We have examined the effect of alpha-SNAP on exocytosis in adrenal chromaffin cells by direct assay of exocytosis using patch-clamp capacitance analysis. Cells were recorded using the whole cell patch-clamp configuration and the cells dialysed with control pipette solution or with a pipette solution containing alpha-SNAP or the deletion mutant alpha-SNAP(41–295). The deletion mutant was found to be unable to bind to syntaxin allowing a test of the requirement for syntaxin-binding for any effect of alpha-SNAP on exocytosis. Following cell dialysis for 10 minutes, cells were depolarised five times at 2 minute intervals. At each depolarisation step cells dialysed with alpha-SNAP showed a significant increase in both the initial rate and extent of exocytosis which was seen as a rise in membrane capacitance. This increase in exocytosis was not observed with alpha-SNAP(41–295) which instead produced some inhibition of the extent but had no effect on the initial rate of exocytosis. These results show directly that alpha-SNAP has a specific and marked stimulatory effect on exocytosis in chromaffin cells.

REFERENCES

REFERENCES
Bajjalieh
S. M.
,
Scheller
R. H.
(
1995
).
The biochemistry of neurotransmitter secretion.
J. Biol. Chem
270
,
1971
1974
Barnard
R.
,
Morgan
A.
,
Burgoyne
R. D.
(
1996
).
Domains of-SNAP required for the stimulation of exocytosis and for NSF-binding and activation.
Mol. Biol. Cell
7
,
693
701
Bittner
M. A.
,
DasGupta
B. R.
,
Holz
R. W.
(
1989
).
Isolated light chains of botulinum neurotoxins inhibit exocytosis.
J. Biol. Chem
264
,
10354
10360
Blasi
J.
,
Chapman
E. R.
,
Yamasaki
S.
,
Binz
T.
,
Niemann
H.
,
Jahn
R.
(
1993
).
Botulinum neurotoxin C1 blocks neurotransmitter release by means of cleaving HPC-1/syntaxin.
EMBO J
12
,
4821
4828
Burgoyne
R. D.
(
1991
).
Control of exocytosis in adrenal chromaffin cells.
Biochim. Biophys. Acta
1071
,
174
202
Burgoyne
R. D.
,
Morgan
A.
(
1993
).
Regulated exocytosis.
Biochem. J
293
,
305
316
Burgoyne
R. D.
(
1995
).
Fast exocytosis and endocytosis triggered by depolarisation in single adrenal chromaffin cells before rapid Ca2+current run-down.
Pflugers Arch
430
,
213
219
Burgoyne
R. D.
,
Morgan
A.
(
1995
).
Ca2+and secretory vesicle dynamics.
Trends Neurosci
18
,
191
196
Calakos
N.
,
Bennett
M. K.
,
Peterson
K. E.
,
Scheller
R. H.
(
1994
).
Protein-protein interactions contributing to the specificity of intracellular vesicular trafficking.
Science
263
,
1146
1149
Chamberlain
L. H.
,
Roth
D.
,
Morgan
A.
,
Burgoyne
R. D.
(
1995
).
Distinct effects of-SNAP, 14–3-3 proteins and calmodulin on priming and triggering of regulated exocytosis.
J. Cell Biol
130
,
1063
1071
Cheek
T. R.
,
Burgoyne
R. D.
(
1986
).
Nicotine-evoked disassembly of cortical actin filaments in adrenal chromaffin cells.
FEBS Lett
207
,
110
114
Chow
R. H.
,
Ruden
L. V.
,
Neher
E.
(
1992
).
Delay in vesicle fusion revealed by electrochemical monitoring of single secretory events in adrenal chromaffin cells.
Nature
356
,
60
63
DeBello
W. M.
,
O'Connor
V.
,
Dresbach
T.
,
Whiteheart
S. W.
,
Wang
S. S.-H.
,
Schweizer
F. E.
,
Betz
H.
