Characteristics of a Ca 2+/calmodulin-dependent binding of the Ca 2+ channel antagonist, nitrendipine, to a postsynaptic density fraction isolated from canine cerebral cortex

Abstract

Synaptic membrane (SM) and postsynaptic density (PSD) fractions isolated from the cerebral cortex (CTX) and cerebellum (CL) of the canine brain were found to contain one class of specific nitrendipine binding sites. The specific binding constants were: CTX-SM, K d = 110 pM ( B max = 126 fmo1/mg protein); CTX-PSD, K d = 207 pM ( B max = 196 fmo1/mg; CL-SM, K d = 100 pM ( B max = 65 fmol/mg); CL-PSD, K d = 189 pM ( B max = 80 fmol/mg). Treatment of the CTX-SM and CTX-PSD fractions with 0.5% deoxycholate and 1.0% N-lauroyl sarcosinate removed 88–91% and 42–51% of the nitrendipine binding, respectively, indicating that the major nitrendipine binding present in the SM fractions are of non-synaptic origin. Moreover, the percentages of total protein and specific nitrendipine binding removed from PSDs by these detergents were similar, indicating no preferential dissociation of the latter, and suggesting that the receptor protein is firmly bound and is probably an intrinsic component of the PSD fraction. Both Ca 2+ and calmodulin were found to be important for the binding of nitrendipine to the CTX-SM and CTX-PSD fractions since: (a) R24571, a calmodulin antagonist, was found to inhibit nitrendipine binding to the CTX-SM and CTX-PSD fractions with IC 50 values of 1.1 μM and 0.9 μM, respectively; (b) removal of Ca 2+ from the CTX-SM and CTX-PSD fractions with 0.2 mM EGTA resulted in losses of specific nitrendipine binding of 80 and 90%, respectively; (c) Ca 2+ alone restored nitrendipine binding to EGTA-pretreated CTX-SM fractions and not to CTX-PSD fractions, with the latter needing both Ca 2+ and calmodulin to restore nitrendipine binding; (d) EGTA treatment removed 14–16% and 89–91% of nitrendipine bound to the CTX-SM and CTX-PSD fractions, respectively, suggesting that calmodulin (but not Ca 2+) is needed to maintain the nitrendipine-nitrendipine receptor-calmodulin complex; (e) Ca 2+-reconstituted EGTA-pretreated CTX-SM fractions and the Ca 2+ plus calmodulin-reconstituted EGTA-pretreated CTX-SM and CTX-PSD fractions were found to have similar binding constants to those for the corresponding native, untreated fractions; and (f) the Ca 2+/calmodulin dependency on nitrendipine binding was similar to the well-known Ca 2+/calmodulin dependency on phosphorylation in EGTA-pretreated PSD fractions. It needed much less Ca 2+ to saturate Ca 2+/calmodulin-dependent phosphorylation of the pretreated CTX-PSD fractions than the nitrendipine binding. Yet, less calmodulin was needed to saturate nitrendipine binding than the phosphorylation. Dithiothreitol was found to inhibit specific binding of nitrendipine to the CTX-SM and CTX-PSD fractions with IC 50 values of 3.6 mM and 3.4 mM, respectively. Verapamil was also inhibitory for the specific binding. However, the benzothiazepine derivatives (±)- cis-diltiazem, (+)- cis-diltiazem and (−)- cis-diltiazem, were found to have no significant effects on either binding of nitrendipine to CTX-SM and CTX-PSD fractions or dissociation by nifedipine of the nitrendipine bound to the subcellular fractions. Previous binding of nitrendipine did not significantly influence the consequent cAMP- or Ca 2+/calmodulin-dependent phosphorylations of CTX-PSD proteins whether performed directly after binding incubation or after removal of excess reagents. However, prior phosphorylation of the CTX-SM fraction in the presence of cAMP, but not of Ca 2+/calmodulin, gave a (≈)40% increase in subsequent specific nitrendipine binding. The results show, for the first time, that a nitrendipine binding site, possibly a voltage-dependent calcium channel, is present in the PSD fraction isolated from the brain.