α-Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Is Expressed in Pancreatic β Cells and Functions in Insulin but Not γ-Aminobutyric Acid Secretion

doi:10.1074/jbc.274.12.8053

α-Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Is Expressed in Pancreatic β Cells and Functions in Insulin but Not γ-Aminobutyric Acid Secretion*

From the Departments of ^‡Biochemistry and ^¶Clinical Pathology, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611 Japan

Abstract

The function of solubleN-ethylmaleimide-sensitive attachment protein-α (α-SNAP) in exocytosis still remains obscure. This study was conducted to determine the physiological role of α-SNAP in the secretion of insulin and γ-aminobutryric acid (GABA) from pancreatic β cells. Reverse transcriptase-polymerase chain reaction analysis of total RNA isolated from rat islets disclosed α-SNAP, but not β-SNAP, mRNA expression, and an immunofluorescence study of rat pancreas showed that α-SNAP was present predominantly in the cytoplasm of the islets of Langerhans. α-SNAP overexpression in rat islets enhanced insulin release relative to the control levels. Anin vitro binding study showed that both wild-type α-SNAP and C-terminal–deleted α-SNAP mutant (1–285) can bind to syntaxin 1A. α-SNAP mutant (1–285) was overexpressed to evaluate its activity as dominant-negative effector on insulin release. Overexpression of α-SNAP mutant (1–285) in rat islets and MIN6 cells decreased glucose-stimulated insulin release to about 50% of the control levels. Suppression of endogeneous α-SNAP in MIN6 cells by treatment with an antisense phosphorothioate oligonucleotide resulted in inhibition of insulin release. In order to examine if α-SNAP functions in exocytosis from synaptic-like microvesicles in pancreatic β cells, the functional role of α-SNAP in GABA release from MIN6 cells was studied. The data showed no effect of α-SNAP mutant (1–285) overexpression on GABA release. We conclude that 1) α-SNAP plays a crucial role in insulin exocytosis via large dense core vesicles, but not GABA released via synaptic-like microvesicles, in pancreatic β cells; and 2) the interaction of α-SNAP and syntaxin 1A may play an important role in the insulin exocytotic process.

Footnotes

↵* This work was supported by a Grant-in-Aid for Scientific Research (B) 08457057 from the Japanese Ministry of Education, Science and Culture, a grant from Research for the Future Program, The Japan Society for the Promotion of Science Grant JSPS-RFTF97I00201, and by a grant from the Japan Private School Promotion Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ To whom correspondence should be addressed: Dept. of Biochemistry, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan. Tel.: 81-422-47-5511 (ext. 3476); Fax: 81-422-47-5538; E-mail: shinya{at}kyorin-u.ac.jp.
↵2 S. Nagamatsu, T. Watanabe, Y. Nakamichi, C. Yamamura, K. Tsuzuki, and S. Matsushima, unpublished results.
Abbreviations:

[Ca²⁺]_i

intracellular calcium

RT-PCR

reverse transcriptase-polymerase chain reaction

NSF

N-ethylmaleimide-sensitive factor

SNAP

soluble NSF attachment protein

SNARE

SNAP receptor

SNAP-25

synaptosomal-associated protein of 25 kDa

GABA

γ-aminobutyric acid

LDCV

large dense core vesicle

SLMV

synaptic-like microvesicle

PAGE

polyacrylamide gel electrophoresis

HPLC

high pressure liquid chromatography

IRI

immunoreactive insulin
- Received October 21, 1998.
- Revision received December 28, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.