The Molecular Architecture of a Reconstituted Calcium Pump

Abstract

If calcium ions are to be accumulated by isolated vesicles of skeletal muscle sarcoplasmic reticulum, the lipids in the membran must have at least two functions. They must support the uptake of Ca²⁺ through the transport protein, and if this Ca²⁺ is to be retained by the vesicle, the lipids must also restrict the non-specific efflux of Ca2 through the membrane. The ATP-dependent accumulation of Ca2 is almost the only membrane function carried out by isolated sarcoplasmic reticulum vesicles, and this singularity of function is matched by the simplicity of the protein composition. About 85% of the protein in the membrane is the calcium transport protein, and this protein stoicheiometrically couples the uptake of two Ca²⁺ to the hdrolysis of one molecule of ATP. If the non-specific efflux of Ca²⁺ from the vesicles is negligible, the initial rate of Ca²⁺ accumulation will be the same as the rate of Ca²⁺ uptake through the protein, and the efficiency of Ca²⁺ accumulation, as measured by the mole ratio of initial Ca²⁺ accumulation to ATP hydrolysis (Ca²⁺ /ATP) will be 2.0, the maximum value possible. Any increase in the non-specific permeability of the vesicles to Ca²⁺ will lower this value since the initial rate of calcium accumulation is the difference between non-specific Ca²⁺ efflux and Ca²⁺ uptake through the protein. We regard the stoicheiometric coupling of ATP hydrolysis to Ca²⁺ uptake by the protein as being unaffected by any of the reconstitution conditions to be described. The stoicheiometry is preserved even when the protein is transporting Ca²⁺ across the membrane of a disrupted sarcoplasmic reticulum vesicle. We consider that a low efficiency of Ca²⁺ accumulation (as measured by the Ca²⁺/ATP ratio) is not the result of a change in the stoicheiometry of coupling but is the result of an increase in the non-specific efflux of Ca²⁺ from the vesicles.