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1 Renal Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA; Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA; Molecular Physiology Unit, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran and Instituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico
2 Molecular Physiology Unit, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran and Instituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico
3 Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA
4 Renal Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA
5 Department of Anesthesiology, Vanderbilt Univerity Medical Center, Nashville, TN, USA
6 Renal Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA; Division of General Internal Medicine, VA Boston Healthcare System, Harvard Medical School, West Roxbury, MA, USA; Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA
* To whom correspondence should be addressed. E-mail: dmount{at}rics.bwh.harvard.edu.
The SLC12A6 gene encoding the K+-Cl- cotransporter KCC3 is expressed in multiple tissues, including kidney. Here we report the molecular characterization of several N-terminal isoforms of human and mouse KCC3, along with intra-renal localization and functional characterization in Xenopus oocytes. Two major isoforms, KCC3a and KCC3b, are generated by transcriptional initiation 5' of two distinct first coding exons. Northern analysis of mouse tissues indicates that KCC3b expression is particularly robust in kidney, which also expresses KCC3a. Western blotting of mouse tissue using an exon 3- specific antibody reveals that kidney is also unique in expressing immunoreactive protein of a lower mass, suggestive evidence that the shorter KCC3b protein predominates in kidney. Immunofluorescence reveals basolateral expression of KCC3 protein along the entire length of the proximal tubule, in both mouse and rat. Removal of the 15-residue exon 2 by alternative splicing generates the KCC3a-x2M and KCC3b-x2M isoforms; other splicing events at an alternative acceptor site within exon 1a generate the KCC3a-S isoform, which is 60 residues shorter than KCC3a. This variation in sequence of N-terminal cytoplasmic domains occurs proximal to a stretch of highly conserved residues and affects the content of putative phosphorylation sites. Kinetic characterization of KCC3a in Xenopus oocytes reveals apparent Km's for Rb+ and Cl- of 10.7 ± 2.5 and 7.3 ± 1.2 mM, respectively, with an anion selectivity of Br- > Cl- > PO4 = I- = SCN- = gluconate. All five N-terminal isoforms are activated by cell swelling (hypotonic conditions), with no activity under isotonic conditions. Although the isoforms do not differ in the osmotic set-point of swelling activation, this activation is more rapid for the KCC3a-x2M and KCC3a-S1 proteins. In summary, there is significant N-terminal heterogeneity of KCC3, with particularly robust expression of KCC3b in kidney. Basolateral swelling-activated K+-Cl- cotransport mediated by KCC3 likely functions in cell volume regulation during the transepithelial transport of both salt and solutes by the proximal tubule.
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