Department of Cell and Systems Physiology University of Occupational and Environmental Health School of Medicine, Kitakyushu 807-8555, Japan.
Department of Cell and Systems Physiology University of Occupational and Environmental Health School of Medicine, Kitakyushu 807-8555, Japan
J Cell Sci. 2019 May 31;132(11):jcs227389. doi: 10.1242/jcs.227389.
Muscarinic receptor stimulation results in activation of nonselective cation (NSC) channels in guinea pig adrenal medullary (AM) cells. The biophysical and pharmacological properties of the NSC channel suggest the involvement of heteromeric channels of TRPC1 with TRPC4 or TRPC5. This possibility was explored in PC12 cells and guinea pig AM cells. Proximity ligation assay (PLA) revealed that when exogenously expressed in PC12 cells, TRPC1 forms a heteromeric channel with TRPC4, but not with TRPC5, in a STIM1-dependent manner. The heteromeric TRPC1-TRPC4 channel was also observed in AM cells and trafficked to the cell periphery in response to muscarine stimulation. To explore whether heteromeric channels are inserted into the cell membrane, tags were attached to the extracellular domains of TRPC1 and TRPC4. PLA products developed between the tags in cells stimulated by muscarine, but not in resting cells, indicating that muscarinic stimulation results in the membrane insertion of channels. This membrane insertion required expression of full-length STIM1. We conclude that muscarinic receptor stimulation results in the insertion of heteromeric TRPC1-TRPC4 channels into the cell membrane in PC12 cells and guinea pig AM cells.
毒蕈碱受体刺激导致豚鼠肾上腺髓质 (AM) 细胞中非选择性阳离子 (NSC) 通道的激活。NSC 通道的生物物理和药理学特性表明涉及 TRPC1 与 TRPC4 或 TRPC5 的异源通道。在 PC12 细胞和豚鼠 AM 细胞中探索了这种可能性。接近连接分析 (PLA) 表明，当在外源性表达于 PC12 细胞时，TRPC1 以 STIM1 依赖性方式与 TRPC4 形成异源通道，但与 TRPC5 不形成。在 AM 细胞中也观察到异源 TRPC1-TRPC4 通道，并在受到毒蕈碱刺激时向细胞外周易位。为了探索异源通道是否插入细胞膜，在 TRPC1 和 TRPC4 的细胞外结构域上附加标签。在受毒蕈碱刺激的细胞中，标签之间形成了 PLA 产物，但在静止细胞中没有，表明毒蕈碱刺激导致通道插入细胞膜。这种膜插入需要全长 STIM1 的表达。我们得出结论，毒蕈碱受体刺激导致 PC12 细胞和豚鼠 AM 细胞中异源 TRPC1-TRPC4 通道插入细胞膜。
