Importance of Non-Selective Cation Channel TRPV4 Interaction with Cytoskeleton and Their Reciprocal Regulations in Cultured Cells

Abstract

BackgroundTRPV4 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 FindingsWe 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 PKCε 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 SignificanceTRPV4 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.

TRPV4-mediated channelopathies

“…17 Indeed, MAP7, a microtubule binding protein and soluble tubulin as well as polymerized microtubule interact with TRPV4, especially at the C-terminal region of TRPV4. 18,19 In a similar manner, close proximity of TRPV4 and actin filaments has been demonstrated by using fluorescence resonance energy transfer (FRET). 20 As TRPV4 is involved in the Charcot-Marie-Tooth disease type 2 (CMT2), it suggests that TRPV4 is genetically linked with other genes that are also involved in the same disease and thus TRPV4 share a special genotype-phenotype correlation with these gene products.…”

“…21 Indeed, our recent study confirmed that Neurofilament protein physically interacts with TRPV4. 19 In the same context, Kif1B, which is also involved in the CMT2 disease and/or peripheral neuropathic pain development, may A better and further understanding of TRPV4 structure, interaction and function can also be derived from studies that have generated and characterized several artificial TRPV4 mutations ( Table 2). All these mutations mostly alter single amino acids or cause deletion of specific regions.…”

TRPV4-mediated mechanotransduction regulates the metabolic response of chondrocytes to dynamic loading

“…Although chondrocyte [Ca 2+ ] i signaling exhibits a high sensitivity to variations in osmolarity or volume (41)(42)(43)(44), there is little or no sensitivity to physiologic levels of other physical stimuli, such as direct membrane stretch, suggesting that membrane connections to the pericellular matrix and/or intracellular cytoskeleton are important for the generation and transduction of [Ca 2+ ] i transients. TRPV4 has known physical interactions with both transmembrane proteins (integrins) (45,46) and cytoskeletal elements (microtubules, actin, tubulin) (47,48), and can transduce a diverse set of environmental signals (49). However, the complete Fig.…”

Right time - right location - right move: TRPs find motors for common functions

“…12 Similarly, TRPV4 is also located at the filopodial and lamelipodial structures; 13 and in a same manner, endogenous TRPV4 is also present in the cholangiocyte cilia.…”