One of the molecular mechanisms capable of regulating the physiological properties of neurones is the phosphorylation of ion channels and other cellular components by cyclic AMP-dependent protein kinase. Another protein kinase present in high concentrations in the mammalian brain is protein kinase C (a calcium/phosphatidylserine/diacylglycerol-dependent protein kinase), but there is no direct evidence, as yet, for the involvement of this enzyme in the control of neuronal excitability. We now present evidence that activation of endogenous protein kinase C by the tumour-promoting phorbol ester TPA (12-O-tetradecanoyl- phorbol-13-acetate), or intracellular injection of the purified enzyme, enhances the voltage-sensitive calcium current in bag cell neurones of the mollusc Aplysia.

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A simple method has been developed for fusing synaptic vesicles into spherical structures 20-50 pzm in diameter. The method has been applied to purified cholinergic synaptic vesicles from Torpedo electric organ, and the membrane properties of these fused structures have been studied by the "cell"-attached version of the patch clamp technique. A large conductance potassium-preferring channel, termed the P channel, was consistently observed in preparations of fused synaptic vesicles. The selectivity of the channel for potassium over sodium was =2.8-fold. Two major conductance levels were observed during P-channel activity, and their relative proportion was dependent on the voltage applied to the membrane through the patch pipette. P channels were not seen in fused preparations of purified Torpedo lipids, nor was the frequency of their occurrence increased in preparations enriched with plasma membrane or nonvesicular membranes. We suggest, therefore, that the P channels are components of the synaptic vesicle membrane. Their function in synaptic transmission physiology is still unknown. (7). Briefly, vesicles were extracted from frozen and crushed electric organ in 0.4 M NaCl/3.5 mM EGTA/10 mM Tris HCl, pH 7.4. They were prepurified on a step sucrose gradient (0.6 M sucrose/0.1 M NaCl/10 mM Tris HCl, pH 7.4; 0.2 M sucrose/0.3 M NaCl/10 mM Tris HCl, pH 7.4). The crude vesicle fraction at the interface (Vb) was collected, mixed with 1 M sucrose to a final sucrose concentration of 0.7-0.8 M, and further purified on a shallow continuous sucrose flotation gradient (sucrose concentrations: 0.7-0.8 M, 0.5 M, 0.45 M, 0.2 M) in either a zonal or swinging bucket rotor. The characterization of the vesicle fraction used in these experiments (Vf; band between 0.5 and 0.2 M sucrose) has been described in detail elsewhere (8). Neither Na/K-ATPase nor 5' nucleotidase was detectable in the purified vesicle preparation.Purification of Torpedo Lipids. Phospholipids were purified from electric organs of T. marmorata by a modification of the method of Bligh and Dyer (9). The frozen organ was thawed, minced, and then extracted by homogenization in chloroform/methanol. The samples were centrifuged (200 x g for 40 min), and the phospholipid-containing chloroform layer was removed and evaporated to dryness. Samples were redissolved in chloroform, loaded onto a column (4 x 80 cm2) of silicic acid, and eluted with chloroform. Fractions were monitored by thin-layer chromatography (Merck; activated plate; 75:25:4, chloroform/methanol/water) for phospholipids; protein was determined by the method of Lowry et al. (10). A protein/lipid ratio of <1:1000 was obtained after one further cycle of chloroform/methanol extraction.Preparation of Plasma Membranes. Plasma membranes were prepared by hypoosmotic lysis of synaptosomes prepared from Torpedo electric organ by the method of Michaelson and Sokolovsky (11). Synaptosomes were stirred for 30 min at 40C in 20 vol of 5 mM potassium glutamate (pH 7.4). Plasma membranes were pelleted by centrifu...