The effect of membrane-fluidizing agents on sodium-pump activity in erythrocytes [proceedings].

in which M is the rate of efflux at a given concentration of internal Na+, “a+],, M,,,,,. the emux at saturating ”a+], (maximal velocity) and K,, the apparent dissociation constant of the Na-site complex. Sodium-pump activity is dependent on the cholesterol content of erythrocytes: its apparent affinity for internal Na+ falls (increase in kk,) and its maximal velocity rises after the cells have been partially depleted of their cholesterol content (Claret et al., 1978) (see Table 1). As removal of cholesterol brings about an increase in membrane fluidity (Cooper, 1978), and as the functions of many membrane-bound enzymes are influenced by the fluidity of lipids in their microenvironment (Sandermann, 1978), the effects of cholesterol depletion on the sodium-pump activity could therefore be mediated through an increase in fluidity. To test this hypothesis, we have measured the pump activity in erythrocytes with a normal cholesterol content, but in the presence of different agents that increase the membrane fluidity: benzyl alcohol, chlorpromazine, imipramine, sodium benzenesulphonate and sodium oleate. The drug-induced changes in ‘microviscosity’ of the ‘ghost’ membranes were measured by the fluorescence-polarization technique with diphenylhexatriene as a probe (Shinitzky & Inbar, 1976). The ouabain-sensitive Na+ emux was measured in drug-containing media as a function of “a+], after the cells have been treated with p-chlorornercuribenzenesulphonate/cysteine to regulate the “a+], (Claret et al., 1978). All the drugs decreased the ‘microviscosity’ of the membrane within 5 min, and the values remained unchanged thereafter. A decrease of 15-22% was obtained at different external concentrations of each drug, which is comparable with that produced by a 25% cholesterol depletion (Table 1). These concentrations were used for the ion-flux experiments. In the presence of sodium benzenesulphonate or sodium oleate, neither the apparent affinity for internal Na+ as shown from the value of KL, nor M,,,,,. were changed (Table 1). In contrast benzyl alcohol, chlorpromazine and imipramine produced a significant increase in KL,. An increase in Mmax, was also observed with benzyl alcohol, but this effect was much lower with imipramine and chlorpromazine. Since fluidity is increased by all of these drugs and since two of them have no effect on the sodium-pump parameters, one interpretation is that the pump activity is not dependent on membrane fluidity. The effects of benzyl alcohol, chlorpromazine and imipramine as well as those of cholesterol depletion could be due to a specific interaction of the drvgs or of cholesterol with the enzyme independently of their effects on membrane fluidity. Another hypothesis would be that both sodium benzenesulphonate and sodium oleate, which bear a negative charge, would not be able to penetrate either the inner negatively charged leaflet (phosphatidylserine) or the microenvironment of the enzyme, because of the same charge effect. Chlorpromazine, imipramine and benzyl alcohol, which are either positively charged or neutral molecules, could reach these areas and produce their effect on the pump activity. This last hypothesis implies that the apparent affinity of the internal sites of the pump is sensitive to the fluidity of its lipid environment at least on the inner half of the membrane.