Item

Abstract

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 ≃ 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+single bondCa²⁺ exchange. We also investigated the K⁺-dependence of the photoreceptor Na⁺single bondCa²⁺ 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⁺=110mM, K₀⁺= 2.5mM) 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 otransports K⁺ under physiological conditions with a stoichiometry of 4 Na⁺; 1 Ca²⁺, 1 K⁺.