THE SODIUM-CALCIUM EXCHANGER OF BOVINE ROD PHOTORECEPTORS - K+-DEPENDENCE OF THE PURIFIED AND RECONSTITUTED PROTEIN

The K+-dependence of the rod photoreceptor sodium-calcium exchanger was investigated using the Ca2+-sensitive dye arsenazo III after reconstitution of the purified protein into proteoliposomes. The uptake of Ca2+ by Na+-loaded liposomes was found to be greatly enhanced by the presence of external K+ (EC50 almost-equal-to 1 mM) in a Michaelis-Menten manner, suggesting that one K+ ion is involved in the transport of one Ca2+ ion. We also found a minimal degree of Ca2+ uptake in the total absence of K+. Other alkali cations, notably Rb+ and, to a lesser extent, Cs+, were also able to stimulate Na+-Ca2+ exchange. We also investigated the K+-dependence of the photoreceptor Na+-Ca2+ exchanger by determining the effects of electrochemical K+ gradients on the Na+-activated Ca2+ efflux from proteoliposomes. We found that, under conditions of membrane voltage clamp with FCCP, inwardly directed electrochemical K+ gradients (i.e., K0+ > K(i)+) inhibited, whereas an outwardly directed electrochemical K+ gradient (i.e., K(i)+ > K0+) enhanced, Na+-dependent Ca2+ efflux, consistent with the notion that K+ is cotransported in the same direction as Ca2+. The investigation of the reconstituted exchanger at physiological (i.e. K(i)+ = 110 mM, K0+ = 2.5 mM) potassium concentrations revealed that the Na+-dependence of Ca2+-efflux was highly cooperative (n = 3.01 from Hill plots), indicating that at least three, but possibly four, Na+ ions are exchanged for one Ca2+ ion. Under these conditions the reconstituted exchanger showed a K(m) for Na+ of 26.1 mM, and a turnover number of 115 Ca2+.s-1 per exchanger molecule. Our results with the purified and reconstituted sodium-calcium exchanger from rod photoreceptors are therefore consistent with previous reports (Cervetto, L., Lagnado, L., Perry, R.J., Robinson, D.W. and McNaughton, P.A. (1989) Nature 337, 740-743; Schnetkamp, P.P.M., Basu, D.K. and Szerencsei, R.T. (1989) Am. J. Physiol. 257, C153-C157) that the sodium-calcium exchanger of rod photoreceptors cotransports K+ under physiological conditions with a stoichiometry of 4 Na+ : 1 Ca2+, 1 K+.