBCe1-A protein-targeted acidic and basic amino acids near or outside putative transmembrane segments and had identified additional inactivating mutations affecting the cotransporter function (8Abuladze N. Azimov R. Newman D. Sassani P. Liu W. Tatishchev S. Pushkin A. Kurtz I. J. Physiol. (Lond.). 2005; 565: 717-730Crossref Scopus (32) Google Scholar). In addition, proposed TM8 was recently analyzed by cysteine-scanning mutagenesis, and several residues were found to be involved in forming the ion translocation pore (9McAlear S.D. Bevensee M.O. J. Biol. Chem. 2006; 281: 32417-32427Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Protein domain swapping studies have suggested the electrogenicity of NBCe1-A is determined by the interaction of TMs in the lipid bilayer (10Choi I. Soo Yang H. Boron W.F. J. Physiol. (Lond.). 2007; 578: 131-142Crossref Scopus (22) Google Scholar). Currently, the structural basis underlying NBC1e1-A transport function remains A of a leucine transporter revealed that of the sodium and translocation A. E. 2005; PubMed Scopus Google Scholar). TM1 is for of the membrane domain of the AE1, a of the SLC4 bicarbonate transport family that NBCe1-A. of AE1 have that of amino acids at the putative of domain and TM1 AE1 H. D. N. Biochemistry. PubMed Scopus Google Scholar), in anion transport PubMed Scopus Google Scholar). The of TM1 in NBCe1-A is suggested by two additional all bicarbonate proteins to SLC4 family a amino stretch at the putative extracellular end of proposed NBCe1-A TM1 (Fig. a amino in TM1 of NBCe1-A proximal renal tubular acidosis and the transport of the cotransporter D. J. N. A. I. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). we that the TM1 region of NBCe1-A be and functionally and be involved in forming the translocation In this study we for the that TM1 of NBCe1-A in forming the ion translocation we have identified the residues in TM1 lining the ion translocation pore by cysteine-scanning mutagenesis with methanethiosulfonate (MTS) Our findings that NBCe1-A TM1 one the translocation and the FGGLLG amino stretch resides in an extracellular small cavity. A of this been an R. Abuladze N. Pushkin A. Liu W. Newman D. Kurtz I. J. 2007; Scholar). mutagenesis were from maleimide, BCECF-AM, and all cell reagents were from MTSEA, MTSET, MTSES, and were from Protein and were from and were from membrane was from kidney of NBCe1-A and AE1 in was used the for Twenty-five cysteine were introduced NBCe1-A at the of amino Gln-424 to and one cysteine was introduced AE1 at the of amino was performed mutagenesis the The of was by Protein NBCe1-A proteins were expressed by of kidney cells cells were in of with and cells were with the with a in that the was 2 of were at in a and cells were with pH and with a NBCe1-A in that the extracellular loop of NBCe1-A of at cells were with and with with in PBS, from for at were times with and in In cells were with 1 of for 2 and the were by a to a Functional of cells on were with NBCe1-A cells were with pH and a I. J. PubMed Scopus Google Scholar). were in 1 1 5 5 pH of intracellular pH were by cells to 1 1 5 pH were to a 1 1 5 pH was in all to the end of was by the The of was in the initial to In the cell was to the HCO3- was the initial of of to the transport with methanethiosulfonate (MTS) reagents, cells were to the reagents MTSEA for 1 for 5 in the the In studies cells were the of function of proteins were a of NBCe1-A for and sensitivity of mutants to were a of in the of of were performed in Functional of cells were on and with AE1 or transport was in a Na+- and the cells were in a Na+- and 1 1 5 pH a was was by the cells in a sodium 2 5 pH was in all to pH was In involving MTSES the cells were with MTSES in a Na+- and to the The of was in the initial of extracellular function and sensitivity to MTSES was a of AE1 of were performed in and cell with biotin maleimide previously (5Zhu Q. Lee D.W. Casey J.R. J. Biol. Chem. 2003; 278: 3112-3120Abstract Full Text Full Text PDF PubMed Scopus (137) Google Q. Casey J.R. 2007; PubMed Scopus Google Scholar). cells were with biotin maleimide in 1 of containing and 1 pH for 10 at the was by a of and cells were with of sodium 10 pH containing and on for 10 was by at for at The was with and and NBCe1-A was by NBCe1-A N terminus D. Pushkin A. Sassani P. Abuladze N. Kurtz I. Am. J. Physiol. Renal Physiol. 2001; 281: PubMed Google Scholar). (5Zhu Q. Lee D.W. Casey J.R. J. Biol. Chem. 2003; 278: 3112-3120Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). and were on and to proteins were by of with in pH containing Protein was by the with (4Tatishchev S. Abuladze N. Pushkin A. Newman D. Liu W. Weeks D. Sachs G. Kurtz I. Biochemistry. 