An aquaporin-4/transient receptor potential vanilloid 4 (AQP4/TRPV4) complex is essential for cell-volume control in astrocytes

Abstract

Regulatory volume decrease (RVD) is a key mechanism for volume control that serves to prevent detrimental swelling in response to hypo-osmotic stress. The molecular basis of RVD is not understood. Here we show that a complex containing aquaporin-4 (AQP4) and transient receptor potential vanilloid 4 (TRPV4) is essential for RVD in astrocytes. Astrocytes from AQP4-KO mice and astrocytes treated with TRPV4 siRNA fail to respond to hypotonic stress by increased intracellular Ca 2+ and RVD. Coimmunoprecipitation and immunohistochemistry analyses show that AQP4 and TRPV4 interact and colocalize. Functional analysis of an astrocyte-derived cell line expressing TRPV4 but not AQP4 shows that RVD and intracellular Ca 2+ response can be reconstituted by transfection with AQP4 but not with aquaporin-1. Our data indicate that astrocytes contain a TRPV4/AQP4 complex that constitutes a key element in the brain's volume homeostasis by acting as an osmosensor that couples osmotic stress to downstream signaling cascades.water channel | glia | brain edema A basic property of any cell type is the ability to resist volume changes in the face of hypotonic stress. Thus, most cells are equipped with mechanisms that help bring cell volume back toward baseline level in the wake of an osmotically induced swelling response. This volume recovery, termed "regulatory volume decrease" (RVD) (1), plays a critical role in the brain, whose functional and structural integrity depends on finely tuned volume homeostasis at the cellular as well as the organ level.A wealth of data indicates that astroglial cells are essential for the maintenance of volume homeostasis in brain (2). Being equipped with AQP4 water channels in their foot processes at the interface between brain and liquid spaces, astrocytes are the first cells to be exposed to osmotic changes and the first cells to swell in response to hypo-osmotic stress (3-5). Further, the proximity of the astroglial processes to the subarachnoidal space and vessels (which act as sinks for excess osmolytes) places astroglia in a unique position for mediating regulatory volume changes, on the part of the astrocytic syncytium and the brain as a whole.A full mechanistic understanding of RVD would pave the way for more sophisticated measures to curtail pathological changes in brain water transport and distribution, as seen in brain tumors, stroke, and several other acute conditions that carry a high morbidity and lethality because of the loss of volume homeostasis. Future drugs affecting AQP4-mediated water transport would be expected to alleviate the acute consequences of inadvertent changes in osmotic driving forces. However, because the lipid bilayer of plasma membranes allows water diffusion (albeit to a restricted extent compared with membranes containing aquaporins), the long-term consequences of osmotic challenges can be offset only by manipulating the osmotic gradients per se. In this context, the RVD mechanisms stand out as targets of potential pharmacological interest (1, 6).Previous studies of t...

TRPV4-AQP4 interactions ‘turbocharge’ astroglial sensitivity to small osmotic gradients

“…Its precise mechanism is cell type-dependent but was suggested to require TRPV4 and AQP4 in cortical astrocytes. 6 We found that AQP4-mediated facilitation of Ca 2C influx through TRPV4 channels facilitates M€ uller cell RVD yet this was observed only under conditions that are unlikely to occur in vivo (i.e., »50% reduction in tonicity). In contrast, TRPV4 activation (by agonists or swelling) itself augmented glial swelling through an unknown mechanism that required an increase in [Ca 2C ] i and, possibly, activation of phospholipase A2.…”

TRPV4 channels stimulate Ca 2 + -induced Ca 2+ release in astrocytic endfeet and amplify neurovascular coupling responses

“…However, immunofluorescence and immunogold electron microscopy evidence suggests that TRPV4 channels-plasma membrane cation channels of the transient receptor potential vanilloid (TRPV) family-are localized in astrocytic endfeet; moreover, 4α-phorbol 12,13-didecanoate (4αPDD), a TRPV4-selective chemical activator, increases [Ca 2+ ] i in cultured cortical astrocytes (11). Also in cultured cortical astrocytes, TRPV4 channels form complexes with aquaporin 4 to regulate cell volume responses to hypoosmotic stress (12). Notably, it has been shown that TRPV4 channels in other preparations are activated by epoxyeicosatrienoic acids (EETs) (13)(14)(15), which have been implicated in NVC (16)(17)(18) and reportedly stimulate Ca 2+ influx in rat cortical astrocytes (19,20).…”

Aquaporins in sperm osmoadaptation: an emerging role for volume regulation

“…Interestingly, the scenario of volume regulation by AQP5/TRPV4 interaction has recently been expanded for AQP4. As demonstrated in mouse astrocytes, the TRPV4/AQP4 complex plays an important role in the initiation of RVD similar to that of the TRPV4/AQP5 complex in salivary gland cells [25] . In this regard, it would npg not be hard to imagine that AQP3 might also form molecular complexes with other ion channels to mediate sperm RVD.…”

“…Despite the observation that AQP3 is important for normal sperm RVD during hypotonic exposure [3] and the increasing body of evidence that several members of AQPs (AQP1, 2, 3, 4, and 5) are actively involved in RVD of diverse cell types [22][23][24][25][26] , the molecular mechanisms by which AQPs take part in RVD are hard to explain using the "simple permeability" theory, which considers aquaporins as inert pores that simply increase the osmotic permeability of plasma membranes. Under such a "simple permeability" model, RVD begins with a hypotonic stress-induced water influx, followed by active solute transport that enables osmolyte efflux and provides the driving force for water to exit [27] , and the participation of AQPs in RVD is merely to facilitate the time it takes to reach osmotic equilibrium.…”

“…Such changes in molecular structure may initiate downstream signaling cascades for RVD events. This model would be particularly suitable in cases when AQPs form functional complexes with other mechanosensors (such as TRPV4 [23,25] ) or directly interact with cytoskeletal components such as actin fi laments (AQP2 has been shown to interact directly with actin [33] ). However, to stringently test such a hypothesis, it would be necessary to create a mutant AQP protein that could not form a tetrameric structure while maintaining the water permeability of each AQP monomer.…”