Studies of the Ca2+ Transport Mechanism of Human Erythrocyte Inside-out Membrane Vesicles. Evidence for the Development of a Positive Interior Membrane Potential

Previous observations on the effects of permeant anions on ATP-dependent calcium transport in inside-out vesicles prepared from human erythrocytes suggested that the calcium pump is electrogenic, generating a positive interior membrane potential. The present work demonstrates the development of a positive interior membrane potential across inside-out vesicle membranes during calcium transport in the absence of permeant anions. Several membrane potential probes, 1-anilino-8-naphthalenesulfonate, 3,3'-dipropylthiodicarbocyanine iodide, and an electron paramagnetic resonant triphenylphosphonium derivative, provide qualitative evidence for the development of a membrane potential. Moreover, a number of parallels are observed between the changes in the membrane potential measured by the probes and calcium transport. These include enhancement by calmodulin, time course of change, similar kinetic properties, and the requirement for intact vesicle membranes. Quantitative measurements of the membrane potential shows a positive interior membrane potential of 26-37 mV using radiolabeled permeant anion distribution and 38-57 mV using 3,3'-dipropylthiodicarbocyanine iodide fluorescence changes. These membrane potentials are of a similar magnitude to those reported for the sarcoplasmic reticulum calcium pump (Zimniak, P., and Racker, E. (1978). J. Biol. Chem. 253, 4631-4637).