THE ROLE OF TRANSMEMBRANE DOMAIN-2 IN CATION-TRANSPORT BY THE NA-K-CLCOTRANSPORTER

The human and shark Na-K-CI cotransporters (NKCC), although 74% identi cal in amino acid sequence, exhibit marked differences in ion transpor t and bumetanide binding, We have utilized shark-human chimeras of NKC C1 to search for regions that confer the kinetic differences. Two chim eras (hs3,1 and its reverse sh3,1) with a junction point located at th e beginning of the third transmembrane domain were examined after stab le transfection in HEK-293 cells. Each carried out bumetanide-sensitiv e Rb-86 influx with cation affinities intermediate between shark and h uman 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 r esidues in this domain revealed that one pair is indeed involved in th e difference in Na affinity, and a second pair is involved in the diff erence in Rb affinity. Substitution of the same residues with correspo nding residues from NKCC2 or the Na-CI cotransporter resulted in catio n affinity changes, consistent with the hypothesis that alternative sp licing of transmembrane domain 2 endows different versions of NKCC2 wi th unique kinetic behaviors. None of the changes in transmembrane doma in 2 was found to substantially affect K-m(Cl), demonstrating that the affinity difference for CI is specified by the region beyond predicte d transmembrane domain 3, Finally, unlike CI, bumetanide binding was s trongly affected by shark-human replacement of transmembrane domain 2, indicating that the bumetanide-binding site is not the same as the CI -binding site.