2003; 42: 755-765Crossref PubMed Scopus (32) Google at a in containing for were performed cells NBCe1-A proteins were times with and with 100 in PBS, pH on of cells were with PBS, and NBCe1-A was analyzed a at and and from and were with a were with were to (5Zhu Q. Lee D.W. Casey J.R. J. Biol. Chem. 2003; 278: 3112-3120Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). were with 10 analysis was performed 10 was used to with NBCe1-A to MTSET, and MTSEA are small reagents that with the of which have been used to residues lining the translocation pore of membrane and A. PubMed Scopus Google Q. Casey J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (52) Google Scholar). an or introduced cysteine residue is in the ion translocation with of reagents the the transport A. PubMed Scopus Google Scholar). NBCe1-A cysteine residues (Fig. 1A). reagents in involving NBCe1-A we performed to the function of the Three cysteine reagents were in MTSEA, MTSES, and the extracellular pH of in the transport a of MTSEA is and Liu G. PubMed Scopus Google Scholar), MTSES a and MTSET a Both of the reagents are 1 of with 1 MTSEA, we in transport (Fig. were with 5 A and of the reagents had an in cells that all three reagents have on transport MTSES (7Kao L. Sassani P. Azimov R. Pushkin A. Abuladze N. Peti-Peterdi J. Liu W. Newman D. Kurtz I. J. Biol. Chem. 2008; 283: 26782-26794Abstract Full Text Full Text PDF PubMed Scopus (52) Google and performed in the (9McAlear S.D. Bevensee M.O. J. Biol. Chem. 2006; 281: 32417-32427Abstract Full Text Full Text PDF PubMed Scopus (35) Google of reagents on cysteine transport cells were with MTSES MTSET or MTSEA by the were expressed a of in the of the and of NBCe1-A that the three reagents had on transport function of cysteine were introduced the at corresponding to amino Gln-424 to which the putative TM1 and the stretch (Fig. of residues in a transmembrane H. A. P. D. J. W. H. and Scholar), we determined of the was and to the plasma the membrane of we an residues in the extracellular loop of NBCe1-A all NBCe1 to NBCe1-A on the plasma membrane cell only the is on the plasma a and shows that cells have membrane cells with have strong membrane (Fig. 1 shows of the proteins expressed on the plasma mutants and the mutants are we the cells with by showed the mutants are expressed but (Fig. all residues are the bicarbonate transporter family residue the the of was analyzed by with C-terminal and was of membrane of NBCe1-A introduced cysteine in a of the sensitivity of to reagents, we the transport of and the to shows that of cysteine mutants, the had 50% that of the transport 5 had that of the function of and to cysteine the transport function of by they are functionally or helical wheel analysis shows the mutants all on one helical face this face of TM1 is functionally by with other Our that transport function a the of to amino NBCe1-A transport function in of to amino D. J. N. A. I. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google of NBCe1-A topology of the C-terminal region of helical wheel for amino acids are to indicate functionally mutants residue MTSEA and residue and all three residues The helical wheel is from the extracellular end of TM1 from residue Thr-442 and the residues in TM1 of NBCe1-A the ion translocation we all the cysteine mutants for the sensitivity to three reagents, MTSES, MTSET, and cells introduced cysteine mutants were with reagents with the transport Functional sensitivity of to reagents was the in function in to the of shows that the transport of T442C and are to the inhibition of all the three reagents and inhibition I441C is to MTSET and MTSEA, and A428C is to MTSEA inhibition. wheel analysis shows that functionally mutants are clustered on one face of TM1 within a 100° I441C (Fig. In to the mutants, mutants showed transport been reported in studies of introduced cysteine mutants of and ion (9McAlear S.D. Bevensee M.O. J. Biol. Chem. 2006; 281: 32417-32427Abstract Full Text Full Text PDF PubMed Scopus (35) Google J. Full Text PDF PubMed Scopus Google S. J. J. PubMed Scopus Google Scholar). of with that the transport function of is by all three reagents, that Thr-442 resides in an aqueous-accessible A mutagenesis study of AE1 proposed the C-terminal end of TM1 is at residue J. J. 2005; PubMed Scopus Google and that the stretch is of the extracellular the of Thr-442 and the stretch of in NBCe1-A we the of introduced