de la Cruz Lizbeth, Puente Erika I, Reyes-Vaca Arturo, Arenas Isabel, Garduño Julieta, Bravo-Martínez Jorge, Garcia David E
Department of Physiology, School of Medicine, Universidad Nacional Autónoma de México (UNAM), Mexico, México.
Department of Physiology, School of Medicine, Universidad Nacional Autónoma de México (UNAM), Mexico, México
Am J Physiol Cell Physiol. 2016 Oct 1;311(4):C630-C640. doi: 10.1152/ajpcell.00111.2016. Epub 2016 Aug 3.
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.
磷脂酰肌醇-4,5-二磷酸（PIP）是一种膜磷酸肌醇，可调节许多离子通道的活性。主要通过电压门控钙（Ca）通道的钙内流促进胰腺β细胞中的胰岛素分泌。然而，Ca通道是否像某些非胰岛素分泌细胞那样受PIP调节尚不清楚。本研究的目的是通过激活氧化震颤素甲碘化物（Oxo-M）诱导的毒蕈碱途径，研究胰腺β细胞中Ca通道是否受PIP耗竭的调节。通过膜片钳技术记录Ca通道电流。在没有动力学变化的情况下，毒蕈碱受体1（MR）的激活降低了Ca电流幅度。Oxo-M诱导的抑制表现出电压非依赖性调节的特征，并且不涉及PKC激活。高浓度的Ca螯合剂可减少一小部分Oxo-M诱导的Ca抑制，而≥50%的这种抑制可通过二辛酰磷脂酰肌醇（diC8-PIP）透析来预防。通过用PH-PLCδ1转染INS-1细胞来检查PIP在质膜中的定位，这揭示了PIP水解与Ca通道抑制之间密切的时间关联。此外，电压敏感磷酸酶引起的PIP耗竭以类似于MR激活后观察到的方式降低了Ca电流。这些结果表明，MR途径的激活通过胰腺β细胞和INS-1细胞中依赖Ca的机制，通过PIP耗竭抑制Ca通道，从而支持膜磷脂通过与离子通道相互作用来调节离子通道活性的假说。
