Voltage-dependent modulation of ion binding and translocation in the cardiac Na⁺-Ca²⁺ exchange system

The transport of Na⁺ and Ca²⁺ ions in the cardiac Na⁺-Ca²⁺ exchanger can be described as separate events (Khananshvili, D. (1990) Biochemistry 29, 2437-2442). Thus, the Na⁺-Na⁺ and Ca²⁺-Ca²⁺ exchange reactions reflect reversible partial reactions of the transport cycle. The effect of diffusion potentials (K⁺-valinomycin) on different modes of the Na⁺-Ca²⁺ exchanger (Na⁺-Ca²⁺, Ca²⁺-Ca²⁺, and Na⁺-Na⁺ exchanges) were tested in reconstituted proteoliposomes, obtained from the Triton X-100 extracts of the cardiac sarcolemmal membranes. The initial rates of the Na_i-dependent ⁴⁵Ca-uptake (t = 1 s) were measured in EGTA-entrapped proteoliposomes at different voltages. At the fixed values of voltage [⁴⁵Ca]_o was varied from 4 to 122 μM, and [Na]_i was saturating (150 mM). Upon varying Δ Ψ from -94 to +91 mV, the V_max values were increased from 9.5 ± 0.5 to 26.5 ± 1.5 nmol · mg^-1·s^-1 and the K_m from 17.8 ± 2.5 to 39.1 ± 5.2 μM, while the V_max/K_m values ranged from only 0.53 ± 0.08 to 0.73 ± 0.17 nmol · mg^-1-s^-1-μm^-1. The equilibrium Ca²⁺-Ca2+ exchange was voltage sensitive at very low [Ca]_o = [Ca]_i = 2 μM, while at saturating [Ca]_o = [Ca]_i = 200 μM the Ca²⁺-Ca²⁺ exchange became voltage-insensitive. The rates of the equilibrium Na⁺Na⁺ exchange appears to be voltage insensitive at saturating [Na]_o = [Na]_i =160 mM. Under the saturating ionic conditions, the rates of the Na⁺-Na⁺ exchange were at least 2-3-fold slower than the Ca²⁺-Ca²⁺ exchange. The following conclusions can be drawn, (a) The near constancy of the V_max/K_m for Na⁺-Ca²⁺ exchange at different voltages is compatible with the ping-pong model proposed previously. (b) The effects of voltage on V_max of Na⁺-Ca²⁺ exchange are consistent with the existence of a single charge carrying transport step, (c) It is not yet possible to clearly assign this step to the Na⁺ or Ca²⁺ transport half of the cycle although it is more likely that 3Na⁺-transport is a charge carrying step. Thus, the unloaded ion-binding domain contains either -2 or -3 charges (presumably carboxyl groups), (d) The binding of Na⁺ and Ca²⁺ appears to be weakly voltage-sensitive. The Ca²⁺-binding site may form a small ion-well (<2-3 Å).