Am J Physiol Renal Physiol. 2007 Dec;293(6):F1768-76. Epub 2007 Oct 3.
Association of VASP with TRPC4 in PKG-mediated inhibition of the store-operated calcium response in mesangial cells.
Wang, X., Pluznick, J. L., Settles, D. C., Sansom, S. C.,
We tested the hypotheses that the NO-cGMP-PKG pathway mediates inhibition of the store-operated cation channel (SOC) in human glomerular mesangial cells (HMC) and that TRPC4, a molecular component of SOC in HMC, is associated with PKG-phosphorylated vasodilator-stimulated phosphoprotein (VASP). Using fura 2 ratiometry, we measured intracellular Ca(2+) concentration [Ca(2+)](i) to determine whether sodium nitroprusside (SNP), an NO donor, and 8-Br-cGMP affected SOC-TRPC4 via PKG. We found that the SOC response in HMC was attenuated in the presence of 100 microM SNP, an NO donor, or 100 microM 8-Br-cGMP. Addition of DT-3 (2.5 microM), a specific PKG-1alpha inhibitor, reversed the effects of 8-Br-cGMP on the SOC response. Application of 100 microM cAMP did not significantly inhibit the SOC response. RT-PCR and Western blotting revealed PKG-1alpha transcript and protein in HMC. Immunocytochemical analysis localized PKG-1alpha to the cytoplasm and plasma membrane of HMC. Previous studies have shown that PKG-mediated phosphorylation of VASP attenuates cellular Ca(2+) entry, resulting in altered growth and proliferation. Therefore, we used Western blotting and immunocytochemistry to determine whether PKG-phosphorylated VASP associates with TRPC4. Western blot analysis revealed that 8-Br-cGMP enhanced the phosphorylation of VASP at serine 239 (Ser239), a known PKG phosphorylation site, in HMC within 5 min. Coimmunoprecipitation and coimmunostaining showed that P-Ser239-VASP associated with TRPC4. However, VASP that was unphosphorylated at Ser239 was not associated with TRPC4. These results indicate that VASP has a role in the NO/PKG-1alpha-mediated inhibition of the TRPC4-SOC response in HMC.
|Experimental screening||Non-experimental screening||Reference|
|TRP channel construct||Interactor source|
|TRP channel||Interactor||Method||Species||Region||Species||Organ/tissue||Sample type|
|Validation: In vivo validation|
|Assay with endogenous proteins||Assay with overexpressed proteins||Reference|
|Cell or tissue||Cell or tissue||TRP channel construct||Interactor construct|
|TRPC4||VASP||Co-immunoprecipitation||Human glomerular mesangial cell||17913834|
|TRPC4||VASP||Co-immunofluorescence staining||Human glomerular mesangial cell||17913834|