Vol. 289, Issue 3, 1509-1516, June 1999

Characterization of a Vertebrate Neuromuscular Junction That Demonstrates Selective Resistance to Botulinum Toxin¹

Julie A. Coffield, Nabil M. Bakry, Andrew B. Maksymowych and Lance L. Simpson

Department of Physiology and Pharmacology (J.A.C.), College of Veterinary Medicine, University of Georgia, Athens, Georgia and Department of Medicine; and Department of Biochemistry and Molecular Pharmacology (N.M.B., A.B.M., L.L.S.), Jefferson Medical College, Philadelphia, Pennsylvania

Botulinum toxin blocks transmitter release by proceeding through a series of four steps: binding to cell surface receptors, penetration of the cell membrane by receptor-mediated endocytosis, penetration of the endosome membrane by pH-induced translocation, and intracellular proteolysis of substrates that govern exocytosis. Each of these steps is essential for toxin action on intact cells. Therefore, alterations in cell structure or cell function that impede any of these steps should confer resistance to toxin. In the present study, screening for susceptibility to four serotypes of botulinum toxin revealed that the cutaneous-pectoris nerve-muscle preparation of Rana pipiens is resistant to type B botulinum toxin. Resistance was demonstrated both by electrophysiologic techniques and by dye-staining techniques. In addition, resistance to serotype B was demonstrated at toxin concentrations that were 2 orders of magnitude higher than those associated with blockade produced by other serotypes. In experiments on broken cell preparations, type B toxin cleaved synaptobrevin from frog brain synaptosomes. However, the toxin did not bind to frog nerve membranes. These findings suggest that resistance is due to an absence of cell surface receptors for botulinum toxin type B. The fact that cutaneous-pectoris preparations were sensitive to other botulinum toxin serotypes (A, C, and D), as well as other neuromuscular blocking agents (-latrotoxin, -bungarotoxin), indicates that botulinum toxin type B receptors are distinct.