NH₂-terminal heterogeneity in the KCC3 K⁺-Cl⁻ cotransporter

Abstract

The SLC12A6 gene encoding the K(+)-Cl(-) cotransporter KCC3 is expressed in multiple tissues, including kidney. Here, we report the molecular characterization of several NH(2)-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 NH(2)-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(m)s for Rb(+) and Cl(-) of 10.7 +/- 2.5 and 7.3 +/- 1.2 mM, respectively, with an anion selectivity of Br(-) > Cl(-) > PO(4) = I(-) = SCN(-) = gluconate. All five NH(2)-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 NH(2)-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.

Similar effects of all WNK3 variants on SLC12 cotransporters

“…The following cDNA clones of the SLC12A family used in this study were previously reported: NCC (3), NKCC1 (9), NKCC2 (3), KCC1 and KCC4 (17), KCC2b (24), KCC3a and KCC3b (18). KCC2a (26) was obtained by cutting and pasting KCC2 exon 1a amplified from the human fetal brain cDNA library in a process similar to that described above.…”

Functional Analysis of a Potassium-Chloride Co-Transporter 3 (SLC12A6) Promoter Polymorphism Leading to an Additional DNA Methylation Site

“…Initially, two first exons denoted by SLC12A6-1 and SLC12A6-2 (formerly KCC3a and KCC3b), which are under the control of separate promoters (Di Fulvio et al, 2001;Mercado et al, 2004), have been described where the transcript possessing alternative exon 1a is predominantly expressed in brain. So far, six alternative transcripts have been identified for SLC12A6 (Mercado et al, 2005). All transcripts except for SLC12A6-2, are regulated by the same promoter.…”

With no lysine L-WNK1 isoforms are negative regulators of the K+-Cl− cotransporters

“…Erythroid function and differentiation (30), cancer cell growth and invasiveness (8,57,58), arterial blood pressure regulation (54), and neuronal excitability (23,33,53) are some of the important roles of these membrane proteins. At the renal level, KCC3 is located in the basolateral membranes of the proximal tubules (PT) (40), while KCC4 is expressed in proximal tubules, in the thick ascending limb of Henle's loop (TALH) (35), and in ␣-intercalated cells of the collecting duct (CD) (6,35). In these zones of the nephron, KCC3 and KCC4 are involved in processes such as glucose and salt reabsorption and acid-base homeostasis (6,35).…”

“…In these zones of the nephron, KCC3 and KCC4 are involved in processes such as glucose and salt reabsorption and acid-base homeostasis (6,35). It is known that KCCs are activated due to an increase in [Cl Ϫ ] i or by cell swelling (39,40,60). This process is related to protein dephosphorylation, and its purpose is to reduce the [Cl Ϫ ] i or mediate the regulatory volume decrease (RVD) (1,14,17).…”