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.
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The authors thank Dr. C. Grewer for critical reading of the manuscript and helpful suggestions.
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Haase, A., Wood, P.G., Pintschovius, V. et al. Time resolved kinetics of the guinea pig Na–Ca exchanger (NCX1) expressed in Xenopus oocytes: voltage and Ca2+ dependence of pre-steady-state current investigated by photolytic Ca2+concentration jumps. Pflugers Arch - Eur J Physiol 454, 1031–1042 (2007). https://doi.org/10.1007/s00424-007-0260-0
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DOI: https://doi.org/10.1007/s00424-007-0260-0