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First published online May 28, 2005
doi: 10.1242/10.1242/jcs.02372

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TRPV4 exhibits a functional role in cell-volume regulation

Daniel Becker^*, Christopher Blase^*, Juergen Bereiter-Hahn and Marina Jendrach

Kinematic Cell Research Group, Johann Wolfgang Goethe University, Marie-Curie-Str. 9, 60439 Frankfurt, Germany

View larger version (41K): [in a new window]   Fig. 1. TRPV4 mRNA is expressed in volume-regulating cells. Total RNA was isolated from HaCaT keratinocytes, distal renal tubule cells (DTC), proximal renal tubule cells (PTC) and CHO cells. A region in the TRPV4 ORF and the glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) ORF were amplified by RT-PCR with gene-specific primers and separated on a 1.5% agarose gel. No PCR product could be detected in CHO cells.

View larger version (119K): [in a new window]   Fig. 2. Membrane localization of EGFP-TRPV4 in transiently transfected CHO cells. CHO cells were transiently transfected with EGFP-TRPV4 and checked for expression by confocal laser-scanning microscopy. The image shows a single xy plane taken slightly above the basal membrane (centre) and the cross-section in xz and yz planes, respectively (top and right). EGFP-TRPV4 is clearly localized to the plasma membrane 72 hours after transfection. Bar, 10 µm.

View larger version (21K): [in a new window]   Fig. 3. TRPV4 is necessary for regulatory volume decrease. Relative volume changes of HaCaT cells and CHO cells exposed to hypotonic or isotonic medium at t=0 seconds. (A) Reduction of extracellular osmolarity led to a rapid increase in cell volume of untreated HaCaT cells (hypo, filled circles, n=18) followed by RVD. Replacement of the medium alone did not affect cell volume (iso, open circles, n=12). Treatment with 100 µM Gd3+ (filled triangles, n=8) or Ca2+-free solution (open squares, n=8) abolished the RVD response and cell volume stayed elevated. (B) Untransfected CHO cells (nt, open circles, n=22) showed no RVD, whereas TRPV4-transfected CHO cells (TRPV4, filled circles, n=25) were able to undergo RVD. Under Ca2+-free conditions (open squares, n=8), no RVD occurred after the initial swelling in transfected cells. Gd3+-pretreated transfected cells also showed no RVD in comparison with untreated cells (filled triangles, n=14). Cell volume was estimated from the central cross-sectional area and normalized to control (t0 seconds) values. Data are means±s.e.m.

View larger version (8K): [in a new window]   Fig. 4. Changes in [Ca2+]i after hypotonic treatment. HaCaT and CHO cells were loaded with the Ca2+ indicator Fluo-4. Peak fluorescence intensities within 90 seconds after hypotonic treatment are plotted as ratios to control conditions (t0 seconds). (A) HaCaT cells exposed to hypotonic (hypo) conditions showed a significant increase in [Ca2+]i compared with isotonic (iso) conditions (P<0.01, ANOVA). By contrast, hypotonically treated cells that were preincubated with 100 µM Gd3+ or kept in Ca2+-free solution showed no significant increase in [Ca2+]i. Data are means±s.e.m. (isotonic, n=12; hypotonic, n=18; Gd3+, n=8; Ca2+-free, n=8). (B) EGFP-TRPV4-transfected CHO cells (TRPV4) exposed to hypotonic conditions showed a significant increase in [Ca2+]i compared with untransfected cells (nt, P<0.01, ANOVA). This increase in [Ca2+]i was absent from transfected cells pretreated with Gd3+ or kept in Ca2+-free solution. Data are means±s.e.m. (untransfected, n=22; EGFP-TRPV4, n=16; Gd3+, n=14; Ca2+-free, n=8).

View larger version (22K): [in a new window]   Fig. 5. Dynamics of [Ca2+]i and cell volume. Individual traces of the relative volume (dashed) and Fluo-4 fluorescence intensity (solid) of single cells exposed to hypotonic conditions at t=0 seconds. Relative values are expressed as ratios to control conditions (t0 seconds). (A-C) HaCaT cells. (D-F) EGFP-TRPV4-transfected CHO cells. Notice that elevation of [Ca2+]i follows cell swelling in both cell models. The duration of the [Ca2+]i peak is in general much shorter in transfected CHO cells than in HaCaT cells, in which [Ca2+]i is at least elevated throughout the maximum swelling phase.