,
Rothman
J. E.
,
Augustine
G. J.
(
1995
).
SNAP-mediated protein-protein interactions essential for neurotransmitter release.
Nature
373
,
626
630
Ferro-Novick
S.
,
Jahn
R.
(
1994
).
Vesicle fusion from yeast to man.
Nature
370
,
191
193
Foran
P.
,
Lawrence
G.
,
Dolly
J. O.
(
1995
).
Blockade by botulinum neurotoxin B of catecholamine release from adrenochromaffin cells correlates with its cleavage of synaptobrevin and a homologue present on the granules.
Biochemistry
34
,
5494
5503
Hanson
P. L.
,
Otto
H.
,
Barton
N.
,
Jahn
R.
(
1995
).
The N-ethylmalemide-sensitive fusion protein and-SNAP induce a conformational change in syntaxin.
J. Biol. Chem
270
,
16955
16961
Hayashi
T.
,
Yamasaki
S.
,
Nauenburg
S.
,
Binz
T.
,
Niemann
H.
(
1995
).
Disassembly of the reconstituted synaptic vesicle membrane complex in vitro.
EMBO J
14
,
2317
2325
Hodel
A.
,
Schafer
T.
,
Gerosa
D.
,
Burger
M. M.
(
1994
).
In chromaffin cells, the mammalian Sec1p homologue is a syntaxin 1A-binding protein associated with chromaffin granules.
J. Biol. Chem
269
,
8623
8626
Hohne-Zell
B.
,
Stecher
B.
,
Gratzl
M.
(
1993
).
Functional characterization of the catalytic site of the tetanus toxin light chain using permeabilised adrenal chromaffin cells.
FEBS Lett
336
,
175
180
Holz
R. W.
,
Bittner
M. A.
,
Peppers
S. C.
,
Senter
R. A.
,
Eberhard
D. A.
(
1989
).
MgATP-independent and MgATP-dependent exocytosis.
J. Biol. Chem
264
,
5412
5419
Kee
Y.
,
Lin
R. C.
,
Hsu
S.-C.
,
Scheller
R. H.
(
1995
).
Distinct domains of syntaxin are required for synaptic vesicle fusion complex formation and dissociation.
Neuron
14
,
991
998
Kibble
A. V.
,
Burgoyne
R. D.
(
1996
).
Calmodulin increases the intial rate of exocytosis in adrenal chromaffin cells.
Pflugers Arch
431
,
464
466
Kiraly-Borri
C. E.
,
Morgan
A.
,
Burgoyne
R. D.
,
Weller
U.
,
Wollheim
C. B.
,
Lang
J.
(
1996
).
Soluble N-methylmaleimide-sensitive factor attachment protein and N-ethylmaleimide-insensitive factors are required for Ca2+-stimulated exocytosis of insulin.
Biochem. J
314
,
199
203
Knight
D. E.
,
Tonge
D. A.
,
Baker
P. F.
(
1985
).
Inhibition of exocytosis in bovine adrenal medullary cells by botulinum toxin type D.
Nature
317
,
719
721
McMahon
H. T.
,
Sudhof
T. C.
(
1995
).
Synaptic core complex of synaptobrevin, syntaxin and SNAP25 forms high affinity-SNAP binding site.
J. Biol. Chem
270
,
2213
2217
Montecucco
C.
,
Schiavo
G.
(
1994
).
Molecular mechanisms of action of bacterial protein toxins.
Mol. Microbiol
13
,
1
8
Morgan
A.
,
Burgoyne
R. D.
(
1995
).
A role for soluble NSF attachment proteins (SNAPs) in regulated exocytosis in adrenal chromaffin cells.
EMBO J
14
,
232
239
Neher
E.
,
Marty
A.
(
1982
).
Discrete change of cell membrane capacitance observed under conditions of enhanced secretion in bovine adrenal chromaffin cells.
Proc. Nat. Acad. Sci. USA
79
,
6712
6716
Pallanck
L.
