Search

A conjugate of alpha-bungarotoxin with horseradish peroxidase was used to visualize alpha-bungarotoxin binding sites at the fine structural level in isolated axonal membrane vesicles from lobster walking leg nerve. These plasma membrane vesicles have previously been shown to exhibit saturable binding of [3H]nicotine and [3H]acetylcholine. Binding of the toxin was identified in the axon plasma membrane and could be blocked by pretreatment with excess free alpha-bungaratoxin or d-tubocurarine. Binding sites for alpha-bungarotoxin were identified by the same technique in sections of intact nerve fibers from both lobster and spider crab and were found to be localized primarily in the axolemma rather than in the Schwann cell membrane.

1. Acetylcholinesterase (AChE) activity of axonal membrane vesicles prepared from lobster (Homarus americanus) walking leg nerve is about 200 mumol ACh/hr/mg protein. 2. The enzyme is a membrane-bound glycoprotein that can be solubilized by lysolecithin and digitonin but is rapidly inactivated by Triton X-100 and other nonionic detergents. 3. The walking leg nerve enzyme contrasts most markedly with postsynaptic AChE in its lack of inhibition by excess substrate and its poor susceptibility to peripheral anionic site ligands. 4. Axonal AChE is competitively blocked by eserine sulfate (1 microM) and by low concentrations of procaine (0.1-0.5 mM). 5. Lidocaine, a tertiary amine, and its quaternary ammonium derivative QX-314 are equipotent non-competitive enzyme inhibitors (Kiapp 2-3 mM), while the primary amine compound GX-HCl is totally inactive. 6. Calcium (10 50 mM), propidium (1 microM) and decamethonium (1 microM), peripheral anionic site ligands in the synaptic enzyme, do not protect the axonal enzyme against inhibition by local anesthetics. 7. Although it is possible that this enzyme is an isozyme of the true AChE of excitable membranes, or that it is an incomplete enzyme fragment, it is also possible that there is a unique membrane-bound AChE present in large quantities in crustacean peripheral nerve.

It has previously been shown that terbium binds to membrane vesicles prepared from the walking leg nerve of the lobster (Homarus americanus) with a high affinity Kd of 2.2 microM. Fluorescence of bound Tb3+ occurs via energy transfer from the aromatic residues of proteins (gamma ex = 280 nm; gamma em = 546 nm), and calcium inhibits Tb3+ binding competitively with a Ki of 1.8 mM. Displacement studies with EDTA demonstrate that more than 95% of the bound Tb3+ is at the vesicle exterior and is not being taken up by the vesicles. To investigate the putative role of Ca2+ in the interaction of local anesthetics with axonal membranes, lidocaine and the analogs GX-HCl and QX-314 were tested as inhibitors of Tb3+ binding. Inhibition by lidocaine is seen only at considerably higher doses (25 mM) than are required for conduction block of intact nerves (5 mM). Inhibition by lidocaine and the primary amine analog GX-HCl is entirely noncompetitive, whereas the quaternary ammonium derivative QX-314 appears to be a mixed competitive-noncompetitive inhibitor of Tb3+ binding. These data are not compatible with the hypothesis that there is a functionally essential cation binding site on the axonal membrane surface for which Ca2+ and local anesthetics compete, although local anesthetic action may be modified indirectly by altered calcium concentrations. Evidence is presented for a mechanism by which local anesthetics indirectly displace Tb3+ by altering the physical state of the axonal membrane.

125I-alpha-Bungarotoxin (alpha-BGT) was used to characterize the binding sites for cholinergic ligands in lobster walking leg nerve membranes. The toxin binding component has been visualized histochemically on the external surfaces of intact axons and isolated axonal membrane fragments. Binding of alpha-BGT to nerve membrane preparations was demonstrated to be saturable and highly reversible (KappD congruent to 1.7 +/- 0.32 X 10(-7) M; Bmax congruent to 249 +/- 46 pmol/mg protein) at pH 7.8, 10 mM-Tris buffer. Binding showed a marked sensitivity to ionic strength that was attributable to the competitive effects of inorganic cations (particularly Ca2+ and Mg2+) in the medium. 125I-alpha-BGT binding could be inhibited by cholinergic drugs (atropine congruent to d-tubocurarine greater than nicotine greater than carbamylcholine congruent to choline) and local anesthetics (procaine greater that tetracaine congruent to lidocaine), but was unaffected by other neuroactive compounds tested (e.g., tetrodotoxin, 4-aminopyridine, quinuclidinyl benzilate, octopamine, bicuculline, haloperidol, ouabain). The pharmacological sensitivity of toxin binding resembles that of nicotine binding to axonal membranes, but differs significantly from nicotinic cholinergic receptors described in neuromuscular junctions, fish electric organs, sympathetic ganglia, and the CNS. The possible physiological relevance of the axonal cholinergic binding component and its relationship to alpha-BGT binding sites in other tissues are discussed.

Isologous local anesthetics containing the ester, thiolester, or selenolester grouping and their quaternary ammonium analogs were studied for their ability to displace [3H]nicotine from plasma membrane fragments of lobster nerve. Tertiary and quaternary analogs were equiactive. The relative ability of oxo, thio, and seleno analogs to displace nicotine was the same as their relative ability to block axonal conduction and synaptic transmission. Among cholinergic ligands, choline and aminocholine, previously shown to be inactive as depolarizing agents, were uniquely unable to displace nicotine. The findings are compatible with the presence of a biopolymer capable of binding cholinergic ligands in axonal membranes and support the postulate that the relative inactivity of quaternary compounds in intact axons is due to permeability barriers.

Using terbium ions as fluorescence probes of calcium-binding sites and osmotic shock to induce trapping of Tb3+ in the vesicle interior, direct binding assays have been developed to study the competition between calcium and local anesthetics for binding sites at the cytoplasmic surface of axonal membrane vesicles. Pharmacologically active concentrations of the membrane-permeable local anesthetic, lidocaine, competitively displace bound Tb3+ in the vesicles, while QX-314, a quaternary ammonium analog of lidocaine that has poor access to the vesicle interior, exhibits no significant displacement of osmotically-loaded, internally-bound Tb3+. These experiments support the hypothesis that local anesthetics may function by displacing Ca2+ from a functionally specific binding site in nerve membranes.