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

The Na-K-Cl cotransporter (NKCC) plays a key role in electrolyte secretion and absorption across polarized epithelia. The structure of the Na-K-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-K-Cl cotransporters (sNKCC1 and hNKCC1). These transporters are 74% identical to one another and have similar transport and regulatory behaviors; however, sNKCC1 differs markedly from hNKCC1 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 86Rb 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 two 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 Vmax rather than Km. 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 sNKCC1 or hNKCC1. Significantly, the well-conserved C terminus does not appear to contain residues involved in the 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.