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. 2011 Feb 8;108(6):2563-8.
doi: 10.1073/pnas.1012867108. Epub 2011 Jan 24.

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

Valentina Benfenati  1 , Marco Caprini, Melania Dovizio, Maria N Mylonakou, Stefano Ferroni, Ole P Ottersen, Mahmood Amiry-Moghaddam
Affiliations

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

Valentina Benfenati et al. Proc Natl Acad Sci U S A. .

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
AQP4 is essential for RVD and for the hypotonicity-induced [Ca2+]i response in primary astroglial cells in primary astroglial cells. (A) Calcein-quenching method for measurement of osmotically induced volume changes in primary astroglial cells from WT (closed symbols, n = 16) and AQP4-KO mice (open symbols, n = 30). The cells were exposed to a hypotonic medium (ΔOsm = 60 mOsm). (B) The initial parts of the curves in A, shown at higher time resolution. Zero time point on the x axis corresponds to 90 s in Fig. 1A. Quantitative analyses of data in A and B are shown in Fig. S1. (C–E) Typical [Ca2+]i dynamics recorded in fura-2–loaded primary astroglial cells from WT mouse (C), AQP4-KO mouse (D), and rat (E). Each line represents the response of an individual cell. (F) Quantitative analyses of the hypotonicity-induced [Ca2+]i response in primary astrocytes. Rat astrocytes (n = 15) are shown by the dark gray bar, WT mouse astrocytes (n = 17) by the light gray bar, and AQP4-KO astrocytes (n = 14) by the white bar. P = 0.0016 for the comparison between AQP4 WT and AQP4-KO astrocytes, independent t-test; P < 0.001 for experiments in presence and absence of [Ca2+]o, paired t test. The 4αPDD response did not differ significantly between WT and AQP4-KO cells (P = 0.9, independent t-test). **P ≤ 0.01; ***P ≤ 0.001.
Fig. 2.
Fig. 2.
TRPV4 knockdown by siRNA significantly reduces the hypotonicity-induced [Ca2+]i response and RVD in rat astrocytes. (A) The TRPV4 antibody recognizes three major bands of ∼90, 100, and 110 kDa. The decrease in TRPV4 protein level, based on densitometric analysis in three different experiments, was most pronounced for siRNA 9 (89% reduction for the 120-kDa band, 61% for the 100-kDa band, and 69% for 90-kDa band) followed by siRNA 7 (89%, 59%, 51%) and siRNA 11 (49%, 54%, 72%). The density of bands of samples treated with control siRNA was set at 100%. The level of actin is not affected by siRNA treatment. (B) Typical [Ca2+]i dynamics recorded in fura-2–loaded primary astroglial cells treated with control (CT) siRNA. (C) Knockdown of TRPV4 by siRNA 9 abolishes the [Ca2+]i response. (D) Quantitative analyses of hypotonicity-induced [Ca2+]i signal in primary astrocytes. Astrocytes treated with CT siRNA (n = 20) are shown by the gray bar and astrocytes treated with TRPV4-specific siRNA (n = 22) by the white bar. P < 0.001 for the comparison between control group and TRPV4 knockdown astrocytes, independent t-test; P < 0.001 for experiment in presence and absence of [Ca2+]o, paired t-test. ***P ≤ 0.001. (E) Calcein-quenching method for measurement of osmotically induced volume changes in primary astroglial cells treated with control siRNA (closed symbols) and TRPV4-specific siRNA 9 (open symbols). The cells were exposed to a hypotonic medium (ΔOsm = 60 mOsm).
Fig. 3.
Fig. 3.
TRPV4 and AQP4 colocalize in astrocytes of adult rat brain. (A–C) Single-plane confocal immunofluorescence images of large-caliber blood vessel of rat occipital cortex. Triple labeling with goat anti-AQP4 (green, A), rabbit anti-TRPV4 (red, B), and mouse anti-GFAP (blue, C) reveals astrocyte processes that are immunopositive for TRPV4 and AQP4. Triple-labeled processes appear in white in D. (Scale bar: 50 μm.) (E–G) Single-plane confocal immunofluorescence images of sagittal section of rat occipital cortex. Triple labeling with goat anti-AQP4 (green, E), rabbit anti-TRPV4 (red, F), and mouse anti-GFAP (blue, G). Triple-labeled processes appear in white in H (merged image, arrow). The triple-labeled processes (white arrows) face the pial surface and surround large-caliber blood vessels. (Scale bars: 50 μm.)
Fig. 4.
Fig. 4.
