Rachel Darman

Recurrent mutations in the spliceosome are observed in several human cancers, but their functional and therapeutic significance remains elusive. SF3B1, the most frequently mutated component of the spliceosome in cancer, is involved in the recognition of the branch point sequence (BPS) during selection of the 3' splice site (ss) in RNA splicing. Here, we report that common and tumor-specific splicing aberrations are induced by SF3B1 mutations and establish aberrant 3' ss selection as the most frequent splicing defect. Strikingly, mutant SF3B1 utilizes a BPS that differs from that used by wild-type SF3B1 and requires the canonical 3' ss to enable aberrant splicing during the second step. Approximately 50% of the aberrantly spliced mRNAs are subjected to nonsense-mediated decay resulting in downregulation of gene and protein expression. These findings ascribe functional significance to the consequences of SF3B1 mutations in cancer.

The secretory Na-K-Cl cotransporter NKCC1 is activated by secretagogues through a phosphorylation-dependent mechanism. We found a phosphorylation stoichiometry of 3.0 ± 0.4 phosphorylated residues/NKCC1 protein harvested from shark rectal gland tubules maximally stimulated with forskolin and calyculin A, showing that at least three sites on the cotransporter are phosphorylated upon stimulation. Three phosphoacceptor sites were identified in the N-terminal domain of the protein (at Thr184, Thr189, and Thr202) using high pressure liquid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry to analyze tryptic fragments of the radiolabeled cotransporter. None of these residues occurs in the context of strong consensus sites for known Ser/Thr kinases. The threonines and the surrounding amino acids are highly conserved between NKCC1 and NKCC2, and similarities are also present in the Na-Cl cotransporter NCC (or TSC). This strongly suggests that the phosphoregulatory mechanism is conserved among isoforms. Through expression of shark NKCC1 mutants in HEK-293 cells, Thr189 was found to be necessary for activation of the protein, whereas phosphorylation at and was with the and that protein to residues in the shark NKCC1 these that the of NKCC1 a phosphoregulatory domain of the The secretory Na-K-Cl cotransporter NKCC1 is activated by secretagogues through a phosphorylation-dependent mechanism. We found a phosphorylation stoichiometry of 3.0 ± 0.4 phosphorylated residues/NKCC1 protein harvested from shark rectal gland tubules maximally stimulated with forskolin and calyculin A, showing that at least three sites on the cotransporter are phosphorylated upon stimulation. Three phosphoacceptor sites were identified in the N-terminal domain of the protein (at Thr184, Thr189, and Thr202) using high pressure liquid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry to analyze tryptic fragments of the radiolabeled cotransporter. None of these residues occurs in the context of strong consensus sites for known Ser/Thr kinases. The threonines and the surrounding amino acids are highly conserved between NKCC1 and NKCC2, and similarities are also present in the Na-Cl cotransporter NCC (or TSC). This strongly suggests that the phosphoregulatory mechanism is conserved among isoforms. Through expression of shark NKCC1 mutants in HEK-293 cells, Thr189 was found to be necessary for activation of the protein, whereas phosphorylation at and was with the and that protein to residues in the shark NKCC1 these that the of NKCC1 a phosphoregulatory domain of the protein matrix-assisted laser desorption ionization time-of-flight high pressure liquid chromatography shark NKCC1 The secretory Na-K-Cl cotransporter NKCC1 is a for of in with the Na-K-Cl cotransporter and the Na-Cl NCC (or NKCC1 to the of the cotransporter also the and of and are are to that are for and and that are for NKCC1 is in and is in the of and This is also a of the of secretory cells, the in the of the is to the of the of is a of from the that the of and be the Na-K-Cl cotransporter is to The cotransporter is in the of secretory the of secretagogues a phosphorylation-dependent activation of the cotransporter on shark rectal gland secretory in phosphorylation at and threonines in to forskolin and the of NKCC1 to be the of at least of the phosphoregulatory sites in cells, in Na-K-Cl cotransporter the cotransporter to be phosphorylated by that the of and phosphorylation of NKCC1 in to in and by rectal gland of the shark to be and for the of the rectal gland to phosphorylation sites on NKCC1 and that the a phosphorylation the three Thr189 is to be for whereas and are necessary for and activation of the stoichiometry of NKCC1 phosphorylation in rectal gland by the of of Na-K-Cl cotransporter a with forskolin and calyculin of 3.0 ± 0.4 of cotransporter was in using the of in This is to that by for cotransporter phosphorylation in the of stoichiometry a of the of phosphoacceptor We that is of the cotransporter and of is in the of and to of were with and were by for NKCC1 is with a between cotransporter phosphorylation and phosphorylated residues in cotransporter