G-protein tonic inhibition of calcium channels in pancreatic β-cells

Abstract

Voltage-gated calcium channels (Ca_V) conduct Ca²⁺ influx promoting neurotransmitters and hormone release. Ca_V 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) Ca²⁺ channels are present in these cells. Thus, autonomic system seems to exert a tonic inhibition on HVA Ca²⁺ channels throughout GPCRs, constitutively preventing hormone secretion. Therefore, this work aimed to investigate noradrenergic and cholinergic inhibition of HVA Ca²⁺ 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 Ca²⁺ 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 (PIP₂). Furthermore, carbachol or intramembrane lipids, such as PIP₂, do not change facilitation ratio according to a voltage-independent regulation. Together, HVA Ca²⁺ channels of pancreatic β-cells are constitutively inhibited by GPCRs, suggesting a natural brake preventing cells from exhaustive insulin secretion.NEW & NOTEWORTHY Our results support the hypothesis that GPCRs tonically inhibit HVA Ca²⁺ channels in pancreatic β-cells. A voltage-clamp protocol with a strong depolarizing prepulse was used to unmask voltage-dependent inhibition of Ca²⁺ channels. The novelty of these results strengthens the critical role of Gβγ's in Ca²⁺ channel regulation, highlighting kinetic slowing and increased facilitation ratio. Together, HVA Ca²⁺ channels of pancreatic β-cells are constitutively inhibited by GPCRs underlying fine-tuning modulation of insulin secretion.

Keywords: G-proteins; calcium currents; intramembrane lipids; pancreatic β-cell.