cysteine mutants to biotin maleimide a that was used previously for AE1 topology analysis (5Zhu Q. Lee D.W. Casey J.R. J. Biol. Chem. 2003; 278: 3112-3120Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). of which are in the N-terminal 10 are in the transmembrane and 2 are in the C-terminal tail (Fig. 1A). shows that is with BM, are aqueous-accessible cysteine residues in the of the proposed intracellular to serine (Fig. indicating the in the lipid bilayer or in a which of introduced In transport the was (Fig. We introduced 14 cysteine at amino positions between and cell of cells introduced cysteine NBCe1-A was performed with at A cysteine residue NBCe1-A and were in and shows is with BM, the is The biotin of introduced cysteine was by of and the corresponding NBCe1-A appears two on the that the and of NBCe1-A (Fig. the of NBCe1-A is the on the the of and is membrane is reported to be by (5Zhu Q. Lee D.W. Casey J.R. J. Biol. Chem. 2003; 278: 3112-3120Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), that the of NBCe1-A resides in a other the we the of whose is the were to which was an and in shows mutants to T442C were mutants and were with mutants and were in the region with residues that were The this region (Fig. of with introduced cysteine mutants were with BM, the of is that 50% that of the we to the of mutants to a is a that can be shows the and and cells had with MTS-TAMRA, cells with T442C, mutants were and with I441C were (Fig. A and that the T442C is to reagents but for we that residue Thr-442 resides at the C-terminal end of the ion translocation pore and that extracellular loop 1 from residue The inhibition of T442C transport by three reagents that it resides in a or structural Thr-442 is involved in the during transport we T442C with at and shows T442C is to to the or of Na+ in or We T442C with MTSES in the shows MTSES can T442C and an NBCe1-A-Thr-442 is involved in the that to an play a critical role in ion and transporter suggested by from and J. A. R. PubMed Scopus Google R. R. PubMed Scopus Google analysis of AE1 Q. Casey J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Our that Thr-442 resides at the extracellular end of TM1 to the of Thr-442, T442C is and by to helical dipole the C terminus of a transmembrane a and the N terminus a J. A. R. PubMed Scopus Google Scholar). of NBCe1-A-Thr-442 to cysteine is to transport amino acids have a δ- helical dipole at the C-terminal end of TM1 is critical for NBCe1-A transport we Thr-442 to amino acids with small or small or and with or function of proteins was shows that of Thr-442 to the residue had but with transport function by of with residue the transport but with the transport function to of to or residues transport function to of the δ- helical dipole at the C-terminal end of TM1 plays a critical role in the NBCe1-A transport and the the of a Our suggested that the for ion transport NBCe1-A is in composed of specific residues in TM1 that are in with the findings can be to other of the SLC4 we the role of the NBCe1-A-Thr-442 corresponding residue in anion exchanger AE1 We with cysteine and sensitivity to function was and with In to was of MTSES reagents on the transport function of a in AE1 (Fig. In addition, to (Fig. a and the of TM1 residues in the that the of TM1 in to other transmembrane segments differs between the two and that is to the Na+ and HCO3- across plasma is the structural basis for NBCe1 In the study we amino acids Gln-424 Gly-448 in the TM1 region of NBCe1-A to residues lining the ion translocation pore by the sensitivity of cysteine mutants to the cysteine we that the TM1 region of NBCe1-A of the ion translocation and the FGGLLG in SLC4 proteins is located in a small aqueous-accessible in NBCe1-A. Our the of A. E. 2005; PubMed Scopus Google and S. A. D. A. J. 2008; PubMed Scopus Google Scholar), which on sodium with in a cotransporter and the transport We have identified two introduced cysteine mutants and in the TM1 region of NBCe1-A that are by three reagents, MTSES, MTSET, and I441C was by MTSET and MTSEA and A428C by MTSEA The of residues have a helical and all on one face of a predicted I441C (Fig. The of inhibition of T442C and by MTSES, MTSET, and MTSEA was 100 and respectively, of the with the findings that Thr-442 and are in an environment that that differs from AE1, which a for Q. Casey J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The basis for inhibition by reagents is their to to a and to the of the ion translocation A. PubMed Scopus Google Scholar). The sensitivity of T442C to of