Ion and bumetanide binding by the Na-K-Cl cotransporter - Importance of transmembrane domains

The Na-K-CI cotransporter (NKCC) plays a key role in electrolyte secretion and absorption across polarized epithelia, The structure of the Na-R-Cl cotransporter transport protein is not known, but from analysis of the primary amino acid sequence and biochemical studies, it has been inferred that the protein has large cytoplasmic N and C termini and a hydrophobic central domain containing 12 transmembrane helices, Both the central domain and the C-terminal domain are highly conserved within the cation-chloride cotransporter family, This paper examines the role of these three domains in interacting with the transported ions and with the inhibitor bumetanide. We have used a chimera approach, exploiting the functional differences between the structurally similar shark and human secretory Na-R-Cl cotransporters (sNKCC1 and hNKCC1). These transporters are 74% identical to one another and have similar transport and regulatory behaviors; however, sNKCCI differs markedly from hNRCC1 with regard to apparent affinities for the cotransported ions and for bumetanide. We prepared six sNKCC1-hNKCC1 chimeras in which N and C termini were interchanged between species, When transfected in HEK-293 cells, each chimera carried out bumetanide-sensitive Rb-86 influx, demonstrating transporter synthesis and cell surface delivery, Monoclonal antibodies J3 and J7 were used to detect the chimeric proteins, and the epitopes for these antibodies were localized to residues 49-196 and 1050-1168, respectively, in the shark sequence, For each of tyro chimeras that were examined, the time course of activation in low Cl- medium was the same as for the parent proteins; activation was found to proceed through a change in V-max rather than K-m. For the six chimeras, the apparent affinities for Na+, K+, Cl-, and bumetanide segregated exactly according-to whether the large hydrophobic central domain was derived from sNKCCI or hNKCCl, Significantly, the well-conserved C terminus does not appear to contain residues involved in She shark-human affinity differences, These results demonstrate that residues involved with ion translocation and inhibitor binding are within the large central domain that contains the 12 predicted transmembrane helices.