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 microM for each [Na]i, the Km and Vmax values increased from 7.7 to 33.5 microM and from 2.3 to 9.0 nmol.mg-1.s-1, respectively. The Vmax/Km values show a +/- 2-10% deviation from the average value of 0.274 nmol.mg-1.s-1.microM-1 over the whole range of [Na]i. These deviations are within the standard 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.mg-1.s-1.microM-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 microM with varying [Na]i = 10-160 mM, the initial rates were found to be [Na]i independent. At a high fixed [45Ca]o = 92 microM the initial rates show a sigmoidal dependence on the [Na]i with Vmax = 13.8 nmol.mg-1.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.

Inactivation of outward Na(+)‐Ca2+ exchange current in guinea‐pig ventricular myocytes.

“…The current amplitude declines at all membrane potentials during Ca2" application, but the decline seems to be more significant at negative membrane potentials. This tendency is consistent with I-V relation changes of the outward exchange current in giant membrane patches in response to an increase of the cytoplasmic Ca2+ concentration or a decrease of cytoplasmic Na+ concentration (Matsuoka & Hilgemann, 1992 (Khananshvili, 1990;Li & Kimura, 1990Hilgemann, Nicoll & Philipson, 1991;Niggli & Lederer, 1991). A consecutive model with one transition to an inactive state (Scheme 1), taking place from the three Na+-loaded exchanger conformation with cytoplasmic orientation of binding sites, described well many experimental results on the inactivation of exchange current in giant patches (Hilgemann et al 1992b).…”

Tet(L) and Tet(K) Tetracycline-Divalent Metal/H + Antiporters: Characterization of Multiple Catalytic Modes and a Mutagenesis Approach to Differences in Their Efflux Substrate and Coupling Ion Preferences

“…If common binding sites, translocation pathways, or both are used, does every substrate use all of the same features of the site or pathway to the same extent, or is there variability among substrates? And is a single translocation pathway used for the efflux substrates and coupling ions in an alternating, ping-pong type of mechanism, as is found for many antiporters (8,16,21,29,40,44), or are the efflux substrates and coupling ions translocated simultaneously through distinct translocation pathways, as in some other antiporters (6,40)? Complete resolution of these major questions will require high-resolution structural data in addition to more detailed studies of binding and transport kinetics and more extensive mutagenesis studies in combination with approaches to provide additional structural information.…”

Na+-Ca2+ Exchanger: Physiology and Pharmacology.

“…2). Kinetic studies using reconstituted proteoliposomes (61,62) and an electrophysiological technique (60,63,64) showed that the apparent affinity of the exchanger for one ion increased as the concentration of the trans ion decreased, indicating a characteristic property of a con secutive reaction cycle. Ion binding sites can be assumed to have a net charge of 2.…”