protein was to and by The Na-K-Cl cotransporter with of a of the of phosphorylation sites on the cotransporter protein to and and with on the phosphorylated were identified by mass in by to from the The of in the were by We were to of the phosphorylated from the with of and to these with of the protein a of the that to the of the phosphorylation from of the are in of phosphorylation of of by Thr189 in a spectrometry of in were and in and to the mass of The in was with to spectrometry of in and The in was with to in the of with a mass of and the with a mass of on mass the the in the domain of the the mass in the a with and these were was with and by the was the was and a at mass of is with the of from The of in the whereas a at a with the of from a with a mass of by the of is that a and mass the of the was found to the in was of the was phosphorylated in the a of the The identified by and in a to that was to a phosphoacceptor of the high in of the of in the of a with a phosphorylation in the at and the at The was to and of The was by in the of and by in the the were with of a from a on the that were three in the with a mass and the to with a mass of a occurs on and the in the is with the The of the was also identified the phosphorylated in the of these phosphorylation sites in of the mutants of the phosphoacceptor sites were in HEK-293 of these was and to the by in The is for shark NKCC1 and the HEK-293 in and is that for of the was in in NKCC1 to be present in the and be in of in HEK-293 are and of of of HEK-293 and The of to is activated in the is of and to in HEK-293 in the Thr189 phosphoacceptor with in a with cotransporter the the activation of the cotransporter by a these mutants the HEK-293 and the with a of Na-K-Cl in and phosphorylation of HEK-293 were in of the of to the to the protein for are ± from to for for the in by the of a phosphoacceptor with the of phosphorylation at that for Thr189 of the Na-K-Cl with The a activation the to be activated in to the is in a of the identified phosphoacceptor sites in a cotransporter also the Thr189 to the consensus for phosphorylation by of the with in by with the Thr189 these the of the cotransporter and was of in by of using forskolin forskolin of in HEK-293 We that the context of Thr189 to that the context of the in the necessary cotransporter of activation of Na-K-Cl in and were for in with forskolin with for the on was in was in in the from a with showing the of the in a The of are from were in at least for to the with Thr189, of the and in that were of activation the activation of these mutants by in of the from in the of is also the from with mutants were to with showing at the A, and This is with a mechanism in phosphorylation at residues for the of phosphorylated with Thr189, phosphorylation at be by with the of were to of with was also found to be to with the was for the at a to the of and was to in with The was to the mutants in activation A, whereas the of was and from that in and the of Thr184, are phosphoacceptor and of are in a context to that these residues also be in activation of the a in the of was to that of with a for the to be activated by a activation the phosphorylation in in in HEK-293 was activated by the was also activated by to of from to in in these forskolin the to the and at least a for the forskolin of at of to the of the and mutants were activated and and at and The is in the of was the activated by activated in was to upon to is that be by a of the to for that were the be in the the of that activation through of phosphorylated the of these found that Thr189 mutants that was that of was at of cotransporter activation This of in in and also with a of in mutants of and in between and is the present are of at the for in the and of in and mutants with HEK-293 and from for are from the of in The are for with for HEK-293 in for of shark rectal gland Na-K-Cl cotransporter protein to a of a phosphorylation stoichiometry and to the of three phosphoacceptor sites (at Thr184, Thr189, and sites are to a in a of the N-terminal conserved between isoforms. The of of the was by mutants of in HEK-293 phosphorylation of Thr189 was found to be necessary and for cotransporter and and were identified The sites to be for activation and for to and phosphorylation stoichiometry is with the that phosphorylation of at least is for activation of the be that of phosphorylation stoichiometry is to be of the of phosphoacceptor sites and be phosphorylation stimulation. This is in is that of the and of protein a of the of the and Thr189 a in that is phosphorylated in to the of stoichiometry for NKCC1 in cotransporter maximally stimulated by of at least phosphoacceptor sites in is also identified three the of in activated NKCC1 from shark and that the N-terminal to is a for phosphorylation by the This is highly conserved between shark and and a of threonines and in a context to that of of of these found that the is from NKCC1 are residues in of are conserved between shark and the residues of be found in tryptic is present in for were to mass of the of are to between shark and with