A NOVEL OMEGA-CONOPEPTIDE FOR THE PRESYNAPTIC LOCALIZATION OF CALCIUMCHANNELS AT THE MAMMALIAN NEUROMUSCULAR-JUNCTION

Voltage-sensitive Ca2+ channels are essential to transmitter release a t the chemical synapse. To demonstrate the localization of voltage-sen sitive Ca2+ channels in relation to the site of transmitter release, m ouse neuromuscular junctions were double-labelled with alpha-bungaroto xin and a novel voltage-sensitive Ca2+ channel probe, SNX-260, a synth etic analog of omega-conopeptide MVIIC. Similar to omega-conopeptide M VIIC, biotinylated SNX-260 blocked nerve-stimulated transmitter releas e at the mouse neuromuscular junction. Fluorescently-tagged biotinylat ed SNX-260 labelled the nerve terminal which appeared thinner than and was outlined by acetylcholine receptor clusters as seen in en face vi ew. This SNX-260 labelling was inhibited by preincubation with unconju gated SNX-260. Side-views of the neuromuscular junction indicated that the SNX-260 labelling was on the synaptic side facing the acetylcholi ne receptor rather than on the nonsynaptic side of the nerve terminal. This presynaptic binding was confirmed by the absence of SNX-260 labe lling in denervated muscles following a nerve cut or disjunction after collagenase treatment. Confocal microscopy revealed spots of SNX-260 labelling that may correlate with active zones. The SNX-260 labelling pattern was not affected by preincubation with unconjugated SNX-111 (o mega-conopeptide MVIIA), an N-type voltage-sensitive Ca2+ channel bloc ker. These findings suggest that SNX-260 is a novel probe for localizi ng non-N type voltage-sensitive Ca2+ channels and that these voltage-s ensitive Ca2+ channels are localized near the transmitter release site s at the mammalian motor nerve terminal membrane. The results are cons istent with the suggestion that non-N, probably P/Q type voltage-sensi tive Ca2+ channels mediate evoked transmitter release at the mammalian neuromuscular junction.