PIP₂ in pancreatic β-cells regulates voltage-gated calcium channels by a voltage-independent pathway

Abstract

Phosphatidylinositol-4,5-bisphosphate (PIP) is a membrane phosphoinositide that regulates the activity of many ion channels. Influx of calcium primarily through voltage-gated calcium (Ca) channels promotes insulin secretion in pancreatic β-cells. However, whether Ca channels are regulated by PIP, as is the case for some non-insulin-secreting cells, is unknown. The purpose of this study was to investigate whether Ca channels are regulated by PIP depletion in pancreatic β-cells through activation of a muscarinic pathway induced by oxotremorine methiodide (Oxo-M). Ca channel currents were recorded by the patch-clamp technique. The Ca current amplitude was reduced by activation of the muscarinic receptor 1 (MR) in the absence of kinetic changes. The Oxo-M-induced inhibition exhibited the hallmarks of voltage-independent regulation and did not involve PKC activation. A small fraction of the Oxo-M-induced Ca inhibition was diminished by a high concentration of Ca chelator, whereas ≥50% of this inhibition was prevented by diC8-PIP dialysis. Localization of PIP in the plasma membrane was examined by transfecting INS-1 cells with PH-PLCδ1, which revealed a close temporal association between PIP hydrolysis and Ca channel inhibition. Furthermore, the depletion of PIP by a voltage-sensitive phosphatase reduced Ca currents in a way similar to that observed following MR activation. These results indicate that activation of the MR pathway inhibits the Ca channel via PIP depletion by a Ca-dependent mechanism in pancreatic β- and INS-1 cells and thereby support the hypothesis that membrane phospholipids regulate ion channel activity by interacting with ion channels.

Potential Therapeutic Applications of Synthetic Conotoxin s-cal14.2b, Derived from Californiconus californicus, for Treating Type 2 Diabetes

“…Thus, isolated pancreatic β-cells were efficiently recorded in primary culture conditions unveiling a significant enhancement of calcium current under s-cal14.2b. Our results reproduce and confirm the primary target on Ca V 1.2/1.3 L-type DHP-sensitive Ca 2+ channels [48]. There was also increased Ca 2+ conductance and Ca 2+ influx through these channels.…”

“…Immunocytochemistry and electrophysiology revealed that s-cal14.2b possibly stimulates insulin secretion by pancreatic β-cells. The dosage-dependent, insulin-like effect of s-cal14.2b in this study suggests that this synthetic peptide could be a new drug candidate for treating T2DM, particularly in the early stages of this disease when pancreatic damage is limited [47,48]. The synthetic cal14.2b peptide is the first conopeptide (less than 20 amino acids) discovered that can modulate the secretion of insulin.…”

Voltage-Sensing Phosphatases: Biophysics, Physiology, and Molecular Engineering

“…All these studies were done with heterologously expressed Ca V in mammalian cells. However, native L-type Ca V channels in INS-1 cells, a line of rat pancreatic ␤-cells, were also found to be sensitive to PtdIns(4,5)P 2 depletion by Dr-VSP, which was heterologously expressed (32). PtdIns(4,5)P 2 -dependent activities of high-voltage-activated Ca v s are of physiological significance, since modulation of neuronal Ca v s through GPCR activation is known to tune many physiological responses (216).…”

Cannabinoid receptors are differentially regulated in the pancreatic islets during the early development of metabolic syndrome

“…For the pancreatic islet obtention and primary cell cultures, we employed a previously described technique. 48 , 49 Briefly, the pancreatic duct was localized and cannulated by insufflation with cold Hanks’ Balanced Salts Solution (HBSS) supplemented with bovine serum albumin (Microlab), NaHCO 3 (4 mM), HEPES (15 mM), and 1% 100X Antibiotic-Antimycotic (Life Technologies-Thermo Fisher Scientific). The pancreas was removed and transferred to a flask containing HBSS supplemented with collagenase P (0.3 g/L) for incubation at 37°C for 12 min with lateral agitation.…”