The Role of Interacting Proteins in TRPV4 Channelopathies

Abstract

Members of the superfamily of transient receptor potential (TRP) channels play a vital role in various physiological systems and are important in the development of numerous channelopathies. Mutations in TRPV4, a polymodal Ca2+-permeable channel, cause a remarkably wide phenotypic spectrum of diseases, classified in skeletal dysplasias (SD), arthropathies and distal motor neuropathies. The majority of the disease causing mutations is located in the N-terminal ankyrin repeat domain of the TRPV4, suggesting an important role of cell-type specific interacting proteins in the development of the diverse diseases. To identify binding partners of the human TRPV4 protein as potential determinants of disease, we performed a MAmmalian Protein-Protein Interaction Trap (MAPPIT) screening using wild type or mutant N-terminus of hTRPV4 as prey. Among the newly identified interacting partners we focused on the zinc-finger domain containing protein ZC4H2. Mutations in the gene encoding for ZC4H2 cause Arthrogryposis Multiplex Congenita (AMC) and Wieacker-wolff syndrome, characterized by congenital joint contractions and developmental defects of the musculoskeletal and nervous system, reminiscent of features observed in TRPV4 channelopathies. Quantitative real time PCR experiments showed expression of ZC4H2 in relevant TRPV4-expressing cell types including primary osteoclasts, osteoblasts and spinal motor neurons. Fura 2-mediated Ca2+-imaging experiments indicate that ZC4H2 increases basal and agonist-evoked activity of hTRPV4 in HEK293-T cells. We further use patch-clamp recordings, subcellular imaging and biochemical analysis to pinpoint the mode of interaction between TRPV4 and ZC4H2, which may shed new light on the etiology of TRPV4-dependent channelopathies.