Deliberate quin2 overload as a method forIn Situ characterization of active calcium extrusion systems and cytoplasmic calcium binding: application to the human platelet

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Abstract
The objectives of the title were accomplished by a four-step experimental procedure followed by a simple graphical and mathematical analysis. Platelets are (i) overloaded with the indicator quin2 to cytoplasmic concentrations of 2.9mm and (ii) are exposed to 2mm external Ca2+ and 1.0 μm ionomycin to rapidly achieve cytoplasmic Ca2+ ([Ca2+]cyt) of ca. 1.5 μm. (iii) The external Ca2+ is removed by EGTA addition, and (iv) the active Ca2+ extrusion process is then monitored as a function of time. Control experiments show that the ionophore shunts dense tubular uptake and does not contribute to the Ca2+ efflux process during phases iii–iv and that the extrusion process is sensitive to metabolic inhibitors. The progress curves for the decline of quin2 fluorescence (resulting from active Ca2+ extrusion) were analyzed as a function of [Ca2+]cyt using a mathematical model involving the probability that an exported Ca2+ was removed from a quin2 complex (vs. a cytoplasmic binding element). The observed rates of decline of quin2 fluorescence at a particular [Ca2+]cyt are dependent upon (i) the absolute rate of the extrusion system (a function of itsK m, Vm and Hill coefficient (n)), (ii) the intrinsic Ca2+ buffer capacity of the cytoplasm (a function of the total site concentration ([B] T ) and itsK d) and (iii) the buffer capacity of the intracytoplasmic quin2 (a function of its concentration andK d). The contribution of (iii) was known and varied and was used to determine (ii) and (i) as a function of [Ca2+]cyt. The Ca2+ binding data were verified by45Ca2+ experimentation. The data fit a single binding site ([B] T =730±200 μm) with an averageK d of 140±10n m. This can be accounted for by platelet-associated calmodulin. The rate of the Ca2+ extrusionvs. [Ca2+]cyt curve can be described by two components: A saturable one withV m=2.3±0.3 nmol min−1 mg-membrane−1,K m=80±10 andn=1.7±0.3 (probably identified with a Ca2+-ATPase pump) and a linear one (probably identified with a Na+/Ca2+ exchanger).