TRPV4 and AQP4 co-IP in rat and mouse brain extracts and in astrocyte-derived cell line DI TNC1. (A) Immunoblots of rat brain proteins precipitated by AQP4 antibody show a TRPV4-immunopositive band at the appropriate molecular weight. Omission of AQP4 antibody (Pre-C) abolishes labeling Controls verify the presence of TRPV4-immunopositive bands in brain extract (Pre-IP). (B) Same experimental design as in A, using TRPV4 antibodies for IP and AQP4 antibodies for immunoblotting. The TRPV4 antibody precipitated an AQP4-immunopositive protein at ∼30 kDa. (C) Immunoblots of rat brain proteins precipitated by anti-AQP4 or anti-TRPV4 antibody do not show any band positive for AQP1. Control verifies the presence of AQP1 in total brain extract (Pre-IP). (D) AQP4-immunopositive band is absent in immunoblots of proteins precipitated by anti-TRPV4 or anti-AQP4 from brains of AQP4-KO mice. (E) The same experimental design as in D, using TRPV4 antibody for immunoblotting. Knockout of AQP4 abolishes the ability of anti-AQP4 to precipitate TRPV4. (F and G) TRPV4 can be immunoprecipitated by AQP4 antibodies (F) and vice versa (G) in primary astroglial cultures. (H) IP experiments on primary astrocytes treated with biotin to label membrane proteins. The AQP4 antibody pulls down a biotinylated protein (BIOT), identified by streptavidin-HRP (STRP-HRP, Left). This protein comigrates with the band immunopositive for TRPV4 (Right). NB, nonbiotinylated sample. (I–L) An AQP4/TRPV4 complex can be reconstituted in the astrocyte-derived cell line DI TNC1. (I) Immunoblots showing that DI TNC1 cells express GFAP (Left) and TRPV4 (Right). Primary astrocytes (ASTRO) are used for reference. (J) AQP4 is not expressed in nontransfected (NT) DI TNC1 cells. Transfected M1 and M23 AQP4 isoforms are precipitated by TRPV4 antibodies. Pre-IP, total cell lysate. (K) Endogenous TRPV4 is precipitated by AQP4 antibodies in cells transfected with M1 or M23 AQP4 isoforms. (L) Overexpression of AQP1 induced an increased signal at the appropriate molecular weight only in AQP1-transfected DI TNC1 cells. Note that AQP1 is not precipitated by endogenous TRPV4.
Fig. 5.
Fig. 5.
Hypotonicity-induced Ca2+ response is reconstituted in AQP4 (M1 or M23)-transfected but not in AQP1-transfected DI TNC1 cells. (A) Traces representative of typical variations in [Ca2+]i observed in fura-2–loaded DI TNC1 nontransfected (NT) cells exposed to hypotonic solution (black bar; ΔOsm = 60 mOsm) in the presence or absence of 5 mM [Ca2+]o (hatched bar) and stimulated with 5 μM 4αPDD (gray line). (B–D) Representative traces depicting [Ca2+]i DI TNC1 cells transfected with EGFP (B), EGFP + M1 (C), or EGFP + M23 (D). The experimental design is as in A. (E) Typical Ca2+ dynamics of three Fluo-4-AM–loaded DI TNC1 cells transfected with AQP1 showing no [Ca2+]i signal upon hypotonicity. Thrombine (10 μM; THB; gray line) was used as positive control. (Inset) Quantitative analyses of maximal variation in fluorescence ratio (ΔF/F) recorded in AQP1 (gray bar; n = 16) and AQP4-M23 (n = 6) transfected cells upon cell exposure to hypotonic solution. (F) Quantitative analysis of data represented in A–D. Mean of variation in fluorescence ratio (ΔF340/F380) recorded upon cell exposure to hypotonic solution in presence (Left) or in absence (Center) of [Ca2+]o (NT, nontransfected, light gray bar, n = 36; EGFP, hatched bar, n = 32; AQP4-M1, black bar, n = 19; AQP4-M23, white bar, n = 13) (ANOVA and post hoc, independent t-test P < 0.001 for AQP4-M1/NT-EGFP and AQP4-M23/NT-EGFP).The increase is abolished in absence of [Ca2+]o. The histogram also includes data obtained by adding gadolinium chloride (Gd3+) to the hypotonic saline to block TRPV cation channels (Right). The application of Gd3+ abolished the [Ca2+]i signal. (paired t-test, P < 0.001 for analyses in presence and absence of [Ca2+ ]o; independent t-test for experiment with Gd3+, P = 0.004 in AQP4-M1 cells, n = 10; P < 0.001 in AQP4-M23 cells, n = 16; P < 0.05 in EGFP cells, n = 6). P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.
Fig. 6.
Fig. 6.
Transfection with M1 or M23 isoforms of AQP4 reconstitutes hypotonicity-induced RVD in DI TNC1 cells. (A) Calcein-quenching method for measurement of osmotically-induced volume changes in cells transfected with AQP4-M1 (black, n = 15), AQP4-M23 (white, n = 22), or AQP1 (gray, n = 17) and in nontransfected cells treated with Lipofectamine only (NT; light gray, n = 46). (B) Direct comparison of the initial phase (the first 200 s following exposure to hypotonic medium) of curves depicted in A. The initial swelling is significantly faster in all the transfected cells than in the nontransfected cells. (C) Quantitative analysis of data presented in A. Ordinate values represent mean rate of volume change at 25 s of hypotonic stress compared with baseline level. (P > 0.001; ANOVA and independent t-test, n as in A). No significant difference is detectable between cells transfected with AQP4-M1 (black; n = 15) and cells transfected with AQP4-M23 (white; n = 22; P = 0.135; independent t-test). **P ≤ 0.01. (D) Ordinate values represent mean volume change calculated as described in Materials and Methods. A decrease in regulatory volume, represented by negative values, is seen after transfection with AQP4 isoforms M1 (black bar) or M23 (white bar) but not after transfection with AQP1 cells (dark gray bar) or in nontransfected (NT) cells (light gray bar). P < 0.01; ANOVA and independent t-test. **P ≤ 0.01.
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