Active Transport of Divalent Cations by the Plasma Membrane Calcium Pump

FERREIRA GOMES, MARIELA S, ROSSI JUAN PABLO F.C., MARTIARENA, JORGE L.

Rosario,Buenos Aires, Argentina

Congreso; XXXV ANNUAL MEETING SOCIEDAD ARGENTINA DE BIOFISICA; 2006

SOCIEDAD ARGENTINA DE BIOFISICA

The plasma membrane calcium pump (PMCA) is a fundamental mechanism to actively remove Ca2+ from the cytoplasm of all eukaryotic cells. PMCAs, like all members of the superfamily of P-type ATPases catalyze the hydrolysis of ATP. The main cation transported by PMCA is Ca2+, although other divalent cations (C2+) also seem to be transported. However, reports about C2+ transport are rather controversial. We reported here a method to measure the active transport of C2+ in inside-out vesicles prepared from human red cell membranes. Inside-out vesicles exposed the ATP and transport sites and therefore, it is possible to measure volume changes due to the transport of divalent cations that drive water into the vesicles. Volume changes were measured recording the intensity of light scattered by a suspension of these vesicles which express mostly the isoform PMCA 4b. Transport of each C2+ was assessed by measuring its concentration in vesicles after 30-50 min pumping by Atomic Absortion. Results show that: (1) Ca2+ is actively transported against the electrochemical gradient. Calcium concentration after 30 min pumping is in the order of 20 mM at steady state. (2) Is ATP dependent (3) Velocity of transport increases hyperbolically with [Ca2+] (4) Ca2+ transport is inhibited by La3+. (5) The gradient is collapsed by Ionophore A23187. (6) PMCA is not specific and can transport different divalent cations with different velocities: Ca2+> Sr2+> Ba2+> Zn2+ =Ni2+ >Pb2+ are actively transported by PMCA with decreasing velocity. (7) This results seems to be correlated with their ionic radii, being the optimal between 75 to 132 pm.(8) Cd2+ and Cu2+ seem to be not transported by PMCA, besides they inhibit the transport of Ca2+ probably by association to -SH residues of amino acids. (9) Small divalent ions like Be2+ and Mg2+ are not transported as all monovalent cations. (10) Trivalent cations like La3+, with an ionic radius similar to Ca2+ (106 pm) inhibits PMCA, blocking both active and passive transport. The methodology here described showed that it is possible: (a) to follow the active transport of Ca2+ measuring the slope of light scattering intensity as a function of time; (b) to measure the influence of inhibitors and activators of PMCA; (c) to measure the transport of divalent cations With grants of ANPCYT, CONICET, NIH and UBACYT.