The residues determining differences in ion affinities among the alternative splice variants F, A, and B of the mammalian renal Na-K-Cl cotransporter (NKCC2).
Three alternatively spliced variants of the renal Na-K-Cl cotransporter (NKCC2) are found in distinct regions of the thick ascending limb of the mammalian kidney; these variants mediate Na(+)K(+)2Cl(-) transport with different ion affinities. Here, we examine the specific residues involved in the variant-specific affinity differences, utilizing a mutagenic approach to change the NKCC2B variant into the A or F variant, with functional expression in Xenopus oocytes. The splice region contains the second transmembrane domain (TM2) and the putative intracellular loop (ICL1) connecting TM2 and TM3. It is found that the B variant is functionally changed to the F variant by replacement of six residues, half of the effect brought about by three TM2 residues and half by three ICL1 residues. The involvement of the ICL1 residues strongly suggests that this region of ICL1 may actually be part of a membrane-embedded domain. Changing six residues is also sufficient to bring about the smaller functional change from the B to the A variant; three residues in TM2 appear to be primarily responsible, two of which correspond to residues involved in the B-to-F changes. A B-variant mutation reported in a mild case of Bartter disease was found to render the cotransporter inactive. These results identify the combination of amino acid variations responsible for the differences among the three splice variants of NKCC2, and they support a model in which a reentrant loop following TM2 contributes to the chloride binding and translocation domains.
Giménez
Ignacio
I
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06511, USA. igimenez@unizar.es
Forbush
Biff
B
D019943:Amino Acid Substitution; D000818:Animals; D001692:Biological Transport; D002713:Chlorine / Q000378:metabolism; D007668:Kidney / Q000737:chemistry*; D008565:Membrane Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D028021:Sodium-Potassium-Chloride Symporters / Q000235:genetics / Q000378:metabolism*; D064466:Solute Carrier Family 12, Member 1
D008565:Membrane Proteins; D020033:Protein Isoforms; C578715:Slc12a1 protein, mouse; D028021:Sodium-Potassium-Chloride Symporters; D064466:Solute Carrier Family 12, Member 1; D002713:Chlorine