Cab45b, a Munc18b-interacting Partner, Regulates Exocytosis in Pancreatic β-Cells

Yi Zhang,You-Hou Kang,Nathan Chang,Patrick P.L Lam,Yunfeng Liu,Vesa M Olkkonen,Herbert Y Gaisano

doi:10.1074/jbc.m109.017467

Abstract

Cab45b is a cytosolic Ca(2+)-binding protein reported to regulate zymogen secretion in pancreatic acini. We now show that Cab45b is also expressed in pancreatic islet beta-cells and interacts there with the Sec1-Munc18 protein Munc18b. We employed patch clamp cell capacitance measurements to show that antibodies against Cab45b inhibited depolarization-evoked membrane capacitance increments, suggesting an impact on beta-cell granule exocytosis, both the readily releasable granule pool and refilling of this pool. Site-specific mutants in the Cab45b EF-hands were used to dissect the molecular interactions involved in Cab45b function. Mutants in EF-hands 2 and 3 had no detectable effects on interaction of Cab45b with Munc18b and did not affect the depolarization-evoked calcium currents, but remarkably, they facilitated the complex formation of Munc18b with syntaxin-2 and -3. As a result, these two EF-hand mutants inhibited beta-cell membrane capacitance increments. This inhibition is mediated via Munc18b because Munc18b silencing with small interfering RNA abolished the effects of these two mutants. The results suggest a mechanism for Cab45b action that involves regulating the dynamic association of Munc18b with SNAREs to impact beta-cell granule exocytosis.

Highlights

It is well established that the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)2 proteins form the core machinery responsible for the fusion of transport vesicles, including secretory granules, with their target membranes
Using cell membrane capacitance measurement, a well established technique for monitoring exocytosis in neurons and neuroendocrine cells [18, 19], we further dissect the functional domains within Cab45b (EF-hands 2 and 3) that impact the association of Munc18b with syntaxins to influence insulin granule exocytosis
The results suggest that Cab45b regulates the association of Munc18b with Syn-2 and Syn-3, controlling the participation of these syntaxins in SNARE complex assembly required for exocytosis

Summary

Introduction

It is well established that the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)2 proteins form the core machinery responsible for the fusion of transport vesicles, including secretory granules, with their target membranes. We show that Cab45b is expressed in pancreatic islet ␤-cells and interacts there with the Sec1-Munc18 protein Munc18b.

Results

Conclusion