Yadav Gaya P, Annamalai Mani, Hagan D Walker, Cui Lina, Mathews Clayton, Jiang Qiu-Xing
bioRxiv. 2024 Dec 24:2024.12.24.630220. doi: 10.1101/2024.12.24.630220.
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 mice exhibited persistent granule deacidification, which suffices to uplift plasma proinsulin level, diminish glucose-induced 2 -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 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 β-cells exhibit persistent granule deacidification, presenting a unifying mechanism for disparate mouse phenotypes: hyperproinsulinemia, near abrogation of 2 phase insulin release after glucose challenge and diminution of monoamine contents in chromaffin granules.
所有真核生物都利用调节性分泌来释放包装在分泌颗粒中的分子信号，用于局部和远程信号传递。近五十年前，首次在牛嗜铬细胞的分泌颗粒中发现了一种阴离子分流电导。这种电导的生化特性仍未明确。十六年前，提出细胞内氯离子/氢离子交换蛋白CLC-3作为候选蛋白，但实验结果对其提出了质疑。在这里，我们表明嗜铬粒蛋白B（CHGB）是培养的和原代神经内分泌细胞中长期寻找的阴离子分流器的核心，其通道功能对于颗粒的正常成熟至关重要。颗粒内pH测量和货物成熟测定表明，神经内分泌细胞中的正常颗粒酸化、胰岛素原向胰岛素的转化以及多巴胺加载均依赖于功能性CHGB+通道。来自小鼠的原代β细胞表现出持续的颗粒脱酸，这足以提高血浆胰岛素原水平、减少葡萄糖诱导的2期胰岛素分泌，并降低敲除小鼠嗜铬颗粒中的单胺含量。来自靶向基因操作、缺乏通道形成部分的缺失突变体的显性负性以及CLC-3/5和ANO-1/2测试的数据均排除了CHGB通道参与分泌颗粒中的阴离子分流。因此，高度保守的CHGB+通道在可能所有脊椎动物的神经元、内分泌、外分泌和干细胞的调节性分泌途径中发挥作用。
CHGB通道功能丧失会损害神经内分泌细胞中的分泌颗粒酸化，这需要阴离子分流传导。CHGBΔMIF是一种无法形成功能性氯离子通道的突变体，对内源性CHGB发挥负显性作用，并导致培养细胞中的颗粒脱酸。CLC-3和-5以及ANO-1和-2均不参与CHGB介导的颗粒酸化。Clcn3基因敲除对调节性分泌的影响可归因于其在内体和内溶酶体区室中的功能。原代β细胞表现出持续的颗粒脱酸，为不同的小鼠表型提供了统一的机制：高胰岛素原血症、葡萄糖刺激后2期胰岛素释放几乎消失以及嗜铬颗粒中单胺含量减少。
