Kinetics of Ca2+ binding to parvalbumin in bovine chromaffin cells: implications for [Ca2+] transients of neuronal dendrites

1. The effect of parvalbumin (PV) on [Ca2+] transients was investigated by perfusing adrenal chromaffin cells with fura-2 and fluorescein isothiocyana te (FITC)-labelled PV. As PV diffused into cells, the decay of [Ca2+] trans ients was transformed from monophasic into biphasic. The proportion of the initial fast decay phase increased in parallel with the fluorescence intens ity of FITC, indicating that PV is responsible for the initial fast decay p hase. 2. The relationship between the fast decay phase and the [Ca2+] level was i nvestigated using depolarizing trains of stimuli. Within a train the relati ve amplitude of the fast decay phase was inversely dependent on the [Ca2+] level preceding a given stimulus. 3. Based on these observations, we estimated the Ca2+ binding ratio of PV ( kappa(P)), the apparent dissociation constant of PV for Ca2+ (K-dc,K-app), and the unbinding rate constant of Ca2+ from PV (k(c-)) in the cytosol of c hromaffin cells. Assuming free [Mg2+] to be 0.14 mM, we obtained values of 51.4 +/- 2.0 nM (n = 3) and 0.95 +/- 0.026 s(-1) (n = 3), for K-dc,K-app an d k(c-), respectively. 4. With the parameters obtained in the perfusion study, we simulated [Ca2+] transients, using two different Ca2+ extrusion rates (gamma) - 20 and 300 s(-1) - which represent typical values for chromaffin cells and neuronal de ndrites, respectively. The simulation indicated that Ca2+ is pumped out bef ore it is equilibrated with PV, when gamma is comparable to the equilibrati on rates between PV and Ca2+, resulting in the fast decay phase of a biexpo nential [Ca2+] transient. 5. From these results we conclude that Ca2+ buffers with slow kinetics, suc h as PV, may cause biexponential decays in [Ca2+] transients, thereby compl icating the analysis of endogenous Ca2+ binding ratios (kappa(s)) based on time constants. Nevertheless, estimates of kappa(s) based on Ca2+ increment s provide reasonable estimates for Ca2+ binding ratios before equilibration with PV.