Flow-induced Ca entry in M1 CCD cells is negatively regulated by cGMP and protein kinase G

• 作 者: Juan Du Wei-Yan Wong Xiaoqiang Yao
• 机 构: School of Biomedical Sciences and Li Ka Shing Institute of Health Sciences,Chinese University of Hon
• 出 处: The 3rd Ion Channel Conference: Ion channels-Structure, Func
• 发表日期: 2011年3期

　　Renal cortical collecting duct (CCD) has important control on renal Na+ absorption and K+ secretion.High tubular flow stimulates CCD epithelial cells to induce Ca2+ rises, which then initiate diverse physiological responses including natriuresis and diuresis.In the present study, we found that cGMP and protein kinase G (PKG) inhibited the flow-induced Ca rise in M1 CCD cells.Co-immunoprecipitation, double immunolabeling and functional study demonstrated that heteromeric TRPV4-P2 channels mediated the Ca2+ responses to flow in M1-CCD cells and TRPV4-P2 co-expressing HEK cells.The mechanism of cGMP and PKG inhibition on TRPV4-P2 was explored.Point mutation at two putative PKG phosphorylation sites (TRPP2T719A and TRPP2S827A) on TRPP2 abolished the cGMP inhibition on heteromeric TRPV4-P2 channels in heterologously expressed HEK cells.In M1 CCD cells, application of fusion peptides TAT-TRPP2s827 + TAT-TRPP2T719, which compete with endogenous PKG phosphorylation sites on TRPP2, abolished the inhibitory effect of cGMP on flow-induced Ca2+ entry.In summary, the present study demonstrates that flow-induced Ca2+ entry in M1-CCD cells is negatively regulated by cGMP and PKG via PKG-mediated action on heteromeric TRPV4-P2 channels.Furthermore, point mutations at two consensus PKG phosphorylation sites on TRPP2 markedly reduced the inhibitory effect of cGMP.In M1 CCD cells, flow-induced calcium influx was also inhibited by 8-Br-cGMP, the effect of which was reversed by KT5823.The cGMP inhibition was absent in M1 CCD cells pretreated with fusion peptides TAT-TRPP2s827 + TAT-TRPP2T719, which compete with endogenous PKG phosphorylation sites on TRPP2.In conclusion, we demonstrated that TRPV4 physically associated with TRPP2 in M1 CCD cells and HEK293 cells over-expression system.This association prolongs the flow-induced Ca2+ influx and enables this influx to be negatively regulated by PKG in M1 CCD cells and HEK293 cells over-expressing TRPV4 and-P2.