Time-dependent binding of paramagnetic and fluorescent hydrophobic ions to the acetylcholine receptor from Torpedo

In receptor-rich vesicles isolated from Torpedo, paramagnetic or fluorescent phosphonium ions bind to both the acetylcholine receptor (AcChR) and the receptor membrane. When added to receptor vesicles, two to three phosphoniums undergo a slow time-dependent binding to the AcChR. The presence of agonist increases the rate but not the extent of binding of the alkylphosphonium nitroxides. Approximately one phosphonium per receptor can be displaced by the addition of saturating concentrations of the high-affinity histrionicotoxin derivative isodihydrohistrionicotoxin or by the addition of phencyclidine or quinacrine mustard. In addition, preincubation of the receptor with these channel blockers prevents approximately one phosphonium from binding to the receptor. When a series of alkyltriphenylphosphonium ions was studied, it was found that the rate of phosphonium binding to the receptor decreased with increasing probe hydrophobicity. This appears to be a function of the partitioning of the probe between membrane and aqueous phases. The phosphonium ions used here promote desensitization of the receptor, as judged by the binding rate of the fluorescent agonist NBDA-C5-acylcholine or alpha-bungarotoxin. Preincubation of the receptor with isodihydrohistrionicotoxin virtually eliminates the phosphonium-mediated desensitization. The rates of the phosphonium-mediated desensitization also appear to be dependent upon the phase partitioning of the probe. These results strongly suggest that the binding sites for the phosphonium ion (and the high-affinity histrionicotoxin blocking site) are accessible only through the aqueous phase. The phosphonium binding and agonist-induced transitions observed here are not observed with a negative hydrophobic ion probe, or a negative surface amphiphile, indicating that modifications in membrane electrostatics do not contribute to the observed changes.(ABSTRACT TRUNCATED AT 250 WORDS)