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First published online 5 August 2008
doi: 10.1242/jcs.023705

Journal of Cell Science 121, 2871-2879 (2008)
Published by The Company of Biologists 2008
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TRPV4 enhances the cellular uptake of aminoglycoside antibiotics

Takatoshi Karasawa1, Qi Wang1, Yi Fu2,3, David M. Cohen2,3 and Peter S. Steyger1,*

1 Oregon Hearing Research Center, Oregon Health and Science University, 3181 Sam Jackson Park Road, Portland, OR 97239, USA
2 Division of Nephrology and Hypertension, Oregon Health and Science University, 3181 Sam Jackson Park Road, Portland, OR 97239, USA
3 Portland Veterans Affairs Medical Center, 3710 SW US Veterans Hospital Road, Portland, OR 97239, USA


Figure 1
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  		Fig. 1. (A-T) TRPV4 expression in kidney and inner ear. In MDCK cells, TRPV4 immunofluorescence was preferentially localized at the basolateral membrane (arrows in A). TRPV4 was also localized at the basolateral membranes (arrows) of wild-type (+/+) murine kidney distal tubule cells, but not in adjacent glomerular regions (g, in B), nor in the kidney of TRPV4-deficient mice (–/–, in C). In wholemounts of the stria vascularis from wild-type mice, TRPV4 was localized at the lumenal surface of marginal cells (m in D; green in E) at the level of the actiniferous tight junctions (red) between adjacent marginal cells (E, same focal plane as D). There was negligible TRPV4 immunoexpression at the lumenal surface of marginal cells of TRPV4-deficient mice (F). In a focal series of the cells shown in D and E, below the actiniferous tight junctions, only weak TRPV4 immunofluorescence occurred in the somata of marginal cells, but was prominent at the periphery of marginal cells (i in G, green in H). There was no TRPV4 expression detected in the somata of marginal cells from TRPV4-deficient mice (I). At lower focal planes than in G and H, strong, characteristic TRPV4 expression (J,M, green in K,N) was localized in intermediate (i) cells. TRPV4 expression could not be detected in the intra-strial tissues of TRPV4-deficient mice (L,O). In z-sections of the stria vascularis, TRPV4 immunolabeling was clearly associated with the lumenal surface (arrows) of the marginal cells (m), between the actiniferous tight junctions, and in the intermediate cells (i) below the weakly-labeled somata of marginal cells of wild-type mice (P,Q). In the organ of Corti, TRPV4 expression was most prominent in the region of tight junctions between adjacent pillar cells (arrowheads), and also between adjacent Deiters' cell phalangeal apices (arrows; R,S). The stereocilia of inner hair cells displayed TRPV4 expression (double arrowheads; R,S). TRPV4 expression was also present in Deiters' cell phalanges (d), but only weakly in OHCs (ohc) and spiral ganglion cells (T). Scale bar in C also applies for A and B. Scale bar in O also applies for D-N, R-T. Scale bar in Q also applies for P.

 

Figure 2
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  		Fig. 2. New kidney cell line generation and TRPV4 expression. By spontaneous immortalization, three cell lines were generated from primary murine kidney cultures. (A,B) Bright-field images show KPT2 cells with a fibroblastic morphology (A), and KDT3 cells with an epithelial (cobblestone-like) appearance and smooth edges to cell clusters (B). (C,D) TRPV4 expression levels were determined by RT-PCR (C) and immunoblotting (D). KDT3 cells showed high levels of TRPV4 whereas KPT1 and KPT2 showed weak TRPV4 expression or possibly non-specific bands. Lower levels of β-actin following RT-PCR (C) and of actin following immunoblotting (D) suggest that KDT3 cells express β-actin and possibly other actin isoforms at lower levels compared with KPT1 and KPT2. (E) Expression of TRPV4 was negligible in KPT2 cells. (F) Immunofluorescence showed TRPV4 localization at the basolateral membrane (arrowheads) of KDT3 cells. (G) Orthogonal view of KDT3 cells shows TRPV4 expression in the basolateral membrane (arrowheads). Scale bars, 20 µm.