the identified Thr184, Thr189, and in a highly conserved of the of The these phosphorylation sites are between shark and NKCC1 by in for of The of three in the of NKCC1 that Thr189 is phosphorylated and is with the of that of the phosphorylation of NKCC1 in gland is found in the N-terminal The identified phosphoregulatory is conserved between NKCC1 and NKCC2, with amino in the to residues The that the domain conserved in in NKCC1 that are activated by the and from strongly for of also is between the the Na-Cl cotransporter NCC to Thr184, Thr189, and in a that NCC is also by in to a of that the of is the domain of the The phosphorylation identified in is amino acids of a protein identified by that the by of the cotransporter This the that the cotransporter is at and is at a that in to None of the phosphoacceptor threonines strong consensus for a known and that NKCC1 phosphorylation is by high is maximally activated with forskolin that and N-terminal is the and is that of these NKCC1 in strongly the that the to that of cotransporter from stimulated with calyculin A, and are and for forskolin and of shark and the that with in the of cotransporter is these mutants were activated by in were activated in activation of This be by a of the This is with the a the that the in a with of is by the of at and the a the at these sites the by phosphorylation at is to the of the to the of the in the the in in the activation of are by the phosphorylation of the This that a of phosphorylation sites on a of the of activation of the in the to a and The is highly for and is to between phosphorylated and with a The of activation in and a of the in of The secretory Na-K-Cl cotransporter NKCC1 is a for of in with the Na-K-Cl cotransporter and the Na-Cl NCC (or NKCC1 to the of the cotransporter also the and of and are are to that are for and and that are for NKCC1 is in and is in the of and This is also a of the of secretory cells, the in the of the is to the of the of is a of from the that the of and be the Na-K-Cl cotransporter is to The cotransporter is in the of secretory the of secretagogues a phosphorylation-dependent activation of the cotransporter on shark rectal gland secretory in phosphorylation at and threonines in to forskolin and the of NKCC1 to be the of at least of the phosphoregulatory sites in cells, in Na-K-Cl cotransporter the cotransporter to be phosphorylated by that the of and phosphorylation of NKCC1 in to in and by The rectal gland of the shark to be and for the of the rectal gland to phosphorylation sites on NKCC1 and that the a phosphorylation the three Thr189 is to be for whereas and are necessary for and activation of the stoichiometry of NKCC1 phosphorylation in rectal gland by the of of Na-K-Cl cotransporter a with forskolin and calyculin of 3.0 ± 0.4 of cotransporter was in using the of in This is to that by for cotransporter phosphorylation in the of stoichiometry a of the of phosphoacceptor We that is of the cotransporter and of is in the of and to of were with and were by for NKCC1 is with a between cotransporter phosphorylation and phosphorylated residues in cotransporter protein was to and by The Na-K-Cl cotransporter with of a of the of phosphorylation sites on the cotransporter protein to and and with on the phosphorylated were identified by mass in by to from the The of in the were by We were to of the phosphorylated from the with of and to these with of the protein a of the that to the of the phosphorylation from of the are in of phosphorylation of of by Thr189 in a spectrometry of in and The in was with to in the of with a mass of and the with a mass of on mass the the in the domain of the the mass in the a with and these were was with and by the was the was and a at mass of is with the of from The of in the whereas a at a with the of from a with a mass of by the of is that a and mass the of the was found to the in was of the was phosphorylated in the a of the The identified by and in a to that was to a phosphoacceptor of the high in of the of in the of a with a phosphorylation in the at and the at The was to and of The was by in the of and by in the the were with of a from a on the that were three in the with a mass and the to with a mass of a occurs on and the in the is with the The of the was also identified the phosphorylated in the of these phosphorylation sites in of the mutants of the phosphoacceptor sites were in HEK-293 of these was and to the by in The is for shark NKCC1 and the HEK-293 in and is that for of the was in in NKCC1 to be present in the and be in of in HEK-293 are and of of of HEK-293 and The of to is activated in the is of and to in HEK-293 in the Thr189 phosphoacceptor with in a with cotransporter the the activation of the cotransporter by a these mutants the HEK-293 and the with a of Na-K-Cl in and phosphorylation of HEK-293 were in of the of to the to the protein for are ± from to for for the in by the of a phosphoacceptor with the of phosphorylation at that for Thr189 of the Na-K-Cl with The a activation the to be activated in to the is in a of the identified phosphoacceptor sites in a