Priming of insulin granules for exocytosis by granular chloride uptake and acidification

Priming of insulin granules for exocytosis by granular chloride uptake and acidification

Field: Other

Authors: Barg, Sebastian
Eliasson, Lena
Thevenod, Frank
Rorsman, Patrik
Renström, Erik

Address of presenting
author: Physiological Sciences, Lund University
Sölvegatan 19
S-22362 Lund

E-mail: sebastian.barg@mphy.lu.se

Phone: 046-2220636

Fax: 046-2227763

Text of abstract: ATP-dependent priming of the secretory vesicles precedes Ca²⁺-induced exocytosis.
According to the prevailing hypothesis, ATP-hydrolysis by N-ethylmaleimide-sensitive
factor (NSF) leads to the disassembly of an exocytotic complex comprised of SNARE
proteins. However, the precise cellular events by which the vesicles become release-competent
remain unestablished. Here we have used insulin-secreting pancreatic B-cells
as a model for Ca²⁺-dependent exocytosis. Exocytosis was measured as whole-cell
capacitance increase, and changes in the intragranular pH monitored using the fluorescent
probe LysoSensor^Ž Red. We found that ATP-hydrolysis controls exocytosis by
energizing a V-type H⁺-ATPase. The activity of the proton pump requires concomitant
influx of Cl^- through ClC3 Cl^--channels and results in intragranular acidification.
Consistent with the idea that acidification is a key event in the priming of the granules for
release, agents that prevent Cl^--uptake or collapse the pH-gradient abolish Ca²⁺-induced
exocytosis by interfering with the refilling of the readily releasable pool of granules
(RRP). The ClC3-channels are reciprocally regulated by ATP and ADP so that their
activity increases when the cytoplasmic ATP:ADP-ratio is high, thus providing a direct
connection between the metabolic state of the cell and its secretory capacity. This
regulation is mediated by a 65 kDa granular protein with a pharmacology similar to that
of the type-1 sulfonylurea receptor (SUR1).

Fulltext / Images

Keywords: exocytosis, granule, chloride, pH, ion channel

	Priming of insulin granules for exocytosis by granular chloride uptake and acidification
Field:	Other
Authors:	Barg, Sebastian Eliasson, Lena Thevenod, Frank Rorsman, Patrik Renström, Erik
Address of presenting author:	Physiological Sciences, Lund University Sölvegatan 19 S-22362 Lund
E-mail:	sebastian.barg@mphy.lu.se
Phone:	046-2220636
Fax:	046-2227763
Text of abstract:	ATP-dependent priming of the secretory vesicles precedes Ca²⁺-induced exocytosis. According to the prevailing hypothesis, ATP-hydrolysis by N-ethylmaleimide-sensitive factor (NSF) leads to the disassembly of an exocytotic complex comprised of SNARE proteins. However, the precise cellular events by which the vesicles become release-competent remain unestablished. Here we have used insulin-secreting pancreatic B-cells as a model for Ca²⁺-dependent exocytosis. Exocytosis was measured as whole-cell capacitance increase, and changes in the intragranular pH monitored using the fluorescent probe LysoSensor^Ž Red. We found that ATP-hydrolysis controls exocytosis by energizing a V-type H⁺-ATPase. The activity of the proton pump requires concomitant influx of Cl^- through ClC3 Cl^--channels and results in intragranular acidification. Consistent with the idea that acidification is a key event in the priming of the granules for release, agents that prevent Cl^--uptake or collapse the pH-gradient abolish Ca²⁺-induced exocytosis by interfering with the refilling of the readily releasable pool of granules (RRP). The ClC3-channels are reciprocally regulated by ATP and ADP so that their activity increases when the cytoplasmic ATP:ADP-ratio is high, thus providing a direct connection between the metabolic state of the cell and its secretory capacity. This regulation is mediated by a 65 kDa granular protein with a pharmacology similar to that of the type-1 sulfonylurea receptor (SUR1).
Fulltext / Images
Keywords:	exocytosis, granule, chloride, pH, ion channel

Index

Created 2000-03-14

Department of Physiological Sciences, Lund University

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