Functional interaction of the cation channel transient receptor potential vanilloid 4 (TRPV4) and actin in volume regulation

Abstract

Many vertebrate cells react to hypotonic conditions with swelling, followed by an active downregulation of the cell volume; a progress called regulatory volume decrease (RVD). While the actual process of volume decrease by loss of osmotically active molecules like K⁺ and Cl⁻, followed by water efflux has been extensively investigated, the signal for activation of RVD still remains obscure. Studies with different cell lines demonstrated a participation of the cation channel transient receptor potential vanilloid 4 (TRPV4) as well as the actin cytoskeleton in volume regulation. Therefore, we analyzed putative links between TRPV4 and F-actin in RVD in HaCaT keratinocytes and CHO cells. Laser scanning microscopy studies revealed a distinct colocalization of TRPV4 and actin in highly dynamic membrane structures, such as microvilli, filopodia and lamellipodia edges. After treatment of cells with the actin-destabilizing reagent latrunculin A, TRPV4 and F-actin no longer colocalized within the membrane. In accordance with these data, close interaction between TRPV4 and F-actin was revealed by FRAP and FRET studies. For functional analysis, CHO cells that endogenously do not express TRPV4, were transfected with recombinant TRPV4, which rendered them RVD-competent. Treatment with latrunculin A abolished both, RVD and the accompanying rise of [Ca²⁺]_i after hypotonic stress in TRPV4-transfected CHO cells. Taken together, our data demonstrate a functional interaction between TRPV4 and F-actin in sensing hypotonicity and the onset of RVD.

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.