Separate Binding and Functional Sites for ω co‐Conotoxin and Nitrendipine Suggest Two Types of Calcium Channels in Bovine Chromaffin Cells

Abstract

Purified adrenomedullary plasma membranes contain two high‐affinity binding sites for ^l25I‐ω‐conotoxin, with K_D values of 7.4 and 364 pM and B_max values of 237 and 1,222 fmol/mg of protein, respectively. Dissociation kinetics showed a biphasic component and a high stability of the toxin‐receptor complex, with a t_1/2 of 81.6 h for the slow dissociation component. Unlabeled ω‐conotoxin inhibited the binding of the radioiodinated toxin, adjusting to a two‐site model with K_i1 of 6.8 and K_i2 of 653 pM. Specific binding was not affected by Ca²⁺ channel blockers or activators, cho‐linoceptor antagonists, adrenoceptor blockers, Na⁺ channel activators, dopaminoceptor blockers, or Na⁺/H⁺ antiport blockers, but divalent cations (Ca²⁺, Sr²⁺, and Ba²⁺) inhibited the toxin binding in a concentration‐dependent manner. The binding of the dihydropyridine [³H]nitrendipine defined a single specific binding site with a K_D of 490 pM and a B_maxof 129 fmol/mg of protein. At 0.25 μM, co‐conotoxin was notable to block depolarization‐evoked Ca²⁺ uptake into cultured bovine adrenal chromaffin cells depolarized with 59 mMK⁺for 30 s, whereas under the same conditions, 1 μM nitrendipine inhibited uptake by ∼60%. When cells were hyper‐polarized with 1.2 mM K⁺ for 5 min and then Ca²⁺ uptake was subsequently measured during additions of 59 mMK⁺, ω‐conotoxin partially inhibited Ca²⁺ uptake in a concentration‐dependent manner. These results suggest that two different types of Ca²⁺ channels might be present in chromaffin cells. However, the molecular identity of ω‐conotoxin binding sites remains to be determined.