cotransporter also the Thr189 to the consensus for phosphorylation by of the with in by with the Thr189 these the of the cotransporter and was of in by of using forskolin forskolin of in HEK-293 We that the context of Thr189 to that the context of the in the necessary cotransporter of activation of Na-K-Cl in and were for in with forskolin with for the on was in was in in the from a with showing the of the in a The of are from were in at least for to the with Thr189, of the and in that were of activation the activation of these mutants by in of the from in the of is also the from with mutants were to with showing at the A, and This is with a mechanism in phosphorylation at residues for the of phosphorylated with Thr189, phosphorylation at be by with the of were to of with was also found to be to with the was for the at a to the of and was to in with The was to the mutants in activation A, whereas the of was and from that in and the of Thr184, are phosphoacceptor and of are in a context to that these residues also be in activation of the a in the of was to that of with a for the to be activated by a activation the phosphorylation in in in HEK-293 was activated by the was also activated by to of from to in in these forskolin the to the and at least a for the forskolin of at of to the of the and mutants were activated and and at and The is in the of was the activated by activated in was to upon to is that be by a of the to for that were the be in the the of that activation through of phosphorylated the of these found that Thr189 mutants that was that of was at of cotransporter activation This of in in and also with a of in mutants of and in between and is the present are of at the for in the and of in and mutants with HEK-293 and from for are from the of in The are for with for HEK-293 in for We the stoichiometry of NKCC1 phosphorylation in rectal gland by the of of Na-K-Cl cotransporter a with forskolin and calyculin of 3.0 ± 0.4 of cotransporter was in using the of in This is to that by for cotransporter phosphorylation in the of stoichiometry a of the of phosphoacceptor We that is of the cotransporter and of is in the of and to of were with and were by for NKCC1 is with a between cotransporter phosphorylation and phosphorylated residues in cotransporter protein was to and by The Na-K-Cl cotransporter with of a of the of phosphorylation sites on the cotransporter protein to and and with on the phosphorylated were identified by mass in by to from the The of in the were by We were to of the phosphorylated from the with of and to these with of the protein a of the that to the of the phosphorylation from of the are in in the of with a mass of and the with a mass of on mass the the in the domain of the the mass in the a with and these were was with and by the was the was and a at mass of is with the of from The of in the whereas a at a with the of from a with a mass of by the of is that a and mass the of the was found to the in was of the was phosphorylated in the a of the The identified by and in a to that was to a phosphoacceptor of the high in of the of in the of a with a phosphorylation in the at and the at The was to and of The was by in the of and by in the the were with of a from a on the that were three in the with a mass and the to with a mass of a occurs on and the in the is with the The of the was also identified the phosphorylated in the of these phosphorylation sites in of the mutants of the phosphoacceptor sites were in HEK-293 of these was and to the by in The is for shark NKCC1 and the HEK-293 in and is that for of the was in in NKCC1 to be present in the and be in NKCC1 is activated in the is of and to in HEK-293 in the Thr189 phosphoacceptor with in a with cotransporter the the activation of the cotransporter by a these mutants the HEK-293 and the with a by the of a phosphoacceptor with the of phosphorylation at that for Thr189 of the Na-K-Cl with The a activation the to be activated in to the is in a of the identified phosphoacceptor sites in a cotransporter We also the Thr189 to the consensus for phosphorylation by of the with in by with the Thr189 these the of the cotransporter and was of in by of using forskolin forskolin of in HEK-293 We that the context of Thr189 to that the context of the in the necessary cotransporter to the with Thr189, of the and in that were of activation the activation of these mutants by in of the from in the of is also the from with mutants were to with showing at the A, and This is with a mechanism in phosphorylation at residues for the of phosphorylated with Thr189, phosphorylation at be by with the of were to of with The was also found to be to with the was for the at a to the of and was to in with The was to the mutants in activation A, whereas the of was and from that in and the of Thr184, are phosphoacceptor and of are in a context to that these residues also be in activation of the a in the of was to that of with a for the to be activated by a activation the phosphorylation in in in HEK-293 was activated by the was also activated by to of from to in in these forskolin the to the and at