Evidence for an external location of the dihydropyridine agonist receptor site on smooth muscle and skeletal muscle calcium channels

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Abstract
1 The location of the binding domain for agonist dihydropyridines (DHP) has been studied by comparing the action of (+)-202 791 and (−)-Bay K 8644 on Ba2+ currents (IBa) in whole cell patch clamp experiments. Drug effects were examined upon internal and external (extracellular) application in A7r5 smooth muscle cells and BC3H1 cells, a cell line expressing Ca channels of the skeletal muscle type. 2 Efficiency of internal drug application in the whole cell studies was demonstrated by inhibition of potassium currents and barium currents (IBa) upon internal perfusion with tetraethylammonium (TEA+) (10 mm) and the permanently charged phenylalkylamine, D 890 (100 μm) respectively. The uncharged DHP, (−)-STBODIPY-DHP (2 μm) was used to estimate the time course of internal perfusion by monitoring its fluorescence. 3 Intracellular application of (+)-202 791 and (−)-Bay K 8644 (5 μm) in patch clamp experiments was ineffective in stimulating Ca2+ channel currents in both cell lines. In contrast a 50 fold lower agonist concentration (0.1 μm (−)-Bay K8644) applied to the external face of the membrane induced typical changes in tail currents and a current increase under conditions when up to 10 μm of the agonist was present in the intracellular perfusion solution. 4 In cell-attached patches in A7r5 cells, (−)-Bay K 8644 increased and (+)-PN 200 110 inhibited single channel activity when applied via the bath solution. This suggests partitioning and lateral diffusion of the DHPs in the lipid of the plasma membrane. 5 We conclude that the binding site for agonist DHPs on Ca2+ channels in A7r5 and BC3H1 cells is located close to the external surface of the membrane. The DHP binding domain can be reached by agonists and antagonists from the extracellular but not from the intracellular face of the membrane.