The effect of transfection with Botulinum neurotoxin C1 light chain on exocytosis measured in cell populations and by single-cell amperometry in PC12cells

We examined the effect on exocytosis in PC12 neuroendocrine cells of transi ent transfection with the specific endoprotease Botulinum neurotoxin C1 lig ht chain (BoNT/C1), which cleaves syntaxin and SNAP-25. The effects of toxi n expression on basal and evoked exocytosis were determined in cell populat ion measurements and also in a single-cell transfection-amperometry assay. Go-expression of BoNT/C1 with human growth hormone (hGH) as a marker of sec retory granules in transfected cells resulted in a 95% inhibition of hGH re lease evoked either by the purinergic agonist ATP or by depolarization with 55 mM K+. In addition, basal hGH release was also inhibited to the same ex tent. The high level of co-transfection efficiency revealed by this extent of inhibition was exploited in a high-resolution single-cell assay based on cell detection by expression of enhanced green fluorescent protein (EGFP) and analysis of evoked dopamine release by amperometry using a carbon fibre microelectrode. Cells expressing EGFP alone showed population responses an d single-cell amperometric responses indistinguishable from those of contro l non-transfected cells. In contrast, co-expression of BoNT/C1 with EGFP re sulted in an almost complete inhibition of current transients due to exocyt osis evoked by ATP. These results establish and validate a single-cell assa y of transfection-amperometry for analysing the effects of specific protein s on exocytosis.