least a for the forskolin of at of stimulation. to the of the and mutants were activated and and at and The is in the of was the activated by activated in was to upon to is that be by a of the to for that were the be in the the of that activation through of phosphorylated the of these found that Thr189 mutants that was that of was at of cotransporter activation This of in in and also with a of in mutants of and in between and is the present are of at the for in the and of of shark rectal gland Na-K-Cl cotransporter protein to a of a phosphorylation stoichiometry and to the of three phosphoacceptor sites (at Thr184, Thr189, and sites are to a in a of the N-terminal conserved between isoforms. The of of the was by mutants of in HEK-293 phosphorylation of Thr189 was found to be necessary and for cotransporter and and were identified The sites to be for activation and for to and phosphorylation stoichiometry is with the that phosphorylation of at least is for activation of the be that of phosphorylation stoichiometry is to be of the of phosphoacceptor sites and be phosphorylation stimulation. This is in is that of the and of protein a of the of the and Thr189 a in that is phosphorylated in to the of stoichiometry for NKCC1 in cotransporter maximally stimulated by of at least phosphoacceptor sites in is also identified three the of in activated NKCC1 from shark and that the N-terminal to is a for phosphorylation by the This is highly conserved between shark and and a of threonines and in a context to that of of of these found that the is from NKCC1 are residues in of are conserved between shark and the residues of be found in tryptic is present in for were to mass the identified Thr184, Thr189, and in a highly conserved of the of The these phosphorylation sites are between shark and NKCC1 by in for of The of three in the of NKCC1 that Thr189 is phosphorylated and is with the of that of the phosphorylation of NKCC1 in gland is found in the N-terminal The identified phosphoregulatory is conserved between NKCC1 and NKCC2, with amino in the to residues The that the domain conserved in in NKCC1 that are activated by the and from strongly for of also is between the the Na-Cl cotransporter NCC to Thr184, Thr189, and in a that NCC is also by in to a of that the of is the domain of the The phosphorylation identified in is amino acids of a protein identified by that the by of the cotransporter This the that the cotransporter is at and is at a that in to None of the phosphoacceptor threonines strong consensus for a known and that NKCC1 phosphorylation is by high is maximally activated with forskolin that and N-terminal is the and is that of these NKCC1 in strongly the that the to that of cotransporter from stimulated with calyculin A, and are and for forskolin and of shark and the that with in the of cotransporter is these mutants were activated by in were activated in activation of This be by a of the This is with the a the that the in a with of is by the of at and the a the at these sites the by phosphorylation at is to the of the to the of the in the the in in the activation of are by the phosphorylation of the This that a of phosphorylation sites on a of the of activation of the in the to a and The is highly for and is to between phosphorylated and with a The of activation in and a of the in of The of shark rectal gland Na-K-Cl cotransporter protein to a of a phosphorylation stoichiometry and to the of three phosphoacceptor sites (at Thr184, Thr189, and sites are to a in a of the N-terminal conserved between isoforms. The of of the was by mutants of in HEK-293 phosphorylation of Thr189 was found to be necessary and for cotransporter and and were identified The sites to be for activation and for to and phosphorylation stoichiometry is with the that phosphorylation of at least is for activation of the be that of phosphorylation stoichiometry is to be of the of phosphoacceptor sites and be phosphorylation stimulation. This is in is that of the and of protein a of the of the and Thr189 a in that is phosphorylated in to the of stoichiometry for NKCC1 in cotransporter maximally stimulated by of at least phosphoacceptor sites in is also identified three the of in activated NKCC1 from shark and We that the N-terminal to is a for phosphorylation by the This is highly conserved between shark and and a of threonines and in a context to that of of of these found that the is from NKCC1 are residues in of are conserved between shark and the residues of be found in tryptic is present in for were to mass the identified Thr184, Thr189, and in a highly conserved of the of The these phosphorylation sites are between shark and NKCC1 by in for of The of three in the of NKCC1 that Thr189 is phosphorylated and is with the of that of the phosphorylation of NKCC1 in gland is found in the N-terminal The identified phosphoregulatory is conserved between NKCC1 and NKCC2, with amino in the to residues The that the domain conserved in in NKCC1 that are activated by the and from strongly for of also is between the the Na-Cl cotransporter NCC to Thr184, Thr189, and in a that