In vitro characterization of botulinum toxin types A, C and D action on human tissues: combined electrophysiologic, pharmacologic and molecular biologic approaches.
Human exposure to botulinum toxin typically occurs in two settings: 1) as an etiologic agent in the disease botulism and 2) as a therapeutic agent for the treatment of dystonia. Epidemiologic studies on botulism suggest that the human nervous system is susceptible to five toxin serotypes (A, B, E, F and G) and resistant to two (C and D). In the past, these epidemiologic findings have been used as the basis for selecting serotypes that should be tested as therapeutic agents for dystonia. Epidemiologic data have been utilized because there are no studies of botulinum neurotoxin action on isolated human nerves. In the present study, electrophysiologic techniques were used to monitor toxin effects on neuromuscular transmission in surgically excised human pyramidalis muscles, ligand binding studies were done to detect and characterize toxin receptors in human nerve membrane preparations, and molecular biologic techniques were used to isolate and sequence a human gene that encodes a substrate for botulinum neurotoxin. The results demonstrated that stable resting membrane potentials (-61.5 mV; S.E.M. +/- 0.7) were maintained in individual fibers of pyramidalis muscle for up to 6 hr at 33 degrees C. The rate of spontaneous miniature endplate potentials was low in physiologic solution (0.14 sec(-1)) but increased in response to elevations in extracellular potassium concentration. In keeping with epidemiologic findings, botulinum toxin type A (10(-8) M) paralyzed transmission in human preparations (ca. 90 min). In contrast to epidemiologic findings, serotype C (10(-8) M) also paralyzed human tissues (ca. 65 min). Iodinated botulinum toxin displayed high-affinity binding to receptors in human nerve membrane preparations (serotype A high-affinity site: K(d) = 0.3 nM, B(max) = 0.78 pmol/mg protein; serotype C high-affinity site: K(d) = 1.96 nM, B(max) = 8.9 pmol/mg protein). In addition, the human nervous system was found to encode polypeptides that are substrates for botulinum neurotoxin types A (synaptosomal-associated protein of M(r) 25,000) and C (syntaxin 1A). These data have important implications bearing on: 1) the development and administration of vaccines against botulism and 2) the testing of toxin serotypes for the treatment of dystonia.
Coffield
J A
JA
Department of Medicine, Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA.
Bakry
N
N
Zhang
R D
RD
Carlson
J
J
Gomella
L G
LG
Simpson
L L
LL
D001905:Botulinum Toxins / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D009045:Motor Endplate / Q000187:drug effects; D018482:Muscle, Skeletal / Q000187:drug effects / Q000294:innervation; D009469:Neuromuscular Junction / Q000187:drug effects*
D001905:Botulinum Toxins