THE INTERACTION OF VASODILATOR‐STIMULATED PHOSPHOPROTEIN (VASP) WITH IP3R AND TRPC CHANNELS IN MADIN‐DARBY CANINE KIDNEY (MDCK) CELLS

Abstract

Several dedifferentiating disorders of tissues are the result of aberrantly elevated [Ca2+]i leading to increased transcription. Although the cGMP‐activated protein kinase (PKG) pathway attenuates increases in [Ca2+]i and dedifferentiation, the mechanism is not understood. We recently found in mesangial cells that when VASP, an actin associated focal adhesion protein, is phosphorylated at Ser239 by PKG, it associates with TRPC4 channels. This was interesting because Homer, a scaffold protein, interacts with IP3R and TRP channels at the Ena/VASP Homology I (EVHI) domain. Because both VASP and Homer contain the EVH1 domains, we hypothesized that VASP forms a similar interaction with TRPC and IP3R in MDCK cells, which are model epithelia for studying diseases such as polycystic kidney disease (PKD). We identified with RT‐PCR and immunocytochemistry the presence of TRPC1 and TRPC4 in MDCK cells. We also demonstrated interaction between VASP and TRPC1 with immunocytochemistry and an interaction between VASP with IP3R using immunocytochemistry and Co‐IP. Moreover, upon PKG phosphorylation of VASP at Ser239, VASP no longer interacted with TRPC1, but instead interacted with TRPC4 on the cell membrane. These results suggest that PKG controls Ca2+ entry in MDCK cells by phosphorylating VASP at Ser239. This may be part of the mechanism by which PKG attenuates the elevated [Ca2+]i in some dedifferentiating diseases.