Solubilization of the bovine cardiac sarcolemmal binding sites for calcium channel blockers
Authors: RUTH Peter , FLOCKERZI Veit , OEKEN Hans‐Jochem , HOFMANN Franz 
Published: March 2005

Nonionic and ionic detergents were used to solubilize the bovine cardiac sarcolemmal binding sites for nimodipine and (−)desmethoxyverapamil in the absence of added ligand. Only Chaps, digitonin and sucrose monolauryl ester were able to solubilize the binding sites in a form that bound radioligands. About 45% of each of the membrane‐bound high‐affinity site was solubilized by 0.4% Chaps (w/v) in the presence of 48% (w/v) glycerol. The solubilized binding sites were destroyed by trypsin or by a 10‐min incubation at 50°C. Calcium stimulated nimodipine binding slightly at 0.3 mM and inhibited (−)desmethoxyverapamil binding completely with an IC50 of 1.2 mM. Nimodipine binding was reduced by 20% in the presence of EGTA. The solubilized receptors sedimented in sucrose density gradients with an apparent s20,W of 21 S. An identical sedimentation value was obtained for the cardiac sarcolemmal and skeletal transverse tubulus receptor which were prelabeled with nitrendipine and solubilized by digitonin.

Solubilization reduced the affinity of nimodipine for its high‐affinity site slightly from 0.35 nM to 1.2 nM and that for its low‐affinity site from 33 nM to 130 nM. Solubilization did not affect significantly the specific density of these sites. Binding of nimodipine to the low‐affinity site was completely abolished by 0.1 μM nitrobenzylthioinosine. After solubilization only the high‐affinity site for (−)desmethoxyverapamil could be measured with tenfold reduced affinity (Kd= 15.3 nM) but unchanged specific density. Binding to the solubilized high‐affinity site for nimodipine and (−)desmethoxyverapamil was stereospecific and showed a similar rank order as the particulate binding sites. Binding of nimodipine was inhibited allosterically by phenylalkylamines. Similarly, (+)PN200‐110 inhibited allosterically (−)desmethoxyverapamil binding. d‐cis‐Diltiazem stimulated nimodipine binding at 20°C 1.2‐fold, reduced the dissociation rate from 0.018 min−1 to 0.0083 min−1 and had no effect on the association rate (0.173 min−1· nM−1). The Kd calculated from the rate constants was 0.1 nM and in close agreement with the value of 0.49 nM measured under equilibrium conditions in the presence of nitrobenzylthioinosine. In contrast, desmethoxyverapamil increased the dissociation rate of nimodipine to 0.03 min−1. The association and dissociation rate constants for (−)desmethoxyverapamil were 0.024 min−1· nM−1 and 0.025 min−1, respectively. d‐cis‐Diltiazem inhibited (−)desmethoxyverapamil binding in an noncompetitive manner with an IC50 value of 9 μM and stimulated 3.4‐fold the dissociation rate of (−)desmethoxyverapamil, suggesting that the solubilized membranes contained a third site specific for diltiazem.

These results suggest that the cardiac sarcolemmal binding sites for nimodipine, (−)desmethoxyverapamil and d‐cis‐diltiazem are solubilized in a form which is kinetically indistinguishable from the membrane‐bound binding sites. They suggest further that these binding sites are localized either on distinct domains of the same protein or on distinct, but tightly bound subunits of the same structure.