Calcium and magnesium binding to rat parvalbumin

Abstract

Ca2+ and Mg2+ binding to rat parvalbumin was measured by means of the fluorescent Ca2+ indicator fluo‐3 using a method developed earlier [Eberhard, M. & Erne, P. (1991) Eur. J. Biochem. 202, 1333–1338]. We demonstrate that rat parvalbumin contains two equivalent Ca2+/Mg2+ binding sites and that Ca2+ and Mg2+ compete for the same sites. Dissociation constants (Kd) for Ca2+ and Mg2+ in Hepes buffer containing 150 mM K+ at 35°C and pH 7.2 are 11.0 ± 1.8 nM and 41 ± 8 μM, respectively. At an ionic strength below 0.2 M, Kd values of Ca2+ binding to rat parvalbumin are approximately proportional to the ion concentration. Kd values of Ca2+ binding were found to be about fourfold larger in the presence of Na+ as compared with K+, indicating that Na+ distinctly influences Ca2+ binding to rat parvalbumin. Both Ca2+ and Mg2+ binding to parvalbumin are exothermic whereas Ca2+ and Mg2+ binding to fluo‐3 are endothermic entropy‐driven processes.

Parvalbumin Is a Mobile Presynaptic Ca2+Buffer in the Calyx of Held that Accelerates the Decay of Ca2+and Short-Term Facilitation

“…The resulting differential equations (Lee et al, 2000) were solved numerically by fourth-order Runge-Kutta integration, using the following parameters for divalent binding of parvalbumin: (Lee et al, 2000). The simulations in Figure 7 were calculated with 100 M of the slow buffer, which corresponds to 50 M parvalbumin, because parvalbumin has two functional Ca 2ϩ -binding sites (Eberhard and Erne, 1994). The total Ca 2ϩ load at the time of stimulation was 16 M, and the baseline [Ca 2ϩ ] i was 50 nM.…”

“…2). Because parvalbumin contains two Ca 2ϩ -binding sites (Haiech et al, 1979;Eberhard and Erne, 1994), one would expect that 50 M parvalbumin should be as effective as 100 M EGTA. The slight (approximately twofold) discrepancy between the expected and the observed efficiency of exogenously added parvalbumin is most likely attributable to technical reasons, such as a lower effective concentration of the recombinant protein at the tip of the patch pipette (see Materials and Methods).…”

Role of the calcium-binding protein parvalbumin in short-term synaptic plasticity

“…In PVϩ͞ϩ paired experiments, a difficulty arises from the fact that the high affinity Ca 2ϩ -binding sites of PV also have a moderately high affinity for Mg 2ϩ such that PV under basal intracellular Ca 2ϩ is largely in the Mg 2ϩ -bound form (28). Fig.…”

Kinetic and Equilibrium Properties of Regulatory Calcium Sensors of NCX1 Protein

“…Ca 2ϩ /Protein Equilibrium Binding Assayed by Fluo-3-The experimental procedures of this assay were previously described (39,40 ] free ϭ xK d , whereas x represents the fractional saturation of Fluo-3 at each given point of the titration curve (x ϭ 1 and x ϭ ϳ0 were determined experimentally with saturating Ca 2ϩ and EDTA, respectively). The [Ca 2ϩ -CBD] levels were derived as follows.…”

“…To resolve this discrepancy as well as to validate the accuracy of our radioactive measurements, the Ca 2ϩ binding properties of CBD1 were quantitatively evaluated by a complementary and independent approach. To this end, the fluorescence assay of Fluo-3 was performed because of its capacity to measure not only the K d values but also the Ca 2ϩ binding stoichiometry (39,40). This The Ca 2ϩ titration curves and data analysis were done as described in the legend of Fig.…”