Probing the extracellular release site of the plasma membrane calcium pump

Abstract

The plasma membrane Ca(2+) pump is known to mediate Ca(2+)/H(+) exchange. Extracellular protons activated (45)Ca(2+) efflux from human red blood cells with a half-maximal inhibition constant of 2 nM when the intracellular pH was fixed. An increase in pH from 7.2 to 8.2 decreased the IC(50) for extracellular Ca(2+) from approximately 33 to approximately 6 mM. Changing the membrane potential by >54 mV had no effect on the IC(50) for extracellular Ca(2+). This argues against Ca(2+) release through a high-field access channel. Extracellular Ni(2+) inhibited Ca(2+) efflux with an IC(50) of 11 mM. Extracellular Cd(2+) inhibited with an IC(50) of 1. 5 mM, >10 times better than Ca(2+). The Cd(2+) IC(50) also decreased when the pH was raised from 7.1 to 8.2, consistent with Ca(2+), Cd(2+), and H(+) competing for the same site. The higher affinity for inhibition by Ni(2+) and Cd(2+) is consistent with a histidine or cysteine as part of the release site. The cysteine reagent 2-(trimethylammonium)ethyl methanethiosulfonate did not inhibit Ca(2+) efflux. Our results are consistent with the notion that the release site contains a histidine.

Dynamics of Calcium Clearance in Mouse Pancreatic β-Cells

“…Because the PMCA exports one cytosolic Ca 2ϩ in exchange for one or two extracellular protons, lowering the proton concentration in the bath slows pumping (18). We found that raising the pH in the bath solution to 8.8 (Na8.8) slowed especially the late phase of Ca 2ϩ clearance (Fig.…”

“…with half-saturating concentration (K PMCA ) of 0.50 mol/l (28) multiplied by a titration function expressing the activation of pumping by protons with a pKa of 7.86 (18). Bidirectional transport by the NCX is computed according to a complex rate equation derived from experiments on electrogenic transport in cardiac myocytes (28).…”

“…Thus, to inhibit the NCX, we used Na ϩ -free solution with Li ϩ replacing Na ϩ (Li7.4). To slow the PMCA pump, we raised the pH 8.8 (Na8.8) (18) or added 200 mol/l LaCl 3 (NaLa7.4) (19). Solutions Li8.8 and LiLa7.4 combined conditions to block two transporters.…”

Characterization of postsynaptic Ca2+signals at theDrosophilalarval NMJ

“…PMCA exchanges extracellular proton(s) for intracellular Ca 2ϩ (Niggli et al 1982;Smallwood et al 1983;Xu and Roufogalis 1988), and it can be inhibited by raising the external pH (Kratje et al 1985;Xu et al 2000). Instead of single APs we used trains of APs since [Ca 2ϩ ] i decayed more slowly after a train of APs and thus it was easier to measure.…”

Ca2+Dynamics along Identified Synaptic Terminals inDrosophilaLarvae

“…Studies of erythrocytes have shown that the PMCA exchanges extracellular proton(s) for intracellular Ca 2ϩ (Niggli et al, 1982;Smallwood et al, 1983;Xu and Roufogalis, 1988), and it can be inhibited by raising the external pH (Kratje et al, 1985;Xu et al, 2000). High pH (8.8) has been used to inhibit the PMCA in squid axons (Dipolo and Beauge, 1982), rat sensory neurons (Benham The boutons were ordered from distal to proximal, with the most distal bouton along a branch assigned number 1.…”