Effect of tetanus toxin on the accumulation of the permeant lipophilic cation tetraphenylphosphonium by guinea pig brain synaptosomes
- Sofia Ramos*,
- Evelyn F. Grollman†,
- Pedro S. Lazo†,
- Sherry A. Dyer†,
- William H. Habig*,
- M. Carolyn Hardegree*,
- H. Ronald Kaback‡, and
- Leonard D. Kohn†
- *Bacterial Toxins Branch, Division of Bacterial Products, Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland 20205
- †Section on Biochemistry of Cell Regulation, Laboratory of Biochemical Pharmacology, National Institute of Arthritis, Metabolism, and Digestive Diseases, Bethesda, Maryland 20205
- ‡Laboratory of Membrane Biochemistry, Roche Institute of Molecular Biology, Nutley, New Jersey 07110
Abstract
Accumulation of the permeant lipophilic cation [3H]tetraphenylphosphonium (TPP+) by synaptosome preparations from guinea pig brain cerebral cortex is inhibited 1:10 by medium containing 193 mM K+ and by veratridine. A further 1:10 to 1:15 decrease in TPP+ uptake occurs under nitrogen and in the presence of mitochondrial inhibitors such as oligomycin, whereas starvation and succinate supplementation have no effect. These data indicate that, in analogy to intact neurons, there is an electrical potential (ΔΨ, interior negative) of -60 to -80 mV across the synaptosomal membrane that is due primarily to a K+ diffusion gradient (K+ in→K+ out). The data also indicate that mitochondria entrapped within the synaptosome but not free mitochondria make a large contribution to the TPP+ concentration gradients observed.
Conditions are defined in which tetanus toxin binds specifically and immediately to synaptosomes in media used to measure TPP+ uptake. Under these conditions tetanus toxin induces dose-dependent changes in TPP+ uptake that are blocked by antitoxin and not mimicked by biologically inactivated toxin preparations. The effect of tetanus toxin on TPP+ uptake is not evident in the presence of 193 mM K+ or veratridine but remains under conditions known to abolish the mitochondrial ΔΨ. Moreover, tetanus toxin has no effect on TPP+ uptake by isolated synaptosomal mitochondria. The results thus define an in vitro action of tetanus toxin on the synaptosomal membrane that can be correlated with biological potency in vivo and is consistent with the in vivo effects of tetanus toxin on neuronal transmission.
