title: Translocation of PKG1α acts on TRPV4-C1 heteromeric channels to inhibit endothelial Ca(2+) entry.
abstract: OBJECTIVE
  TRPV4-C1 heteromeric channels contribute to store-operated Ca(2+) entry in vascular endothelial cells. However, the negative regulation of these channels is not fully understood. This study was conducted to investigate the inhibitory effect of PKG1α on TRPV4-C1 heteromeric channels.
  
  
  METHODS
  Immuno-fluorescence resonance energy transfer (FRET) was used to explore the spatial proximity of PKG1α and TRPC1. Phosphorylation of endogenous TRPC1 was tested by phosphorylation assay. [Ca(2+)]i transients and cation current in MAECs were assessed with Fura-2 fluorescence and whole-cell recording, respectively. In addition, rat mesenteric arteries segments were prepared, and vascular relaxation was examined with wire myography.
  
  
  RESULTS
  In immuno-FRET experiments, after exposure of these cells to 8-Br-cGMP, more PKG1α was observed in the plasma membrane, and PKG1α and TRPC1 were observed to be in closer proximity. TAT-TRPC1(S172) and TAT-TRPC1(T313) peptide fragments, which contain the PKG targeted residues Ser172 and Thr313, respectively, were introduced into isolated endothelial cells to abrogate the translocation of PKG1α. Furthermore, a phosphorylation assay demonstrated that PKG directly phosphorylates TRPC1 at Ser172 and Thr313 in endothelial cells. In addition, PKG activator 8-Br-cGMP markedly reduced the magnitude of the 4αPDD-induced and 11,12-EET-induced [Ca(2+)]i transients, the cation current and vascular relaxation.
  
  
  CONCLUSIONS
  This study uncovers a novel mechanism by which PKG negatively regulates endothelial heteromeric TRPV4-C1 channels through increasing the spatial proximity of TRPV4-C1 to PKG1α via translocation and through phosphorylating Ser172 and Thr313 of TRPC1.
authors.affiliation: School of Pharmaceutical Sciences, Jiangnan University, Wuxi 214122, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Zhang
authors.affiliation: School of Pharmaceutical Sciences, Jiangnan University, Wuxi 214122, China.
authors.forename: Ai-Qin
authors.initials: AQ
authors.lastname: Mao
authors.affiliation: School of Pharmaceutical Sciences, Jiangnan University, Wuxi 214122, China.
authors.forename: Chun-Yuan
authors.initials: CY
authors.lastname: Sun
authors.affiliation: School of Pharmaceutical Sciences, Jiangnan University, Wuxi 214122, China.
authors.forename: Xiao-Dong
authors.initials: XD
authors.lastname: Zhang
authors.affiliation: School of Pharmaceutical Sciences, Jiangnan University, Wuxi 214122, China.
authors.forename: Qiong-Xi
authors.initials: QX
authors.lastname: Pan
authors.affiliation: School of Pharmaceutical Sciences, Jiangnan University, Wuxi 214122, China.
authors.forename: Dan-Tong
authors.initials: DT
authors.lastname: Yang
authors.affiliation: School of Pharmaceutical Sciences, Jiangnan University, Wuxi 214122, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Jin
authors.affiliation: School of Pharmaceutical Sciences, Jiangnan University, Wuxi 214122, China.
authors.forename: Chun-Lei
authors.initials: CL
authors.lastname: Tang
authors.affiliation: Wuxi People's Hospital Heart Centre, Jiangnan University, Wuxi 214122, China.
authors.forename: Zhen-Yu
authors.initials: ZY
authors.lastname: Yang
authors.affiliation: School of Biomedical Sciences, the Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China.
authors.forename: Xiao-Qiang
authors.initials: XQ
authors.lastname: Yao
authors.affiliation: Wuxi Neuroscience Center of Jiangnan University & Department of Neurosurgery, Wuxi Second Hospital Affiliated Nanjiang Medical University & Wuxi Second People's Hospital of Jiangsu Province, Wuxi 214002, China.
authors.forename: Xiao-Jie
authors.initials: XJ
authors.lastname: Lu
authors.affiliation: School of Pharmaceutical Sciences, Jiangnan University, Wuxi 214122, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Ma
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D018929:Cell Culture Techniques; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D062965:Cyclic GMP-Dependent Protein Kinase Type I / Q000378:metabolism*; D042783:Endothelial Cells / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D066298:In Vitro Techniques; D008564:Membrane Potentials / Q000187:drug effects; D008638:Mesenteric Arteries / Q000378:metabolism*; D018345:Mice, Knockout; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000494:pharmacology; D010766:Phosphorylation; D021381:Protein Transport; D017207:Rats, Sprague-Dawley; D050052:TRPC Cation Channels / Q000235:genetics / Q000378:metabolism*; D014664:Vasodilation
chemical_list: D015220:Calcium Channels; D010446:Peptide Fragments; D050052:TRPC Cation Channels; C096351:transient receptor potential cation channel, subfamily C, member 1; C016276:8-bromocyclic GMP; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D006152:Cyclic GMP; D002118:Calcium