Item

Abstract

Kinetic properties of the Na–Ca exchanger (guinea pig NCX1) expressed in Xenopus oocytes were investigated by patch clamp techniques and photolytic Ca²⁺ concentration jumps. Current measured in oocyte membranes expressing NCX1 is almost indistinguishable from current measured in patches derived from cardiac myocytes. In the Ca–Ca exchange mode, a transient inward current is observed, whereas in the Na–Ca exchange mode, current either rises to a plateau, or at higher Ca²⁺ concentration jumps, an initial transient is followed by a plateau. No comparable current was observed in membrane patches not expressing NCX1, indicating that photolytic Ca²⁺ concentrations jumps activate Na–Ca exchange current. Electrical currents generated by NCX1 expressed in Xenopus oocytes are about four times larger than those obtained from cardiac myocyte membranes enabling current recording with smaller concentration jumps and/or higher time resolution. The apparent affinity for Ca²⁺ of nonstationary exchange currents (0.1 mM) is much lower than that of stationary currents (6 μM). Measurement of the Ca²⁺ dependence of the rising phase provides direct evidence that the association rate constant for Ca²⁺ is about 5 × 108 M⁻¹ s⁻¹ and voltage independent. In both transport modes, the transient current decays with a voltage independent but Ca²⁺-dependent rate constant, which is about 9,000 s⁻¹ at saturating Ca²⁺ concentrations. The voltage independence of this relaxation is maintained for Ca²⁺ concentrations far below saturation. In the Ca–Ca exchange mode, the amount of charge translocated after a concentration jump is independent of the magnitude of the jump but voltage dependent, increasing at negative voltages. The slope of the charge–voltage relation is independent of the Ca²⁺ concentration. Major conclusions are: (1) Photolytic Ca²⁺ concentration jumps generate current related to NCX1. (2) The dissociation constant for Ca²⁺ at the cytoplasmic transport binding site is about 0.1 mM. (3) The association rate constant of Ca²⁺ at the cytoplasmic transport sites is high (5 × 10⁻⁸ M⁻¹s⁻¹) and voltage independent. (4) The minimal five-state model (voltage independent binding reactions, one voltage independent conformational transition and one very fast voltage dependent conformational transition) used before to describe Ca²⁺ translocation at saturating Ca²⁺ concentrations is valid for Ca²⁺ concentrations far below saturation.