Stdies of the Ca²⁺ transport mechanism of human erythrocyte inside-out plasma membrane vesicles. II. Stimulation of the Ca²⁺ pump by phosphate

The possible role of phosphate in the calcium translocation process in human erythrocyte inside-out vesicles (IOV) has been investigated. Millimolar phosphate was found to stimulate the initial velocity of calcium transport 3- to 4-fold and increase the K(m) (Ca²⁺) of calcium transport 2- to 3-fold. The stimulation of calcium transport by phosphate was due to an active, calcium-dependent, calmodulin-stimulated uptake of phosphate into IOV. The rates of phosphate transported (+calmodulin, 9.5 nmol/min/unit of acetylcholinesterase; -calmodulin 2.98 nmol/min/unit of acetylcholinesterase) suggest a 1:1 stoichiometry of phosphate transported with calcium. Furthermore, the addition of calcium ionophore A23187 to IOV resulted in a concomitant release of accumulated calcium and phosphate. The K(m) (P(i)) for phosphate transport was 0.6 mM, compared to a K(m) (P(i)) of 1.2 mM for the phosphate-stimulated Ca²⁺ transport. The K(m) (Ca²⁺) for phosphate transport was 1.2 mM in the presence or absence of calmodulin; 50 ng/ml of calmodulin was required to half-maximally activate phosphate transport. Inhibition of phosphate transport by the band III inhibitors, 4-acetamido-4'-isothiocyanostilbene-2'-disulfonic acid and N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate, suggests that net transport of phosphate is mediated by the band III protein. The similar potency of the band III inhibitors on both calcium and phosphate suggests a close relationship between these transport processes. These results are consistent with a model in which the active transport of calcium into human erythrocyte IOV develops a positive membrane potential (inside positive) which facilitates net accumulation of phosphate into the vesicles, reducing the electrochemical gradient against which the calcium pump operates, and stimulating calcium transport.