The functional properties of alternatively spliced isoforms of the mouse apical Na⁺-K⁺-2Cl cotransporter (mBSC1) were examined, using expression in Xenopus oocytes and measurement of ²²Na⁺ or ⁸⁶Rb⁺ uptake. A total of six isoforms, generated by the combinatorial association of three 5' exon cassettes (A, B, and F) with two alternative 3' ends, are expressed in mouse thick ascending limb (TAL) [see companion article, D. B. Mount, A. Baekgaard, A. E. Hall, C. Plata, J. Xu, D. R. Beier, G. Gamba, and S. C. Hebert. Am. J. Physiol. 276 (Renal Physiol. 45): F347-F358, 1999]. The two 3' ends predict COOH-terminal cytoplasmic domains of 129 amino acids (the C4 COOH terminus) and 457 amino acids (the C9 terminus). The three C9 isoforms (mBSC1-A9/F9/B9) all express Na⁺-K⁺-2Cl cotransport activity, whereas C4 isoforms are nonfunctional in Xenopus oocytes. Activation or inhibition of protein kinase A (PKA) does not affect the activity of the C9 isoforms. The coinjection of mBSC1-A4 with mBSC1-F9 reduces tracer uptake, compared with mBSC1-F9 alone, an effect of C4 isoforms that is partially reversed by the addition of cAMP-IBMX to the uptake medium. The inhibitory effect of C4 isoforms is a dose-dependent function of the alternatively spliced COOH terminus. Isoforms with a C4 COOH terminus thus exert a dominant negative effect on Na⁺-K⁺-2Cl cotransport, a property that is reversed by the activation of PKA. This interaction between coexpressed COOH-terminal isoforms of mBSC1 may account for the regulation of Na⁺-K⁺-2Cl cotransport in the mouse TAL by hormones that generate cAMP.

sodium-potassium-chloride cotransporter; bumetanide; protein kinase A; adenosine 3',5'-cyclic monophosphate; thick ascending limb of Henle; alternative splicing

This article has been cited by other articles:

				H. Castrop and J. Schnermann Isoforms of renal Na-K-2Cl cotransporter NKCC2: expression and functional significance Am J Physiol Renal Physiol, October 1, 2008; 295(4): F859 - F866. [Abstract] [Full Text] [PDF]

				P. Welker, A. Bohlick, K. Mutig, M. Salanova, T. Kahl, H. Schluter, D. Blottner, J. Ponce-Coria, G. Gamba, and S. Bachmann Renal Na+-K+-Cl- cotransporter activity and vasopressin-induced trafficking are lipid raft-dependent Am J Physiol Renal Physiol, September 1, 2008; 295(3): F789 - F802. [Abstract] [Full Text] [PDF]

				E. Moreno, P. S. Cristobal, M. Rivera, N. Vazquez, N. A. Bobadilla, and G. Gamba Affinity-defining Domains in the Na-Cl Cotransporter: A DIFFERENT LOCATION FOR Cl- AND THIAZIDE BINDING J. Biol. Chem., June 23, 2006; 281(25): 17266 - 17275. [Abstract] [Full Text] [PDF]

				A. Paredes, C. Plata, M. Rivera, E. Moreno, N. Vazquez, R. Munoz-Clares, S. C. Hebert, and G. Gamba Activity of the renal Na+-K+-2Cl- cotransporter is reduced by mutagenesis of N-glycosylation sites: role for protein surface charge in Cl- transport Am J Physiol Renal Physiol, May 1, 2006; 290(5): F1094 - F1102. [Abstract] [Full Text] [PDF]

				D. B. Mount Membrane trafficking and the regulation of NKCC2 Am J Physiol Renal Physiol, March 1, 2006; 290(3): F606 - F607. [Full Text] [PDF]

				G. Gamba Molecular Physiology and Pathophysiology of Electroneutral Cation-Chloride Cotransporters Physiol Rev, April 1, 2005; 85(2): 423 - 493. [Abstract] [Full Text] [PDF]

				C. Tovar-Palacio, N. A. Bobadilla, P. Cortes, C. Plata, P. de los Heros, N. Vazquez, and G. Gamba Ion and diuretic specificity of chimeric proteins between apical Na+-K+-2Cl- and Na+-Cl- cotransporters Am J Physiol Renal Physiol, September 1, 2004; 287(3): F570 - F577. [Abstract] [Full Text] [PDF]

				P. Meade, R. S. Hoover, C. Plata, N. Vazquez, N. A. Bobadilla, G. Gamba, and S. C. Hebert cAMP-dependent activation of the renal-specific Na+-K+-2Cl- cotransporter is mediated by regulation of cotransporter trafficking Am J Physiol Renal Physiol, June 1, 2003; 284(6): F1145 - F1154. [Abstract] [Full Text] [PDF]

				E. Gagnon, B. Forbush, A. W. Flemmer, I. Gimenez, L. Caron, and P. Isenring Functional and molecular characterization of the shark renal Na-K-Cl cotransporter: novel aspects Am J Physiol Renal Physiol, November 1, 2002; 283(5): F1046 - F1055. [Abstract] [Full Text] [PDF]

				C. Plata, P. Meade, N. Vazquez, S. C. Hebert, and G. Gamba Functional Properties of the Apical Na+-K+-2Cl- Cotransporter Isoforms J. Biol. Chem., March 22, 2002; 277(13): 11004 - 11012. [Abstract] [Full Text] [PDF]

				G. Gamba Alternative splicing and diversity of renal transporters Am J Physiol Renal Physiol, November 1, 2001; 281(5): F781 - F794. [Abstract] [Full Text] [PDF]

				A. Mercado, P. de los Heros, N. Vazquez, P. Meade, D. B. Mount, and G. Gamba Functional and molecular characterization of the K-Cl cotransporter of Xenopus laevis oocytes Am J Physiol Cell Physiol, August 1, 2001; 281(2): C670 - C680. [Abstract] [Full Text] [PDF]

				C. Plata, P. Meade, A. Hall, R. C. Welch, N. Vazquez, S. C. Hebert, and G. Gamba Alternatively spliced isoform of apical Na+-K+-Cl{-} cotransporter gene encodes a furosemide-sensitive Na+-Cl{-}cotransporter Am J Physiol Renal Physiol, April 1, 2001; 280(4): F574 - F582. [Abstract] [Full Text] [PDF]

				E. Feraille and A. Doucet Sodium-Potassium-Adenosinetriphosphatase-Dependent Sodium Transport in the Kidney: Hormonal Control Physiol Rev, January 1, 2001; 81(1): 345 - 418. [Abstract] [Full Text] [PDF]

				A. Attmane-Elakeb, H. Amlal, and M. Bichara Ammonium carriers in medullary thick ascending limb Am J Physiol Renal Physiol, January 1, 2001; 280(1): F1 - F9. [Abstract] [Full Text] [PDF]

				D. B. Mount, A. Baekgaard, A. E. Hall, C. Plata, J. Xu, D. R. Beier, G. Gamba, and S. C. Hebert Isoforms of the Na-K-2Cl cotransporter in murine TAL I. Molecular characterization and intrarenal localization Am J Physiol Renal Physiol, March 1, 1999; 276(3): F347 - F358. [Abstract] [Full Text] [PDF]