Distribution of Ca2+ channels on frog motor nerve terminals revealed by fluorescent ω-conotoxin

Abstract

Tetramethylrhodamine-conjugated ω-conotoxin was used as a fluorescent stain (Jones et al., 1989) to determine the spatial distribution of voltage-gated Ca2+ channels along frog motor nerve terminals. Like native ω-conotoxin, the fluorescent toxin blocked neuromuscular transmission irreversibly. The fluorescent staining was confined to the neuromuscular junction and consisted of a series of narrow bands (in face views) or dots (in side views) approximately 1 μm apart. This characteristic staining pattern was prevented by pretreatment with ω-conotoxin and by prior denervation for 5-7 d. Combined fluorescence and phase-contrast optics indicated that the stain was on the synaptic rather than the nonsynaptic side of the nerve terminal. The bands and dots of stain proved to be in spatial register with the postsynaptic junctional folds, as revealed by combined staining of ACh receptors. It is concluded that the voltage-gated Ca2+ channels on frog motor nerve terminals are concentrated at active zones. The findings are consistent with the suggestion (Heuser et al., 1974; Pumplin et al., 1981) that the large intramembranous particles seen at freeze-fractured active zones are voltage-gated Ca2+ channels.