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

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 2 ^nd -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.

Loss of CHGB channel functions impairs secretory granule acidification in neuroendocrine cells, which necessitates anion shunt conduction. CHGBΔMIF, a mutant unable to form a functional Cl ^- channel, exerts negative dominance on endogenous CHGB and results in granule deacidification in cultured cells. Neither CLC-3 & -5 nor ANO-1 & -2 participate in the CHGB-mediated granule acidification. Clcn3 knockout effects on regulated secretion can be attributed to its functions in endosomal and endolysosomal compartments. Primary Chgb-/- β-cells exhibit persistent granule deacidification, presenting a unifying mechanism for disparate mouse phenotypes: hyperproinsulinemia, near abrogation of 2 ^nd phase insulin release after glucose challenge and diminution of monoamine contents in chromaffin granules.