The properties of the intracellular potassium binding site of the Na+:Ca2+,K+ exchanger were investigated in detached rod outer segments (OS) perfused with high Ca2+i and recorded under whole-cell voltage-clamp conditions. The OS were loaded with known amounts of K+ with the aid of a few Ca2+-activated K+ channels while perfusing with high external K+ (K+o). The substitution of K+o with Na+o elicited an inward, transient exchange current (forward mode: Na+ intruded per Ca2+ and K+ extruded) whose time integral was equal to the amount of K+ charges loaded in the OS, thus demonstrating that K+ is transported by the exchanger with a stoichiometry consistent with one K+ ion extruded per charge imported. The activation of forward exchange by intracellular K+ (K+i) was fitted by first-order kinetics with a Michaelis constant, Kapp(K+i)1.6 mM and a maximal current, Imax-65 pA. [Na+]i larger than 100 mM had little effect on Kapp(K+i) and Imax, indicating that Na+i does not compete with K+i for exchange sites under physiological conditions, and that Na+ release at the intracellular side is not a rate limiting step for the exchange process.
Intracellular K binding site properties of the Na-Ca,K exchanger of vertebrate photoreceptors.
RISPOLI, Giorgio
1997
Abstract
The properties of the intracellular potassium binding site of the Na+:Ca2+,K+ exchanger were investigated in detached rod outer segments (OS) perfused with high Ca2+i and recorded under whole-cell voltage-clamp conditions. The OS were loaded with known amounts of K+ with the aid of a few Ca2+-activated K+ channels while perfusing with high external K+ (K+o). The substitution of K+o with Na+o elicited an inward, transient exchange current (forward mode: Na+ intruded per Ca2+ and K+ extruded) whose time integral was equal to the amount of K+ charges loaded in the OS, thus demonstrating that K+ is transported by the exchanger with a stoichiometry consistent with one K+ ion extruded per charge imported. The activation of forward exchange by intracellular K+ (K+i) was fitted by first-order kinetics with a Michaelis constant, Kapp(K+i)1.6 mM and a maximal current, Imax-65 pA. [Na+]i larger than 100 mM had little effect on Kapp(K+i) and Imax, indicating that Na+i does not compete with K+i for exchange sites under physiological conditions, and that Na+ release at the intracellular side is not a rate limiting step for the exchange process.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
