Local anesthetics noncompetitively inhibit terbium binding to the exterior surface of nerve membrane vesicles

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 K_d of 2.2 μM. Fluorescence of bound Tb³⁺ occurs via energy transfer from the aromatic residues of proteins (γ_ex = 280 nm; γ_em = 546 nm), and calcium inhibits Tb³⁺ binding competitively with a K_i 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.