 

Figure 3
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  		Fig. 3. GTTR uptake is enhanced in the absence of extracellular Ca2+. (A,B) In the presence of extracellular 1.25 mM Ca2+, KPT2 and KDT3 cells had modest and low levels of intracellular GTTR fluorescence, respectively, after 30 seconds of GTTR treatment. (C,D) In the absence of extracellular Ca2+, KPT2 cells showed a modest increase in GTTR fluorescence (C), whereas KDT3 cells showed substantially enhanced GTTR uptake (D). In both KPT2 and KDT3 cells, GTTR fluorescence was diffusely distributed in the cytoplasm, with small puncta (arrowheads) within the nucleus. (E) Fluorescence intensity of cytoplasmic GTTR in KDT3 cells was significantly lower than in KPT2 cells in the presence of extracellular Ca2+ (**P<0.01). Removal of extracellular Ca2+ greatly enhanced GTTR fluorescence in both KPT2 and KDT3 cells compared with 1.25 mM Ca2+ condition (P<0.001). The enhanced GTTR levels by extracellular Ca2+ removal were similar between KPT2 and KDT3 cells. Fluorescence intensity data are shown as mean ± s.d. Scale bar, 20 µm.

 

Figure 4
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  		Fig. 4. Generation of KPT2-derived cell lines that express TRPV4. (A) In immunoblots, three empty vector control clones (KPT2-pBabe) did not express TRPV4, whereas three TRPV4-expressing clones (KPT2-TRPV4) highly expressed TRPV4 with two or three bands of ~110 kDa. Actin bands at 42 kDa confirmed that there were similar amounts of total protein in the samples. (B) There was negligible TRPV4 expression in KPT2-pBabe cells. (C) In KPT2-TRPV4 cells, immunofluorescence showed uniform localization of TRPV4 at the cell membrane. (D) Orthogonal view of KPT2-TRPV4 shows that TRPV4 protein expression is uniform in the membrane, including the apical membrane. Scale bar in B also applies for C and D (20 µm). (E) TRPV4-dependent Ca2+ entry in response to hypotonic stress or the TRPV4 agonist 4{alpha}-PDD was observed in KPT2-TRPV4 cells, but not in KPT2-pBabe cells. Fura-2 fluorescence was monitored as an index of intracellular Ca2+ concentration (see Materials and Methods), and the Fura-2 ratio (in arbitrary unit; U) is depicted as a function of time. Arrowhead denotes onset of hypotonic stress or addition of 4{alpha}-PDD.

 

Figure 5
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  		Fig. 5. (A-E) KPT2-TRPV4 cell lines show enhanced GTTR uptake in the absence of extracellular Ca2+. After 30 seconds of GTTR treatment, there was little difference in GTTR uptake between KPT2-TRPV4 and KPT2-pBabe cells in the presence of extracellular Ca2+ (A,B), with no significant difference in fluorescence intensity of cytoplasmic GTTR (E). Enhanced GTTR uptake was observed in KPT2-TRPV4 cells in the absence of extracellular Ca2+ compared with KPT2-pBabe cells (C,D). Similar results were obtained for all the three KPT2-TRPV4 and three KPT2-pBabe cell lines. Fluorescence intensity of cytoplasmic GTTR in KPT2-TRPV4 cells was significantly higher than in KPT2-pBabe cells under conditions of 0 mM or 0.05 mM extracellular Ca2+ (E). Fluorescence intensity data are shown as mean ± s.d. **P<0.01. Bar in C also applies for A, B and D (20 µm).

 

Figure 6
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  		Fig. 6. GTTR uptake by TRPV4 is dependent on M680 residue within the channel pore. (A,B) KPT2-derived cell lines that express the M680D TRPV4 mutant were generated and tested for GTTR uptake. Immunofluorescence confirmed that the M680D TRPV4 mutant had normal membrane localization in the cells.(C,D) Immunoblotting showed similar protein expression levels of TRPV4 wild-type and the M680D mutant in KPT2-TRPV4 and the two KPT2-M680D clones (M680D#1 and #2), respectively. After 30 seconds of GTTR treatment, there was no significant enhancement in GTTR uptake by extracellular Ca2+ removal in KPT2-M680D cells, and similar results were obtained for the two KPT2-M680D cell lines (D). Fluorescence intensity data are shown as mean ± s.d. **P<0.01. Scale bar, 20 µm.

 

Figure 7
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  		Fig. 7. TRPV4 contributes to GTTR retention in KPT2-TRPV4 cells in an endolymph-like extracellular environment. After 1 minute of GTTR treatment in PBS containing Ca2+ at 1.25 mM, KPT2-pBabe and KPT2-TRPV4 cells had similar levels of GTTR (A,D). After incubation in clearance buffer, KPT2-pBabe cells had lower GTTR fluorescence levels compared with KPT2-TRPV4 cells (B,C,E,F). Treatment with 10 µM RR to block inward currents accelerated GTTR clearance from KPT2-TRPV4 cells (G,H). Fluorescence intensity of cytoplasmic GTTR was quantified and statistically analyzed, and data are shown as mean ± s.d. (I). **P<0.01. Scale bar, 20 µm.

 

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© The Company of Biologists Ltd 2008