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EDITORIAL FOCUS

Association of VASP with TRPC4 in PKG-mediated inhibition of the store-operated calcium response in mesangial cells

¹Department of Nephrology, No. 6 Hospital, Shanghai Jiaotong University, Shanghai, China; ²Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut; and ³Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska

Submitted 3 August 2007 ; accepted in final form 2 October 2007

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 Ca²⁺ concentration [Ca²⁺]_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 µM SNP, an NO donor, or 100 µM 8-Br-cGMP. Addition of DT-3 (2.5 µM), a specific PKG-1 inhibitor, reversed the effects of 8-Br-cGMP on the SOC response. Application of 100 µM cAMP did not significantly inhibit the SOC response. RT-PCR and Western blotting revealed PKG-1 transcript and protein in HMC. Immunocytochemical analysis localized PKG-1 to the cytoplasm and plasma membrane of HMC. Previous studies have shown that PKG-mediated phosphorylation of VASP attenuates cellular Ca²⁺ 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-1-mediated inhibition of the TRPC4-SOC response in HMC.

cGMP; SOC; nitric oxide; SNP

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