their is with three for the Thr-442 residue in NBCe1-A that one of the ion sites in a in TM1 that is critical for during transport and a narrow region of a pore involving The is the of reagents on the T442C is by the of Na+ or HCO3- The of a of T442C with or MTSES the We Thr-442 at the of the ion translocation a narrow region to a and of a small the ion the that inhibition to reagents, it resides the lipid is by the that Thr-442 resides at the C-terminal end of TM1 and that of the or of the residue at this the transport function of NBCe1-A, indicating the δ- helical dipole involving Thr-442 contributes to the formation of the reagents have a of 6 in and 10 in A. PubMed Scopus Google Scholar). The of NBCe1-A transport function by reagents the at is 10 is by the that of Thr-442 with or amino acids that have or had with or whose are large that of transport function to 50% that of the of the that an to the extracellular end of TM1 a with an thereby the extracellular of the A. E. 2005; PubMed Scopus Google Scholar). The I441C was by MTSET and MTSEA by but insensitive to have two lining the ion translocation pore a environment is for MTSES and aqueous-accessible but lining the ion translocation the helical wheel resides at the C-terminal end of 100° from Thr-442, Ala-435, and which it that the ion translocation We that of MTSES the δ- helical dipole at the C-terminal end of of MTSET or MTSEA δ- helical dipole the transport I441C is to the of MTSET and MTSEA by to or MTS-TAMRA, it resides in a region to Ala-428 is by MTSEA but by In the pH of the a of MTSEA is and to the lipid The of MTSEA by the pore from the The of reagents to A428C transport function that a to in NBCe1-A, which is for Na+ and the helical wheel A428C is located within a 100° from T442C and A435C, it the ion translocation pore the intracellular Our that the FGGLLG is to but to this stretch of residues in a small aqueous-accessible cavity. maleimide is a with a of it with cysteine to a biotin is composed of a and with which the small the The in can be by the that the of to is that mutations in a segment can cause and to the plasma In this study we mutations in TM1 NBCe1-A intracellular of which two in the The of in this region it have an In AE1, and are expressed in the and the at a of J. J. 2005; PubMed Scopus Google Scholar). We that this region a for the of TMs during of to NBCe1-A transport function and proximal renal tubular acidosis D. J. N. A. I. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). we that of to 50% NBC1e1-A transport Our is with the the of to NBCe1-A transport function D. J. N. A. I. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is that the of the residue at this is for cotransporter the corresponding residue of in AE1 in the stretch of residues P. J. D. K. P. S. Liu U. S. A. PubMed Scopus Google Scholar). Our that residue in AE1 to the cause of The structural the SLC4 that for their ion transport are Our are the a structural between the two of the SLC4 the bicarbonate transporter NBCe1-A and bicarbonate exchanger The of in AE1 to MTSES inhibition that AE1 a with NBCe1-A. residues in NBCe1-A TM8 were previously reported to the ion translocation pore in AE1, the of NBCe1-A TM8 cysteine mutants to MTSEA from AE1 (9McAlear S.D. Bevensee M.O. J. Biol. Chem. 2006; 281: 32417-32427Abstract Full Text Full Text PDF PubMed Scopus (35) Google D. Casey J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (55) Google Scholar). studies are to the and interaction between TM1 and TM8 in NBCe1-A. The anion translocation pore of AE1 been and is an with on of the membrane to a narrow translocation in the of the lipid bilayer Q. Casey J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (52) Google D. Casey J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Our that the of the three reagents, MTSEA, the ion translocation in NBCe1-A, the on AE1 mutants was Q. Casey J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (52) Google D. Casey J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (55) Google Scholar). findings NBCe1-A an ion translocation from AE1, the leucine In summary, we have identified three residues on one helical face of TM1 lining the ion translocation of NBCe1-A. residue Thr-442 marks the C-terminal end of TM1 and is involved in forming a narrow region for the ion the translocation The FGGLLG resides in a small aqueous-accessible on the extracellular of NBCe1-A Our that the of the NBCe1-A ion translocation of NBCe1-A is from that of We of for the AE1

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