Distinction between the two basic mechanisms of cation transport in the cardiac sodium-calcium exchange system

Abstract

In order to distinguish between the Ping-Pong and sequential mechanisms of cation transport in the cardiac Na+-Ca2+ exchange system, the initial rates of the Nai-dependent 45Ca uptake (t = 1 s) were measured in reconstituted proteoliposomes, loaded with a Ca chelator. Under "zero-trans" conditions ([Na]o = [Ca]i = 0) at a fixed [Na]i = 10-160 mM with varying [45Ca]o = 2.5-122 .mu.M for each [Na]i, the Km and Vmax values increased from 7.7 to 33.5 .mu.M and from 2.3 to 9.0 nmol .cntdot. mg-1 .cntdot. s-1, respectively. The Vmax/Km values show a .+-.2-10% deviation from the average value of 0.274 nmol .cntdot. mg-1 .cntdot. s-1 .cntdot. .mu.M-1 over the whole range of [Na)i. These deviations are within the standared error of Vmax (.+-.3-7%), Km(.+-.11-17%), and Vmax/Km (.+-.11-19%). This suggests that, under conditions in which Vmax and Km are [Na]i dependent and vary 4-5-fold, the Vmax/Km values are constant within the experimental error. In the presence of K+-valinomycin the Vmax/Km values are 0.85 .+-. 0.17 and 1.08 .+-. 0.18 nmol .cntdot. mg-1 .cntdot. s-1 .cntdot. .mu.M-1 at [Na]i = 20 and 160 mM, respectively, suggesting that under conditions of "short circuit" of the membrane potential the Vmax/Km values still exhibit the [Na]i independence. At a very low fixed [45Ca]o = 1.1 .mu.M with varying [Na]i = 10-160 mM, the initial rates were found to be [Na]i independent. At a high fixed [45Ca]o = 92 .mu.M the initial rates show a sigmoidal dependence on the [NA]i with Vmax = 13.8 nmol .cntdot. mg-1 .cntdot. s-1, KmNa = 21 mM, and Hill coefficient nH = 1.5. The presented data support a Ping-Pong (consecutive) mechanism of cation transport in the Na+-Ca2+ exchanger.