Effects of Various Enzymes and Chemical Modification Reagents on the Na⁺ and Cl^--dependent Responses of the Ganglion Cells to Acetylcholine

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Using the abdominal ganglion cells of Aplysia, we analyzed the effects of various enzymes and chemical modification reagents on the acetylcholine (ACh)-induced responses of the excitatory (Na⁺-dependent) and inhibitory (Cl^--dependent) types. (1) Phospholipase A (2mg/ml) caused no appreciable effects on either type of response. (2) Phospholipase C (2mg/ml) markedly depressed both types of response. These suggested that the phosphoryl group of the phospholipid is an important site related to the binding of ACh, common to both types of ACh-receptors. (3) Carboxypeptidase A (10mg/ml) caused no observable effects on either type of response. (4) Carboxypeptidase B (10mg/ml) depressed the inhibitory type of response without affecting the excitatory one. (5) Pyridoxal-5′-phosphate (1mM) also depressed the inhibitory response without affecting the excitatory one. These findings (4, 5) suggested that the Cl^--channel in the inhibitory ACh-receptor complex includes a C-terminal lysine which may play an active role in the movement of Cl^- across the receptor membrane. (6) L-Leucine aminopeptidase (1mg/ml) depressed the excitatory response without altering the inhibitory one. (7) p-Nitrothiophenol (1mM) also depressed the excitatory response without affecting the inhibitory one. These findings (6, 7) suggested the presence of a certain N-terminal amino acid near a glutamate or aspartate residue within a molecular moiety of Na⁺-channel included in the excitatory ACh-receptor complex.