Fig. 1. Ca2+ induces changes in the conformation of SNAREs in
permeabilised chromaffin cells: acquisition of resistance to trypsin
correlates with the extent of exocytosis. Chromaffin cells permeabilised using
20 µM digitonin in KGEP without (
) or with 2 mM MgATP ([UNK]) and the
indicated buffered, free [Ca2+]. After 15 minutes, aliquots were
assayed (±s.d, n=4; some error bars are obscured by symbols)
for released catecholamine (A). The cells were maintained for a further 30
minutes, 2 mM PMSF was added and a membrane fraction prepared from four wells;
after boiling for 2 minutes in 1% SDS, the samples were subjected to SDS-PAGE
and western blotting, with antibodies reactive with the specified proteins
(B). Only the relevant track positions are shown. (C) Western blots of
membranes from cells treated as above, except that 100 µg/ml trypsin was
added to the KGEP immediately after the removal of aliquots for the
catecholamine assay; thus, the cells were exposed to the protease for 30
minutes. (D,E) The intensity of all the immunosignals, in cells exposed to
trypsin in the absence of MgATP, were computed from digitised images (see
Materials and Methods). For each protein, the values were normalised as a
percentage of the highest intensity signal, then plotted against
[Ca2+]; values for DßH are plotted in (D) while synaptotagmin
I (), syntaxin (
), SNAP-25 (
) and synaptobrevin (
) are
shown in (E). Plotted data are representative of results obtained on at least
three separate occasions.
