Mechanism, regulation and physiological significance of the loop diuretic-sensitive NaCl/KCl symport system in animal cells

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Abstract
Investigations in numerous laboratories have characterized a salt transport system, present in many animal cell types, which catalyzes the transmembrane transport of NaCl and KCI in a tightly coupled process. The system is inhibited by loop diuretics such as furosemide and bumetanide. This transport system has been designated the loop diuretic-sensitive NaCl/KCl symporter. It has been implicated in transepithelial salt secretion and absorption as well as in cell volume regulation, and it may be defective in patients suffering from essential hypertension. This review serves to evaluate research conducted to date regarding the mechanism, mode of regulation, and physiological significance of the transport system. Ion binding specificities and absolute binding constants for all three naturally occurring ions have been determined in one cell system, the MDCK kidney epithelial cell line. In that same cell line, substrate binding was shown to exhibit apparent positive cooperativity. Although a few reports suggest unidirectional transport of ions via this system under certain conditions, the consensus of reports indicates fully reversible, bidirectional salt transport with the direction of net flux determined by the magnitudes of the gradients of the three transported ions. Growth of cells in media containing a low concentration of K+ (<0.25 mM) allows selection of mutants lacking or defective in the symporter. Kinetic analyses with the MDCK cell line have shown that the symporter catalyzes accelerative exchange transport. However, exchange transport of one ion in the absence of one of the other two ionic substrates has not been documented. Comparison with other well-characterized transmembrane transport systems has shown that the characteristics of the NaCl/KCl symporter most resemble those of two-species facilitators (chemiosmotically-coupled symporters) found in prokaryotes and eukaryotes alike. These two-species facilitators consist of a single transmembrane protein and may function by a carrier-type mechanism as originally proposed by Peter Mitchell. A molecular model for the NaCl/KCl symporter is presented and discussed. Activation of symport activity requires ATP and probably occurs by a protein kinase-catalyzed mechanism. In some cell types activation is cyclic AMP dependent. ATP hydrolysis is not stoichiometric with transport. Phosphorylation of an integral membrane protein with an apparent size of 240 000 daltons correlates with activation of transport. It is postulated that this protein is the loop diuretic-sensitive NaCl/KCl symporter.