Functional interaction of the cation channel transient receptor potential vanilloid 4 (TRPV4) and actin in volume regulation
Introduction
Many mammalian cell types, like renal tubule cells, bronchial epithelia, keratinocytes, spermatocytes or erythrocytes, encounter in their environment varying osmolarities. The ability of these cells to adapt to changing osmotic conditions is essential for cellular homeostasis, and disequilibrium can lead to dramatic events like apoptosis and necrosis (Liedtke et al., 2003). Therefore, many cell types have evolved specialized mechanisms of volume regulation to counteract damage induced by either cell swelling or shrinking. Under hypotonic conditions, cells increase their volume by uptake of water due to the osmotic gradient. The reduction of the volume to its former value is achieved by a process called regulatory volume decrease (RVD), which enables these cells to sustain in a hypotonic environment (Okada et al., 2001).
Recently, the direct participation of the cation channel transient receptor potential vanilloid 4 (TRPV4) in RVD at cellular level and systemic osmosensing in organism has been revealed (Liedtke and Friedman, 2003; Arniges et al., 2004; Becker et al., 2005; Wu et al., 2007). TRPV4 is a member of the vanilloid subfamily of transient receptor potential (TRP) channels and acts as a Ca2+-permeable, non-selective cation channel. Interestingly, TRPV4 can be activated by diverse stimuli; apart from hypotonicity, these stimuli encompass moderate heat, synthetic ligands like 4α-phorbol 12,13-didecanoate (4αPDD) and also endogenous agonists like arachidonic acid (AA) (Güler et al., 2002; Watanabe et al., 2002a, Watanabe et al., 2002b, Watanabe et al., 2003). The predicted TRPV4 structure contains six membrane-spanning domains with a pore loop, the N-terminal domain with at least three ankyrin repeats as well as the C-terminal domain residue within the cytoplasm (Liedtke et al., 2000; Wissenbach et al., 2000). TRPV4 is expressed in various tissues and is involved among others in epithelial permeability (Reiter et al., 2006), cystic fibrosis (Arniges et al., 2004), nociception (Alessandri-Haber et al., 2005), bladder voiding (Gevaert et al., 2007) and thermosensation (Lee et al., 2005).
Under hypotonic conditions, swelling of HaCaT keratinocytes, human airway epithelia and TRPV4-transfected CHO cells lead to an activation of TRPV4, resulting in an influx of Ca2+ followed by RVD (Arniges et al., 2004; Becker et al., 2005). While the role of Cl− and K+ channels in RVD has been well studied, the nature of the signal that leads to the initiation of RVD is still unclear. An intact cytoskeleton, mainly microfilaments, is required for the swelling-induced Ca2+ entry and RVD in several cell types (Bibby and McCulloch, 1994; Shen et al., 1999; Ebner et al., 2005; Liu et al., 2006; Blase et al., 2009). Furthermore, interaction between TRPV4 and a microtubule-associated protein (MAP7), which possibly binds not only to microtubules but also to actin, has been proposed (Suzuki et al., 2003). Hence, it is tempting to suggest that an interaction between TRPV4 and actin triggers RVD in TRPV4-expressing cells.
To address a putative functional interaction of TRPV4 and actin in osmoregulation, the localization of the two proteins, their interaction and the functional relevance were investigated. Taken together, our data indicate that TRPV4–actin interaction is a prerequisite for the activation of TRPV4 by hypotonicity and TRPV4-mediated RVD.
Section snippets
Cell culture
HaCaT keratinocyte cells (passage 47–72) (Boukamp et al., 1988) were cultivated in keratinocyte SFM medium (Invitrogen, Karlsruhe, Germany), containing 10% fetal calf serum (FCS), 10 mM Hepes (Invitrogen) at 37 °C in 5% CO2 atmosphere. CHO-K1 cells (passage 28–50) were cultivated in F-12 HAMs cell medium (Invitrogen), containing 10% FCS and 10 mM Hepes at 37 °C in 5% CO2 atmosphere.
Constructs and transfection
Construction of TRPV4-GFP starting from HaCaT keratinocyte mRNA had been described before (Becker et al., 2005). For
Colocalization of TRPV4 and F-actin is altered by latrunculin A treatment
The data of Blase et al. (2009), stress the importance of actin in RVD of HaCaT keratinocytes. HaCaT cells express the cation channel TRPV4 endogenously, and blocking of TRPV4 by inhibitors Ruthenium Red or Gd3+ abolished Ca2+ influx as well as RVD after hypotonic shock, identifying TRPV4 as a necessary component in volume regulation (Becker et al., 2005). To investigate a putative interaction of TRPV4 and actin, we first analyzed the localization of both proteins in HaCaT keratinocytes and CHO
Discussion
The mechanism underlying the activation of the cation channel TRPV4 during hypotonic stress and the functional relationship between TRPV4 and F-actin were investigated. CHO cells that do not possess endogenous TRPV4 (Becker et al., 2005; Suzuki et al., 2003) were transfected with TRPV4 and then treated with latrunculin A, which inhibits actin polymerization (Spector et al., 1983; Coue et al., 1987). The subsequent loss of F-actin after latrunculin A treatment was confirmed by LSM. In
Acknowledgements
We gratefully acknowledge Prof. Dr. Herbert Zimmermann (Johann Wolfgang Goethe University, Frankfurt/Main, Germany) for the possibility to perform electroporation in his Laboratory. Furthermore, many thanks go to Marga Müller for technical support in operating the ratio imager. For critical reading of the manuscript, we are indebted to Chris Blase. This work was supported by the SFB 628 “Functional Membrane Proteomics” (P9) and the Center for Membrane Proteomics (CPM) Frankfurt/Main Germany.
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