Existence of a Ca 2+-dependent K + channel in synaptic membrane and postsynaptic density fractions isolated from canine cerebral cortex and cerebellum, as determined by apamin binding

Abstract

Apamin, a 18-amino acid neurotoxin isolated from bee venom, is a specific blocker of one class of the Ca 2+-dependent K + channels. The monoiodo derivative of the toxin with high specific radioactivity (1600 Ci/mmol) has been used to study its binding to synaptic. membrane (SM) and postsynaptic density (PSD) fractions isolated from cerebral cortex (CTX) and cerebellum (CL) of canine brains. The B max (30.2 fmol/mg protein) for CTX-PSD is about twice that for CTX-SM (17.3 fmol/mg protein), suggesting a concentration of the apamin receptor protein in CTX-PSD over CTX-SM fractions. The lower value of B max for CL-PSD (12.3 fmol/mg protein), and the higher K d value (51 pM) than for CTX-SM (33 pM), CTX-PSD (24 pM), and and CL-SM (39 pM), may reflect the disruptive effect of Triton X-100 on these thin structures. The values of B max and K d for CTX-SM are similar to those (22.0 fmol/mg protein and 33 pM) for rat CTX-SM 33. Both Ca 2+ and NA + inhibit apamin binding to CTX-PSD with K 0.5 values of 14 and 31 mM, respectively, while the optimum concentration of KCl for activation is 5 mM. All these values are similar to those found for rat synaptosomes 30. Covalent labeling of the apamin binding protein, using the non-cleavable cross-linker, disuccinimidyl suberate, reveals an apamin binding polypeptide of 27 kdaltons under reducing and denaturing conditions in both the CTX-SM and CTX-PSD preparations, similar to that (28 kdaltons) reported for rat CTX-SM fractions 33. Prior phosphorylation of isolated CTX-PSD had no effect on apamin binding, nor did apamin binding influence subsequent phosphorylation of CTX-PSD. Calmodulin, an intrinsic PSD protein, may not play a role in apamin binding to PSD, since addition of calmodulin, or removal of the calmodulin by EGTA treatment, resulted in no change in the binding capacity of the PSD. The apamin binding protein seems to be bound quite firmly in the CTX-PSD fraction since treatments with 0.5% deoxycholate, 1% N-lauroyl sarcosinate, 4 M guanidine-HCl, pH 7.0, 0.5 M KCl, could only remove the apamin-receptor complexes from CTX-PSD by 40, 55, 52, 12 and 15%, respectively. These results contrast with the findings that the two detergents mentioned solubilize 80–93% of the receptor from the synaptosomal or synaptic membrane fractions, indicating that a good deal of the receptor in these fractions is membrane-bound and not connected to the PSD. The results suggest that the apamin binding protein, which spans the postsynaptic membrane, is an intrinsic PSD component. This finding is the first instance of a channel protein being found in a PSD fraction. The present results, together with previous ones showing the presence of GABA, flunitrazepam, and glutamate binding sites in the isolated PSD fraction, indicate that the PSDs are involved in not only the anchoring of membrane receptor proteins, but also in the anchoring of membrane channel proteins, and suggest a mediating role for the PSD in synaptic events.