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

摘要

class="enumerated" style="list-style-type:decimal">The effect of parvalbumin (PV) on [Ca²⁺] transients was investigated by perfusing adrenal chromaffin cells with fura-2 and fluorescein isothiocyanate (FITC)-labelled PV. As PV diffused into cells, the decay of [Ca²⁺] transients was transformed from monophasic into biphasic. The proportion of the initial fast decay phase increased in parallel with the fluorescence intensity of FITC, indicating that PV is responsible for the initial fast decay phase.The relationship between the fast decay phase and the [Ca²⁺] level was investigated using depolarizing trains of stimuli. Within a train the relative amplitude of the fast decay phase was inversely dependent on the [Ca²⁺] level preceding a given stimulus.Based on these observations, we estimated the Ca²⁺ binding ratio of PV (κP), the apparent dissociation constant of PV for Ca²⁺ (Kdc,app), and the unbinding rate constant of Ca²⁺ from PV (kc-) in the cytosol of chromaffin 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 Kdc,app and kc-, respectively.With the parameters obtained in the perfusion study, we simulated [Ca²⁺] transients, using two different Ca²⁺ extrusion rates (γ) – 20 and 300 s⁻¹– which represent typical values for chromaffin cells and neuronal dendrites, respectively. The simulation indicated that Ca²⁺ is pumped out before it is equilibrated with PV, when γ is comparable to the equilibration rates between PV and Ca²⁺, resulting in the fast decay phase of a biexponential [Ca²⁺] transient.From these results we conclude that Ca²⁺ buffers with slow kinetics, such as PV, may cause biexponential decays in [Ca²⁺] transients, thereby complicating the analysis of endogenous Ca²⁺ binding ratios (κS) based on time constants. Nevertheless, estimates of κS based on Ca²⁺ increments provide reasonable estimates for Ca²⁺ binding ratios before equilibration with PV.