title: The Ca(2+)-extruding ATPase of the human platelet creates and responds to cytoplasmic pH changes, consistent with a 2 Ca2+/nH+ exchange mechanism.
abstract: The Ca(2+)-extruding ATPase pump of the human platelet was studied in situ by measuring Ca2+ extrusion from quin2-overloaded platelets (Johansson, J.S., Haynes, D.H. 1988. J. Membrane Biol. 104:147-163). Cytoplasmic pH (pHcyt) was measured by BCECF fluorescence in parallel experiments. The pump was studied by raising the cytoplasmic free Ca2+ to 2.5 microM and monitoring active Ca2+ extrusion into a Ca(2+)-free medium. The pump was shown to perturb pHcyt, to not respond to changes in membrane potential and to respond to imposed changes in pHcyt in a manner consistent with the Ca2+ pump acting as a 2 Ca2+/nH+ exchanger. (i) Raising the external pH (pHext) from 7.40 to 7.60 lowers the Vmax of the pump in basal condition (Vmax,1) from 110 +/- 18 to 73 +/- 12 microM/min (= mumol/liter cell volume/min). (ii) Lowering pHext to 7.13 raised Vmax,1 to 150 +/- 15 microM/min. (iii) In an N-methyl-D-glucamine (NMDG+) medium, the pump operation against high [Ca2+]cyt acidifies the cytoplasm by -0.36 +/- 0.10 pH units, and the pump becomes self-inhibited. (iv) Use of nigericin to drive pHcyt down to 6.23 reduces the Vmax,1 to 18 +/- 11 microM/min. (v) Alkalinization of the cytoplasm by monensin in the presence of Na+ raises the Vmax,1 (basal state with Km,1 = 80 nM) to 136 +/- 24 microM/min, but also activates the pump fourfold (Vmax,2 = 280 +/- 28 microM/min; Km,2 = 502 +/- 36 nM). (vi) Transient elevation of pHcyt by NH4Cl at high [Ca2+]cyt activates the pump eightfold (Vmax,2 > or = 671 +/- 350 microM/min). The large activation by alkaline pHcyt at high [Ca2+]cyt can be explained by Ca(2+)-calmodulin activation of the pump (Valant, P.A., Adjei, P.N., Haynes, D.H. 1992. J. Membrane Biol. 130:63-82) and by increased Ca2+ affinity of calmodulin at high pH.
authors.affiliation: Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Florida 33101.
authors.forename: P A
authors.initials: PA
authors.lastname: Valant
authors.forename: D H
authors.initials: DH
authors.lastname: Haynes
mesh_terms: D001792:Blood Platelets / Q000201:enzymology* / Q000502:physiology; D002118:Calcium / Q000097:blood / Q000502:physiology; D015220:Calcium Channels / Q000097:blood / Q000502:physiology*; D000252:Calcium-Transporting ATPases / Q000097:blood / Q000502:physiology*; D003593:Cytoplasm / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D006859:Hydrogen / Q000378:metabolism / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics
chemical_list: D015220:Calcium Channels; D006859:Hydrogen; D000252:Calcium-Transporting ATPases; D002118:Calcium