Department of Pharmacology and Toxicology, Centre for Molecular Biosciences, University of Innsbruck, Innsbruck, Austria.
Department of Drug Science, NIS Centre, University of Torino, Torino, Italy.
J Physiol. 2024 Aug;602(15):3793-3814. doi: 10.1113/JP285681. Epub 2024 Jul 14.
High voltage-gated Ca channels (HVCCs) shape the electrical activity and control hormone release in most endocrine cells. HVCCs are multi-subunit protein complexes formed by the pore-forming α and the auxiliary β, αδ and γ subunits. Four genes code for the αδ isoforms. At the mRNA level, mouse chromaffin cells (MCCs) express predominantly the CACNA2D1 gene coding for the αδ-1 isoform. Here we show that αδ-1 deletion led to ∼60% reduced HVCC Ca influx with slower inactivation kinetics. Pharmacological dissection showed that HVCC composition remained similar in αδ-1 MCCs compared to wild-type (WT), demonstrating that αδ-1 exerts similar functional effects on all HVCC isoforms. Consistent with reduced HVCC Ca influx, αδ-1 MCCs showed reduced spontaneous electrical activity with action potentials (APs) having a shorter half-maximal duration caused by faster rising and decay slopes. However, the induced electrical activity showed opposite effects with αδ-1 MCCs displaying significantly higher AP frequency in the tonic firing mode as well as an increase in the number of cells firing AP bursts compared to WT. This gain-of-function phenotype was caused by reduced functional activation of Ca-dependent K currents. Additionally, despite the reduced HVCC Ca influx, the intracellular Ca transients and vesicle exocytosis or endocytosis were unaltered in αδ-1 MCCs compared to WT during sustained stimulation. In conclusion, our study shows that αδ-1 genetic deletion reduces Ca influx in cultured MCCs but leads to a paradoxical increase in catecholamine secretion due to increased excitability. KEY POINTS: Deletion of the αδ-1 high voltage-gated Ca channel (HVCC) subunit reduces mouse chromaffin cell (MCC) Ca influx by ∼60% but causes a paradoxical increase in induced excitability. MCC intracellular Ca transients are unaffected by the reduced HVCC Ca influx. Deletion of αδ-1 reduces the immediately releasable pool vesicle exocytosis but has no effect on catecholamine (CA) release in response to sustained stimuli. The increased electrical activity and CA release from MCCs might contribute to the previously reported cardiovascular phenotype of patients carrying αδ-1 loss-of-function mutations.
高电压门控钙通道（HVCCs）调节大多数内分泌细胞的电活动和激素释放。HVCCs 是由孔形成的α和辅助β、αδ和γ亚基组成的多亚基蛋白复合物。四个基因编码αδ同工型。在 mRNA 水平上，小鼠嗜铬细胞（MCCs）主要表达编码αδ-1同工型的 CACNA2D1 基因。在这里，我们发现αδ-1 缺失导致 HVCC Ca 内流减少约 60%，失活动力学减慢。药理学分析表明，与野生型（WT）相比，αδ-1 MCCs 中 HVCC 的组成仍然相似，表明αδ-1 对所有 HVCC 同工型都具有相似的功能影响。与 HVCC Ca 内流减少一致，αδ-1 MCCs 的自发性电活动减少，动作电位（AP）的半最大值持续时间缩短，原因是上升和下降斜率更快。然而，诱导的电活动表现出相反的效果，αδ-1 MCCs 在强直放电模式下显示出更高的 AP 频率，并且与 WT 相比，更多的细胞产生 AP 爆发。这种功能获得表型是由 Ca 依赖性 K 电流的功能激活减少引起的。此外，尽管 HVCC Ca 内流减少，但与 WT 相比，在持续刺激期间，αδ-1 MCCs 中的细胞内 Ca 瞬变和囊泡胞吐或内吞作用没有改变。总之，我们的研究表明，αδ-1 基因缺失会减少培养的 MCC 中的 Ca 内流，但由于兴奋性增加，导致儿茶酚胺（CA）分泌的悖论性增加。关键点：αδ-1 高电压门控钙通道（HVCC）亚基缺失可使小鼠嗜铬细胞（MCC）Ca 内流减少约 60%，但导致诱导性兴奋性增加。MCC 细胞内 Ca 瞬变不受 HVCC Ca 内流减少的影响。αδ-1 的缺失减少了即刻可释放的囊泡胞吐作用，但对持续刺激时 CA 的释放没有影响。MCC 中电活动和 CA 释放的增加可能有助于以前报道的携带αδ-1 功能丧失突变的患者的心血管表型。