NCC is also by in The to a of that the of is the domain of the The phosphorylation identified in is amino acids of a protein identified by that the by of the cotransporter This the that the cotransporter is at and is at a The that in to None of the phosphoacceptor threonines strong consensus for a known and that NKCC1 phosphorylation is by high is maximally activated with forskolin that and N-terminal is the and is that of these NKCC1 in strongly the that the to that of cotransporter from stimulated with calyculin A, and are and for forskolin and of shark and the that with in the of cotransporter is these mutants were activated by in were activated in activation of This be by a of the This is with the a the that the in a with of is by the of at and the a the at these sites the by phosphorylation at is to the of the to the of the in the the in in the activation of are by the phosphorylation of the This that a The of phosphorylation sites on a of the of activation of the in the to a and The is highly for and is to between phosphorylated and with a The of activation in and a of the in of We and for protein amino and mass for and for of We are to for and to and for on the

The Na-K-Cl cotransporter NKCC1 is activated by phosphorylation of a regulatory domain in its N terminus. In the accompanying paper (Darman, R. B., and Forbush, B. (2002) J. Biol. Chem. 277, 37542-37550), we identify three phosphothreonines important in this process. Using a phospho-specific antibody (anti-phospho-NKCC1 antibody R5) raised against a diphosphopeptide containing Thr(212) and Thr(217) of human NKCC, we were readily able to monitor the cotransporter activation state. In (32)P phosphorylation experiments with rectal gland tubules, we show that the R5 antibody signal is proportional to the amount of (32)P incorporated into NKCC1; and in experiments with NKCC1-transfected HEK-293 cells, we demonstrate that R5-detected phosphorylation directly mirrors functional activation. Immunofluorescence analysis of shark rectal gland shows activation-dependent R5 antibody staining along the basolateral membrane. In perfused rat parotid glands, isoproterenol induced staining of both acinar and ductal cells along the basolateral membrane. Isoproterenol also induced basolateral staining of the epithelial cells in rat trachea, whereas basal cells in the subepithelial tissue displayed heavy, non-polarized staining of the cell membrane. In rat colon, agonist stimulation induced staining along the basolateral membrane of crypt cells. These data provide direct evidence of NKCC1 regulation in these tissues, and they further link phosphorylation of NKCC1 with its activation in transfected cells and native tissue. The high conservation of the regulatory threonine residues among NKCC1, NKCC2, and NCC family members, together with the fact that tissues from divergent vertebrate species exhibit similar R5-binding profiles, lends further support to the role of this regulatory locus in vivo.

The unusually low 78% amino acid identity between the orthologous human SLC26A6 and mouse slc26a6 polypeptides prompted systematic comparison of their anion transport functions in Xenopus oocytes. Multiple human SLC26A6 variant polypeptides were also functionally compared. Transport was studied as unidirectional fluxes of (36)Cl(-), [(14)C]oxalate, and [(35)S]sulfate; as net fluxes of HCO(3)(-) by fluorescence ratio measurement of intracellular pH; as current by two-electrode voltage clamp; and as net Cl(-) flux by fluorescence intensity measurement of relative changes in extracellular and intracellular [Cl(-)]. Four human SLC26A6 polypeptide variants each exhibited rates of bidirectional [(14)C]oxalate flux, Cl(-)/HCO(3)(-) exchange, and Cl(-)/OH(-) exchange nearly equivalent to those of mouse slc26a6. Cl(-)/HCO(3)(-) exchange by both orthologs was cAMP-sensitive, further enhanced by coexpressed wild type cystic fibrosis transmembrane regulator but inhibited by cystic fibrosis transmembrane regulator DeltaF508. However, the very low rates of (36)Cl(-) and [(35)S]sulfate transport by all active human SLC26A6 isoforms contrasted with the high rates of the mouse ortholog. Human and mouse orthologs also differed in patterns of acute regulation. Studies of human-mouse chimeras revealed cosegregation of the high (36)Cl(-) transport phenotype with the transmembrane domain of mouse slc26a6. Mouse slc26a6 and human SLC26A6 each mediated electroneutral Cl(-)/HCO(3)(-) and Cl(-)/OH(-) exchange. In contrast, whereas Cl(-)/oxalate exchange by mouse slc26a6 was electrogenic, that mediated by human SLC26A6 appeared electroneutral. The increased currents observed in oocytes expressing either mouse or human ortholog were pharmacologically distinct from the accompanying monovalent anion exchange activities. The human SLC26A6 polypeptide variants SLC26A6c and SLC26A6d were inactive as transporters of oxalate, sulfate, and chloride. Thus, the orthologous mouse and human SLC26A6 proteins differ in anion selectivity, transport mechanism, and acute regulation, but both mediate electroneutral Cl(-)/HCO(3)(-) exchange.