Muscarinic receptor stimulation induces TASK1 channel endocytosis through a PKC-Pyk2-Src pathway in PC12 cells

Muscarinic receptor stimulation or protein kinase C (PKC) activation in rat adrenal medullary and PC12 cells rapidly induces tyrosine phosphorylation of TWIK-related-acid-sensitive K⁺ 1 (TASK1) channels with the subsequent clathrin-dependent endocytosis. Our previous study suggested that the muscarinic signal is transmitted to the non-receptor tyrosine kinase Src through PKC and Pyk2. Although PKC activation is known to stimulate Pyk2 in certain types of cells, its molecular mechanism remains unclear. In this study, proximity ligation assay (PLA) and other molecular biological approaches were used to elucidate the details of this muscarinic signaling in PC12 cells. When green fluorescent protein (GFP)-TASK1 was expressed, the majority of GFP-TASK1 was located at the cell periphery. However, the simultaneous expression of GFP-TASK1 and PKCα, but not PKCδ, led to GFP-TASK1 internalization. Muscarinic receptor stimulation resulted in transient co-localization of Pyk2 and Src at the cell periphery, and expression of kinase dead (KD) Pyk2 and Src, but not Pyk2 and KD Src, resulted in GFP-TASK1 internalization. PLA analysis revealed that in response to muscarine, PKCαactivates Pyk2 through phosphorylating its serine residues. These results indicate that muscarinic receptor stimulation induces TASK1 channel endocytosis sequentially through PKCα, Pyk2, and Src, and PKCα activates Pyk2 through phosphorylation.