Molecular requirements of chromogranin B for the long-sought anion shunter of regulated secretion

Abstract

All eukaryotes utilize regulated secretion to release molecular signals packaged in secretory granules for local and remote signaling. An anion shunt conductance was first suggested in secretory granules of bovine chromaffin cells nearly five decades ago. Biochemical identity of this conductance remains undefined. CLC-3, an intracellular Cl^-/H⁺ exchanger, was proposed as a candidate sixteen years ago, which, however, was contested experimentally. Here, we show that chromogranin B (CHGB) makes the kernel of the long-sought anion shunter in cultured and primary neuroendocrine cells and its channel functions are essential to proper granule maturation. Intragranular pH measurements and cargo maturation assays revealed that normal granular acidification, proinsulin-insulin conversion, and dopamine-loading in neuroendocrine cells all rely on functional CHGB+ channels. Primary β-cells from Chgb-/- mice exhibited persistent granule deacidification, which suffices to uplift plasma proinsulin level, diminish glucose-induced 2nd-phase insulin secretion and dwindle monoamine content in chromaffin granules from the knockout mice. Data from targeted genetic manipulations, dominant negativity of a deletion mutant lacking channel-forming parts and tests of CLC-3/5 and ANO-1/2 all exclude CHGB-less channels from anion shunting in secretory granules. The highly conserved CHGB+ channels thus function in regulated secretory pathways in neuronal, endocrine, exocrine and stem cells of probably all vertebrates.

Keywords: Anion shunt pathway; Cargo maturation; Dopamine loading; Granule acidification; Hyperproinsulinemia; Neurodegenerative diseases; Neuroendocrine cancer; Proinsulin-to-insulin conversion; Regulated secretory pathways; Secretory granules; Type 2 diabetes (T2DM).