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NH₂-terminal heterogeneity in the KCC3 K⁺-Cl^– cotransporter

¹Renal Division, Brigham and Women’s Hospital, ²Division of General Internal Medicine, Veterans Affairs Boston Healthcare System, Harvard Medical School, Boston, Massachusetts; Departments of ³Medicine and ⁴Anesthesiology, Vanderbilt University Medical Center, Nashville, Tennessee; and ⁵Molecular Physiology Unit, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán and Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico

Submitted 22 December 2004 ; accepted in final form 21 July 2005

The SLC12A6 gene encoding the K⁺-Cl^– cotransporter KCC3 is expressed in multiple tissues, including kidney. Here, we report the molecular characterization of several NH₂-terminal isoforms of human and mouse KCC3, along with intrarenal localization and functional characterization in Xenopus laevis oocytes. Two major isoforms, KCC3a and KCC3b, are generated by transcriptional initiation 5' of two distinct first coding exons. Northern blot analysis of mouse tissues indicates that KCC3b expression is particularly robust in the kidney, which also expresses KCC3a. Western blotting of mouse tissue using an exon 3-specific antibody reveals that the 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 the 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 NH₂-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 X. laevis oocytes reveals apparent K_ms 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 NH₂-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-S proteins. In summary, there is significant NH₂-terminal heterogeneity of KCC3, with particularly robust expression of KCC3b in the 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.

K-Cl cotransporter; cell volume; SLC12

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