Relationship between H +, anion, and monovalent cation movements and Ca 2+ transport in sarcoplasmic reticulum: Further proof of a cation exchange mechanism for the Ca 2+-Mg 2+-ATPase pump

Abstract

Two spectroscopic probes of free internal Ca 2+ were used to determine the influence of H + and anion permeation on the active transport of Ca 2+ by skeletal sarcoplasmic reticulum. The studies were carried out on a well-characterized Ca 2+-Mg 2+-ATPase-rich sarcoplasmic reticulum fraction. Studies of D. McKinley and G. Meissner (1977, FEBS Lett., 82, 47–50) show that this fraction consists of two populations of vesicles: type I which has an electrically active monovalent cation (M +) permeability and type II which lacks it. The present study distinguishes between electrically active (charge-carrying) and electrically silent (e.g., countertransport) mechanisms of ion permeation in the two vesicles and shows how the active transport of Ca 2+ is influenced by these permeabilities. The major results are as follows: (1) Both type I and II vesicles have an electrically active H + permeability. (2) Type I vesicles have electrically active anion (A −) permeabilities; type II vesicles do not. (3) At low concentrations of nonpenetrating buffers, ion imbalances across the membrane can create pH imbalances. This is due to the coupling of M + and A − movements with H + movements. Following a jump in KCl concentration internal acidification is observed in type I vesicles while internal alkalinization is observed in type II vesicles. These pH gradients are dissipated on a time scale of seconds and tens of minutes for type I and II vesicles, respectively. (4) Tris(hydroxymethyl)aminomethane (Tris) was shown to be effective in dissipating pH gradients in type II vesicles. A model is proposed whereby HCl is equilibrated across the membrane by a Tris-catalyzed transport cycle involving transport of an ion pair between Tris-H + and Cl − and return of the unprotonated form of the buffer. (5) The permeabilities of several physiological and nonphysiological anions were determined for type I and II vesicles. Electrically active permeability was demonstrated for Cl − and phosphate in type I vesicles. Type II vesicles lacked electrically active mechanisms for these two anions. Evidence is given for slow Cl − OH − exchange and for rapid Cl − HCO 3 − exchange in type II vesicles. Electrically silent phosphate influx probably occurs by H 2PO 4 − OH − exchange. (6) Under normal conditions the Ca 2+ uptake of type II vesicles is masked. It can be unmasked by addition of nigericin in the presence of Tris. The combination of ionophore and penetrating buffer render the type II vesicles KCl permeable, allowing the replenishment of internal K + during active transport. The results are analyzed and shown to be in agreement with the Ca 2+-Mg 2+-ATPase pump acting as a Ca 2+ K + exchanger. The results are shown to be in disagreement with electrogenic models of pump function.