The role of transmembrane domain 2 in cation transport by the Na-K-Cl cotransporter

The human and shark Na-K-CI cotransporters (NKCC), although 74% identical in amino acid sequence, exhibit marked differences in ion transport and bumetanide binding, We have utilized shark-human chimeras of NKCC1 to search for regions that confer the kinetic differences. Two chimeras (hs3,1 and its reverse sh3,1) with a junction point located at the beginning of the third transmembrane domain were examined after stable transfection in HEK-293 cells. Each carried out bumetanide-sensitive Rb-86 influx with cation affinities intermediate between shark and human cotransporters. In conjunction with the previous finding that the N and C termini are not responsible for differences in ion transport, the current observations identify the second transmembrane domain as playing an important role, Site-specific mutagenesis of two pairs of residues in this domain revealed that one pair is indeed involved in the difference in Na affinity, and a second pair is involved in the difference in Rb affinity. Substitution of the same residues with corresponding residues from NKCC2 or the Na-CI cotransporter resulted in cation affinity changes, consistent with the hypothesis that alternative splicing of transmembrane domain 2 endows different versions of NKCC2 with unique kinetic behaviors. None of the changes in transmembrane domain 2 was found to substantially affect K-m(Cl), demonstrating that the affinity difference for CI is specified by the region beyond predicted transmembrane domain 3, Finally, unlike CI, bumetanide binding was strongly affected by shark-human replacement of transmembrane domain 2, indicating that the bumetanide-binding site is not the same as the CI-binding site.