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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