Inositol 3,4,5,6-tetrakisphosphate inhibits insulin granule acidification and fusogenic potential

Abstract

ClC Cl- channels in endosomes, synaptosomes, lysosomes, and β-cell insulin granules provide charge neutralization support for the functionally indispensable acidification of the luminal interior by electrogenic H+-ATPases (Jentsch, T. J., Stein, V., Weinreich, F., and Zdebik, A. A. (2002) Physiol. Rev. 82, 503-568). Regulation of ClC activity is, therefore, of widespread biological significance (Forgac, M. (1999) J. Biol. Chem. 274, 12951-12954). We now ascribe just such a regulatory function to the increases in cellular levels of inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P4) that inevitably accompany activation of the ubiquitous Ins(1,4,5)P3 signaling pathway. We used confocal imaging to record insulin granule acidification in single mouse pancreatic β-cells. Granule acidification was reduced by perfusion of single cells with 10 μM Ins(3,4,5,6)P4 (the concentration following receptor activation), whereas at 1 μM ("resting" levels), Ins(3,4,5,6)P4 was ineffective. This response to Ins(3,4,5,6)P4 was not mimicked by 100 μM Ins(1,4,5,6)P4 or by 100 μM Ins(1,3,4,5,6)P5. Ins(3,4,5,6)P4 did not affect granular H+-ATPase activity or H+ leak, indicating that Ins(3,4,5,6)P4 instead inhibited charge neutralization by ClC. The Ins (3,4,5,6)P4-mediated inhibition of vesicle acidification reduced exocytic release of insulin as determined by whole-cell capacitance recordings. This may impinge upon type 2 diabetes etiology. Regulatory control over vesicle acidification by this negative signaling pathway in other cell types should be considered.