Binding Kinetics of Calbindin-D28k Determined by Flash Photolysis of Caged Ca2+

Abstract

We have used UV flash photolysis of DM-nitrophen in combination with model-based analysis of Oregon Green 488 BAPTA-5N fluorescence transients to study the kinetics of Ca(2+) binding to calbindin-D(28K). The experiments used saturated DM-nitrophen at a [Ca(2+)] of 1.5 microM. Under these conditions, UV laser flashes produced rapid steplike increases in [Ca(2+)] in the absence of calbindin-D(28K), and in its presence the decay of the flash-induced fluorescence was due solely to the Ca(2+) buffering by the protein. We developed a novel method for kinetic parameter derivation and used the synthetic Ca(2+) buffer EGTA to confirm its validity. We provide evidence that calbindin-D(28K) binds Ca(2+) in at least two distinct kinetic patterns, one arising from high-affinity sites that bind Ca(2+) with a k(on) comparable to that of EGTA (i.e., approximately 1 x 10(7) M(-1) s(-1)) and another with lower affinity and an approximately eightfold faster k(on). In view of the inability of conventional approaches to adequately resolve rapid Ca(2+) binding kinetics of Ca(2+) buffers, this method promises to be highly valuable for studying the Ca(2+) binding properties of other biologically important Ca(2+) binding proteins.

Cytosolic Ca2+ Buffers

“…The ratio between high:intermediate affinity sites is either 3:1 or 2:2. The experimental data could be equally well modeled with either stochiometry for the two types of binding sites (Nagerl et al 2000). For determining CR's kinetic Ca 2þ -binding properties in vitro, cooperativity was included Amino acids X, Y, Z, and -Z provide side-chain oxygen ligands, Ã provides the backbone carbonyl oxygen, and at -X, a water molecule is hydrogen-bonded to a loop residue.…”

“…EF-hand 2 is nonfunctional and under physiological conditions EF6 most likely is as well. The four medium/high affinity sites (Nagerl et al 2000) are considered Ca 2þ -specific, albeit low affinity Mg 2þ binding (K D,Mg % 700 mM) to the same sites at physiological [Mg 2þ ] i decreases the apparent Ca 2þ affinity approximately two-fold; additionally, Mg 2þ binding increases the cooperativity of Ca 2þ binding (Berggard et al 2002a). The NMR solution structure of Ca 2þ -bound rat CB-D28k reveals that it consists of a single, almost globular (ellipsoid) fold with six distinguishable EFhand domains (Kojetin et al 2006) (Fig.…”

“…f CB-D28k has high-affinity (h) and medium-affinity (m) sites, the stochiometry h/m is either 2/2 or 3/1 (Nagerl et al 2000). g For details, see text and Faas et al (2007).…”

Deficiency in parvalbumin, but not in calbindin D-28k upregulates mitochondrial volume and decreases smooth endoplasmic reticulum surface selectively in a peripheral, subplasmalemmal region in the soma of Purkinje cells

“…The two proteins differ in their number of Ca 2ϩ -binding sites; two for PV and four for CB, but probably more importantly in their Ca 2ϩ -binding kinetics . PV is a slow-onset Ca 2ϩ buffer (Lee et al, 2000), while Ca 2ϩ -binding to CB is faster (Nagerl et al, 2000). Both proteins contribute to the modulation of parallel fiberevoked Ca 2ϩ transients in PC dendrites (Schmidt et al, 2003b).…”

A Sensitive Period of Mice Inhibitory System to Neonatal GABA Enhancement by Vigabatrin is Brain Region Dependent

“…The increase in CB levels in the hippocampi of GVG-treated newborn and adult mice, without any corresponding alteration in the density or size of CB-IR neurons, might indicate an increase in cellular levels of this protein. The CB protein serves as an endogenous fast Ca 2 + buffer (Nägerl et al, 2000) and thus it regulates activity-dependent processes in synapses (Blatow et al, 2003;Schmidt et al, 2005) and protein activation (Berggard et al, 2002). Therefore, modified cellular CB levels in GVG-treated mice may result in a change in synaptic strength, excitability, and modulation of signaling pathways involving Ca 2 + .…”