In vivo labeling of L-type Ca2+ channels by fluorescent dihydropyridines: evidence for a functional, extracellular heparin-binding site.

Abstract

We have synthesized and charted fluorescently labeled dihydropyridines (DHos) as probes for L-type Ca2+ channels. Racemic as well as (+)-and (-)-1,4-dihydro-2,6-dlmethryl4-(2-trifluoromethylphenyl)-3,5-pyrdlhncrboxylic acid 2-(aminoethyl)ethyl ester hydrochlorides were coupled to boron dipyrromethane (Bodipy) derivatives. (4,4-Diluoro-5,7-dlmethyl-4-bora-3a,4a-diaza)-3-(s-andaceuepropn add (DMBodipy)-DHP and (4,4-difluoro-7-styryl-4bora-3a Radloligand Biding. Rabbit skeletal muscle transversetubule or guinea pig-cortex membranes were prepared as described (30). Rabbit skeletal muscle L-type Ca2+ channels were partially purified by solubilization and chromatography on wheat germ agglutinin-Sepharose (31). Fluorescent DHPs and other drugs were diluted and added in dimethyl sulfoxide

Effect of aging on calcium signaling in C57Bl6J mouse cerebral arteries

“…The decrease of fluorescent DHP binding found in our study could reflect a decrease in the expression of L-type Ca 2+ channels rather than a post-transductional change that would result more in a change in ligand affinity [28]. Thus, the decrease of depolarisation-induced Ca 2+ signal and contraction could be attributed to the decrease of CaV1.2 expression.…”

Calcium channels: The β‐subunit increases the affinity of dihydropyridine and Ca2+ binding sites of the α1‐subunit

“…It has previously been shown that ligandchannel complexes are destabilized when free Ca*' is reduced below micromolar concentrations, resulting in a time-and temperature-dependent ligand dissociation, that is determined by the conversion rate of the channel to a very low affinity binding state [24]. This process is reversed by readdition of Ca*' [22,24] and the conversion rates, monitored by ligand dissociation from the low affinity state, have been suggested to reflect Ca*' dissociation from the channel [27]. In order to compare the conversion rate constants of a, and a# we performed kinetic experiments where dissociation of (+)-[3HJisradipine binding was induced by reducing the free Ca*' concentration below 10 nM by the addition of 10 mM EDTA.…”

Transgenic Overexpression of the Ca2+-binding Protein S100A1 in the Heart Leads to Increased in Vivo Myocardial Contractile Performance

“…Assessment of ␣ 1 -DHPR-Cardiac membrane preparations (10-g each) from TGS100A1 and control mice were incubated with 30 nM DM-BODIPY-dihydropyridine (Molecular Probes D-7443) (17) for 60 min at room temperature in equilibration buffer composed of 20 mM HEPES, pH 7.0, 150 mM NaCl, 1 mM EDTA, and 0.5% Tween 20. Samples were centrifuged at 100,000 ϫ g for 20 min, washed three times with equilibration buffer, and resuspended in 1 ml of the buffer.…”

“…1B). In addition to the ␣ 2 -DHPR, levels of the ␣ 1 -DHPR subunit, which is the major channel protein of the L-type voltage-dependent calcium channel, were assessed in TG S100A1 and NLC sarcolemmal membrane preparations by quantification of fluorescence DM-BODIPY-dihydropyridine binding (17). This revealed no significant difference between the groups (TG S100A1, 0.30 Ϯ 0.03 versus NLC, 0.31 Ϯ 0.06; n ϭ 6; p ϭ NS.…”