Reduction of K+ efflux in cultured mouse fibroblasts, by mutation or by diuretics, permits growth in K+-deficient medium.

Abstract

The mouse fibroblastic cell line LM(TK-) is unable to grow at external K+ concentrations below a threshold value of 0.4 mM. At subthreshold K+ concentrations, LM(TK-) cells rapidly lose intracellular K+ and eventually lyse. We have analyzed the pathwayprimarily responsible for K+ efflux under these experimental conditions and report its specific inhibition by two diuretics, furosemide and bumetanide. Bumetanide, an analog of furosemide, was a more potent inhibitor (by several orders of magnitude) than was furosemide itself. The effects of ouabain and bumetanide were additive, suggesting independence of diureticsensitive K+ efflux from Na+/K+ pump-mediated fluxes. Characterization of K+ efflux in LTK-5, a mutant derived from LM(TK-) and selected for its ability to grow at 0.2 mM K+, indicated that the mutant had lost the diuretic-sensitive K+ efflux pathway. Net Mammalian cells maintain a high steady-state K+ concentration and a low steady-state Na+ concentration relative to their surrounding physiological fluids. The transmembrane electrochemical gradients established by these two cations have been implicated in several physiological responses, including the regulation of cell volume (1, 2), nutrient cotransport (3-6), and membrane excitability (7-10). Because the steady-state K+ concentration reflects the sum of influx and efflux components, both active and passive, an alteration in the activity of one component must be accompanied by a compensating alteration in another if the cationic steady state is to be maintained (11). Should a particular flux become altered so that antagonistic fluxes are unable to compensate, the resulting change in intracellular cation concentration may be lethal to the cell. Such considerations were the basis for the selection of two K+ transport mutants that gained the ability to grow at a K+ concentration below that minimally required for parental cell growth (12). Both mutants proved capable of maintaining high intracellular K+ concentrations at reduced external K+ through changes in ouabain-insensitive K+ transport.One of the mutants, LTK-5, exhibited altered activity of a Na+-K+ cotransport system (12, 13) similar to that described in human (14, 15) and avian (16) erythrocytes and in Ehrlich cells (17-21) and murine fibroblasts (22,23). Specifically, in characterizing the mutant relative to its parent strain under conditions of K+ depletion and high external K+, Gargus and Slayman (13) noted that LTK-5 demonstrated enhanced furosemidesensitive, Na+-dependent K+ influx. For two reasons, however, it seemed unlikely that the increase in K+ influx was causally related to the ability of the mutant to survive selection. (i) When furosemide-sensitive K+ influx was measured as a function of the extracellular K+ concentration in both parent and mutant cells, it reached a half-maximal rate at 6 mM K+, considerably higher than the Km for the ouabain-sensitive Na+/K+ pump (1.3 mM). At 0.2 mM K+, the concentration used in the selection, furosemide-sensitive influx was barely detecta...

The Right Place at the Right Time

“…If the loss of this system were responsible for the ability of LTK-5 to grow at 0.2M K, then inhibiting the system with diuretics should confer a similar ability on the parent strain, LMTK − . We showed that this was indeed the case: thus, the reduction of K efflux either by mutation or by diuretics inhibition permitted growth in K + deficient medium (39). In a later paper, we showed that the diuretic-sensitive system requires both Na + and Cl − , and can be described as a K + Na + Cl − cotransport system (40).…”

Atrial natriuretic factor stimulates Na/K/Cl cotransport in vascular smooth muscle cells.

“…Although Na/K/Cl cotransport is a bidirectional transport system (10) and the directionality of the net flux in VSMC is not known, preliminary data suggests that this system mediates net efflux under physiological conditions, as has been shown to be true in MDCK cells (22) and L cells (23). On this basis, if it is assumed that the present experimental conditions mimic the physiological state, an increase in activity of Na/K/Cl cotransport could actually lead to a net decrease in intracellular Na.…”

“…These peptides contain approximately [20][21][22][23][24][25][26][27][28][29][30] amino acids, and the amino acid sequence and the cDNA sequence have been identified (2). ANF has been shown to cause vasorelaxation as well as diuresis and natriuresis (3).…”

Sodium-lithium exchange and sodium-potassium cotransport in human erythrocytes. Part 1: Evaluation of a simple uptake test to assess the activity of the two transport systems.

“…2 " 4 There are no data available to prove this assumption, however, and other functions may be more important, for instance, a K + inward transport driven by the inwardly directed Na + gradient. 23 We have studied the relation between outward transport of Na + and K + and inward transport of K + , using Rb + as a tracer, and have found that the transport rates in the two directions are closely linked to each other ( fig. 11).…”

“…10), indicating that the activity of the cotransport system is either genetically determined 23 or governed by a plasma factor that remains unchanged over long periods of time.…”