,
Ordway
R. W.
,
Ganetzky
B.
(
1995
).
A Drosophila NSF mutant.
Nature
376
,
25
–.
Parsons
T. D.
,
Coorssen
J. R.
,
Horstmann
H.
,
Almers
W.
(
1995
).
Docked granules, the exocytic burst, and the need for ATP hydrolysis in endocrine cells.
Neuron
15
,
1085
1096
Pryer
N. K.
,
Wuestehube
L. J.
,
Sheckman
R.
(
1992
).
Vesicle-mediated protein sorting.
Annu. Rev. Biochem
61
,
471
516
Pusch
M.
,
Neher
E.
(
1988
).
Rates of diffusional exchange between small cells and a measuring patch pipette.
Pflugers Arch
411
,
204
211
Regazzi
R.
,
Wollheim
C. B.
,
Lang
J.
,
Theler
J.-M.
,
Rossetto
O.
,
Montecucco
C.
,
Sadoul
K.
,
Weller
U.
,
Palmer
M.
,
Thorens
B.
(
1995
).
VAMP-2 and cellubrevin are expressed in pancreatic B-cells and are essential for Ca2+but not for GTPS-induced insulin secretion.
EMBO J
14
,
2723
2730
Roth
D.
,
Burgoyne
R. D.
(
1994
).
SNAP-25 is present in a SNARE complex in adrenal chromaffin cells.
FEBS Lett
351
,
207
210
Roth
D.
,
Burgoyne
R. D.
(
1995
).
Stimulation of catecholamine secretion from adrenal chromaffin cells by 14–3-3 proteins is due to reorganisation of the cortical actin network.
FEBS Lett
374
,
77
81
Rothman
J. E.
(
1994
).
Mechanisms of intracellular protein transport.
Nature
372
,
55
62
Sadoul
K.
,
Lang
J.
,
Montecucco
C.
,
Weller
U.
,
Regazzi
R.
,
Catsicas
S.
,
Wollheim
C. B.
,
Halban
P. A.
(
1995
).
SNAP-25 is expressed in Islets of Langerhans and is involved in insulin release.
J. Cell Biol
128
,
1019
1028
Schiavo
G.
,
Shone
C. C.
,
Bennett
M. K.
,
Scheller
R. H.
,
Montecucco
C.
(
1995
).
Botulinum neurotoxin type C cleaves a single lys-ala bond within the carboxyl-terminal region of syntaxins.
J. Biol. Chem
270
,
10566
10570
Sollner
T.
,
Bennett
M. K.
,
Whiteheart
S. W.
,
Scheller
R.
,
Rothman
J.
(
1993
).
A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion.
Cell
75
,
409
418
Sollner
T.
,
Whiteheart
S. W.
,
Brunner
M.
,
Erdjument-Bromage
H.
,
Geromanos
S.
,
Tempst
P.
,
Rothman
J. E.
(
1993
).
SNAP receptors implicated in vesicle targeting and fusion.
Nature
362
,
318
324
Sudhof
T. C.
(
1995
).
The synaptic vesicle cycle: a cascade of protein-protein interactions.
Nature
375
,
645
653
Vitale
M. L.
,
Seward
E. P.
,
Trifaro
J. M.
(
1995
).
Chromaffin cell cortical actin network dynamics control the size of the release-ready vesicle pool and the initial rate of exocytosis.
Neuron
14
,
353
363
von Ruden
L.
,
Neher
E.
(
1993
).
A Ca-dependent early step in the release of catecholamines from adrenal chromaffin cells.
Science
262
,
1061
1065
Whiteheart
S. W.
,
Griff
I. C.
,
Brunner
M.
,
Clary
D. O.
,
Mayer
T.
,
Buhrow
S. A.
,
Rothman
J. E.
(
1993
).
SNAP family of NSF attachment proteins includes a brain-specific isoform.
Nature
362
,
353
355
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