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

Abstract

The human and shark Na-K-Cl 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 86 Rb inf lux 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-Cl 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 Cl is specified by the region beyond predicted transmembrane domain 3. Finally, unlike Cl, 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 Cl-binding site.

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)

“…The current study and those of others (16,17) have highlighted residues that determine ion affinities in NKCC2 and are presumed to be part of the translocation pocket, complementing related findings in NKCC1 (2).…”

“…In fact the situation was unlikely to have been that simple, because we previously found that two sets of residues in the beginning of TM2, upstream of the B-to-AF residues (corresponding to GV232 and GL237 in the NKCC2B sequence, Fig. 1), mediate affinity differences for Na ϩ and K ϩ between shark and human NKCC1s (2). Considered together, the NKCC1 and NKCC2 findings argue either: (a) that at least some of the observed K m differences are due to long range effects, or (b) that TM2/ICL1 is not one extended helix, but has at least one hairpin enabling more than one set of amino acid residues to interact with the same transported ion or ion pair.…”

“…The Na-K-Cl cotransporter (NKCC) 2 carries out the coupled movement of 1 Na ϩ , 1 K ϩ , and 2 Cl Ϫ ions across the cell membrane, taking advantage of the sodium electrochemical gradient to accumulate Cl Ϫ . Two isoforms are encoded by separate genes: NKCC1 (Slc12a2), a nearly ubiquitous isoform with an important role in chloride secretory epithelia, and NKCC2 (Slc12a1), a kidney-specific isoform that is the target of the present investigation.…”

“…The transmembrane region is responsible for ion binding and translocation (2), whereas the N-terminal cytosolic domain contains the regulatory phosphoacceptors (3) as well as a binding site for the regulatory kinase (4,5) and, in NKCC1, a binding site for the regulatory phosphatase (6). Three TMs have been shown to be involved in ion translocation, as determined with a chimera and single residue mutation approach (7,8); one of these is TM2, which is encoded by the NKCC2 splice region.…”

NKCC2 Surface Expression in Mammalian Cells

“…As a consequence, considerably less is known about the cell biology and post-transcriptional regulation of NKCC2 than the other key renal transport proteins. Interestingly, Isenring et al (35,63) were able to express an NKCC2-NKCC-1 chimera, in which the 5Ј-untranslated region and cDNA encoding the first 104 amino acids of rabbit NKCC2A were replaced with the corresponding region from human NKCC1. In addition, Payne and Forbush (34) reported that when the proximal region of the NKCC2 N terminus (the first 105 AA) was deleted, immunoreactive proteins were detected in transiently transfected cells.…”