Department of Physiology, Faculty of Medicine, Universidad Nacional Autónoma de México, UNAM, Ciudad de México, Mexico.
Am J Physiol Cell Physiol. 2023 Sep 1;325(3):C592-C598. doi: 10.1152/ajpcell.00447.2022. Epub 2023 Jul 17.
Voltage-gated calcium channels (Ca) conduct Ca influx promoting neurotransmitters and hormone release. Ca are finely regulated by voltage-dependent and independent pathways either by G-protein-coupled receptors (GPCRs) or intramembrane lipids, respectively, in neurons and glands. Interestingly, pancreatic β-cells are abundantly innervated by both sympathetic and parasympathetic neurons, while a variety of high-voltage activated (HVA) Ca channels are present in these cells. Thus, autonomic system seems to exert a tonic inhibition on HVA Ca channels throughout GPCRs, constitutively preventing hormone secretion. Therefore, this work aimed to investigate noradrenergic and cholinergic inhibition of HVA Ca channels in pancreatic β-cells. Experiments were conducted in pancreatic β-cells of rat by using patch-clamping methods, immunocytochemistry, pharmacological probes, and biochemical reagents. A voltage-clamp protocol with a strong depolarizing prepulse was used to unmask tonic inhibition. Herein, we consistently find a basal tonic inhibition of HVA Ca channels according to a GPCRs regulation. Facilitation ratio is enhanced by noradrenaline (NA) according to a voltage-dependent regulation and a membrane-delimited mechanism, while no facilitation changes are observed with carbachol or phosphatidylinositol 4,5-bisphosphate (PIP). Furthermore, carbachol or intramembrane lipids, such as PIP, do not change facilitation ratio according to a voltage-independent regulation. Together, HVA Ca channels of pancreatic β-cells are constitutively inhibited by GPCRs, suggesting a natural brake preventing cells from exhaustive insulin secretion. Our results support the hypothesis that GPCRs tonically inhibit HVA Ca channels in pancreatic β-cells. A voltage-clamp protocol with a strong depolarizing prepulse was used to unmask voltage-dependent inhibition of Ca channels. The novelty of these results strengthens the critical role of Gβγ's in Ca channel regulation, highlighting kinetic slowing and increased facilitation ratio. Together, HVA Ca channels of pancreatic β-cells are constitutively inhibited by GPCRs underlying fine-tuning modulation of insulin secretion.
电压门控钙通道（Ca）介导 Ca 内流，促进神经递质和激素释放。在神经元和腺体中，Ca 受到电压依赖性和非依赖性途径的精细调节，分别通过 G 蛋白偶联受体（GPCRs）或跨膜脂质调节。有趣的是，胰岛β细胞被交感和副交感神经元大量支配，而这些细胞中存在多种高电压激活（HVA）Ca 通道。因此，自主神经系统似乎通过 GPCRs 对 HVA Ca 通道发挥持续的抑制作用，从而阻止激素分泌。因此，本工作旨在研究胰岛β细胞中去甲肾上腺素能和胆碱能对 HVA Ca 通道的抑制作用。实验在大鼠胰岛β细胞中通过膜片钳技术、免疫细胞化学、药理学探针和生化试剂进行。使用具有强去极化预脉冲的电压钳实验方案来揭示持续抑制作用。在此，我们一致发现根据 GPCRs 调节存在 HVA Ca 通道的基础持续抑制。去甲肾上腺素（NA）根据电压依赖性调节和膜限制机制增强易化比，而用卡巴胆碱或磷脂酰肌醇 4,5-二磷酸（PIP）观察不到易化变化。此外，根据非电压依赖性调节，卡巴胆碱或跨膜脂质（如 PIP）不改变易化比。总之，胰岛β细胞的 HVA Ca 通道受 GPCRs 持续抑制，提示存在一种天然的刹车机制，防止细胞进行过度的胰岛素分泌。我们的结果支持 GPCRs 持续抑制胰岛β细胞中 HVA Ca 通道的假说。使用具有强去极化预脉冲的电压钳实验方案来揭示 Ca 通道的电压依赖性抑制。这些结果的新颖性增强了 Gβγ 在 Ca 通道调节中的关键作用，突出了动力学减慢和易化比增加。总之，胰岛β细胞的 HVA Ca 通道受 GPCRs 持续抑制，精细调节胰岛素分泌。
