Importance of Non-Selective Cation Channel TRPV4 Interaction with Cytoskeleton and Their Reciprocal Regulations in Cultured Cells Chandan Goswami1,2*, Julia Kuhn1, Paul A. Heppenstall3¤, Tim Hucho1 1 Signal Transduction in Pain and Mental Retardation, Department for Molecular Human Genetics Max Planck Institute for Molecular Genetics, Berlin, Germany, 2 National Institute of Science Education and Research, Bhubaneswar, India, 3 Klinik fu¨ r Anaesthesiologie und Operative Intensivmedizin, Charite´ Universita¨tsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany Abstract Background: TRPV4 and the cellular cytoskeleton have each been reported to influence cellular mechanosensitive processes as well as the development of mechanical hyperalgesia. If and how TRPV4 interacts with the microtubule and actin cytoskeleton at a molecular and functional level is not known. Methodology and Principal Findings: We investigated the interaction of TRPV4 with cytoskeletal components biochemically, cell biologically by observing morphological changes of DRG-neurons and DRG-neuron-derived F-11 cells, as well as functionally with calcium imaging. We find that TRPV4 physically interacts with tubulin, actin and neurofilament proteins as well as the nociceptive molecules PKCe and CamKII. The C-terminus of TRPV4 is sufficient for the direct interaction with tubulin and actin, both with their soluble and their polymeric forms. Actin and tubulin compete for binding. The interaction with TRPV4 stabilizes microtubules even under depolymerizing conditions in vitro. Accordingly, in cellular systems TRPV4 colocalizes with actin and microtubules enriched structures at submembranous regions. Both expression and activation of TRPV4 induces striking morphological changes affecting lamellipodial, filopodial, growth cone, and neurite structures in non-neuronal cells, in DRG-neuron derived F11 cells, and also in IB4-positive DRG neurons. The functional interaction of TRPV4 and the cytoskeleton is mutual as Taxol, a microtubule stabilizer, reduces the Ca2+-influx via TRPV4. Conclusions and Significance: TRPV4 acts as a regulator for both, the microtubule and the actin. In turn, we describe that microtubule dynamics are an important regulator of TRPV4 activity. TRPV4 forms a supra-molecular complex containing cytoskeletal proteins and regulatory kinases. Thereby it can integrate signaling of various intracellular second messengers and signaling cascades, as well as cytoskeletal dynamics. This study points out the existence of cross-talks between non- selective cation channels and cytoskeleton at multiple levels. These cross talks may help us to understand the molecular basis of the Taxol-induced neuropathic pain development commonly observed in cancer patients. Citation: Goswami C, Kuhn J, Heppenstall PA, Hucho T (2010) Importance of Non-Selective Cation Channel TRPV4 Interaction with Cytoskeleton and Their Reciprocal Regulations in Cultured Cells. PLoS ONE 5(7): e11654. doi:10.1371/journal.pone.0011654 Editor: Hiroaki Matsunami, Duke University, United States of America Received December 3, 2009; Accepted June 15, 2010; Published July 19, 2010 Copyright: 2010 Goswami et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding from Max Planck Institute for Molecular Genetics (Berlin, Germany) is gratefully acknowledged. CG is now supported by National Institute of Science Education and Research, India. The authors declare that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: goswami@molgen.mpg.de ¤ Current address: EMBL Monterotondo, Adriano Buzzati-Traverso Campus, Monterotondo, Italy Introduction Transient receptor potential vanilloid sub type 4 (TRPV4) is a member of TRP super family of Ca2+-permeable non-selective cation channels. This polymodal receptor is involved in cellular processes such as mechanosensation, osmosensation and thermo- sensation [1–5]. Some of these sensory functions are well conserved in different species. For example, mammalian TRPV4 can rescue mechanosensitive defects observed in C.elegans OSM-9 mutants [5]. In higher organisms TRPV4 is endogenously expressed in nociceptive dorsal root ganglion (DRG) neurons but also in many non-neuronal tissues and cells such as skin, kidney corneal epithelial cells [6], cerebral microvascular endothelial cells [7], cortical astrocytes [8], tracheal epithelial cells [9], keratinocyte cell lines [10] and in other cells. The widespread distribution of TRPV4 is indicative of its involvement in various physiological functions. Indeed, TRPV4 is of importance in shear stress-induced vasodila- tion [11] as well as in auditory functions [12–13]. Recently TRPV4 gained importance as it has been linked with the development of different pathophysiological conditions such as neuropathic pain, cystic fibrosis, brachyolmia and cancer [14–18]. From several reports, the involvement of cytoskeleton can be correlated with the localization and function of TRPV4. For example, TRPV4 is found in structures like cilia in various tissues and cells [9,19–21] and in lamellipodia, where it regulates the dynamics of cytoskeleton [22–23]. Many cellular functions involving TRPV4 are known to require active participation of the cytoskeleton. For example, TRPV4 activity is central to PLoS ONE | www.plosone.org 1 July 2010 | Volume 5 | Issue 7 | e11654