Microfilament-associated Protein 7 Increases the Membrane Expression of Transient Receptor Potential Vanilloid 4 (TRPV4)

The molecular mechanism of the transmission of changes in the shape of the cell surface to ion channels remains obscure. Ca2+ influx induced by cell deformity is inhibited by actin-freezing reagents, suggesting that the actin microfilament couples with an ion channel. Transient receptor potential vanilloid 4 (TRPV4) is a candidate in the calcium-permeable, swelling-activated mechanosensitive channel in heterogeneously expressed cells. To investigate the mechanosensitive molecular complex, we found that microtubule-associated protein 7 (MAP7) is the mouse TRPV4 C-terminal binding protein. MAP7 was coimmunoprecipitated with TRPV4. The results of a pull-down assay demonstrated that the alignment of amino acids 798-809 of TRPV4 was important in this interaction. TRPV4 and MAP7 colocalized in the lung and kidney. The coexpression of these two molecules resulted in the redistribution of TRPV4 toward the membrane and increased its functional expression. The alignment of amino acids 798-809 of TRPV4 was also important in the functional expression. The activated current was abolished by actin-freezing but not by microtubule-freezing reagents. We therefore believe that MAP7 may enhance the membrane expression of TRPV4 and possibly link cytoskeletal microfilaments. The molecular mechanism of the transmission of changes in the shape of the cell surface to ion channels remains obscure. Ca2+ influx induced by cell deformity is inhibited by actin-freezing reagents, suggesting that the actin microfilament couples with an ion channel. Transient receptor potential vanilloid 4 (TRPV4) is a candidate in the calcium-permeable, swelling-activated mechanosensitive channel in heterogeneously expressed cells. To investigate the mechanosensitive molecular complex, we found that microtubule-associated protein 7 (MAP7) is the mouse TRPV4 C-terminal binding protein. MAP7 was coimmunoprecipitated with TRPV4. The results of a pull-down assay demonstrated that the alignment of amino acids 798-809 of TRPV4 was important in this interaction. TRPV4 and MAP7 colocalized in the lung and kidney. The coexpression of these two molecules resulted in the redistribution of TRPV4 toward the membrane and increased its functional expression. The alignment of amino acids 798-809 of TRPV4 was also important in the functional expression. The activated current was abolished by actin-freezing but not by microtubule-freezing reagents. We therefore believe that MAP7 may enhance the membrane expression of TRPV4 and possibly link cytoskeletal microfilaments. Microtubule-associated protein 7 increases the membrane expression of transient receptor potential vanilloid 4 (TRPV4). Vol. 278 (2003)51448–51453Journal of Biological ChemistryVol. 280Issue 27Preview“Microtubule-associated” in the title was printed as associated as “Microfilament-associated.” The correct title is listed above. Also, the definition for MAP7 in the abbreviation footnote should read “microtubule-associated protein 7” instead of “microfilament-associated protein 7.” Full-Text PDF Open Access Cells experience a wide variety of mechanical stresses and require a sensitive mechanism to regulate the deformity of the cell surface. The molecular structure that transmits cell surface deformities to ion channels (the mechanosensitive ion channel) has been shown by a number of findings to be in bacterial MscL (1Martinec B. Buechner M. Delcour A.H. Adler J. Kung C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2297-2301Crossref PubMed Scopus (528) Google Scholar), yeast Mid1 (2Iida H Nakamura H. Ono T. Okamura M. Anraku Y. Mol. Cell. Biol. 1994; 14: 8259-8271Crossref PubMed Scopus (194) Google Scholar), mammalian DEG/MEC (3Overnos J.G. Corey D.P. Annu. Rev. Neurosci. 1997; 20: 567-594Crossref PubMed Scopus (140) Google Scholar), and TREK (4Patel A. Honore E. MainGret F. Lesage F. Fink M. Duprat F. Lazdunski M. EMBO J. 1998; 17: 4283-4290Crossref PubMed Scopus (533) Google Scholar) and their derivatives (5Hamill O.P. Martinac B. Physiol. Rev. 2001; 81: 687-740Crossref Scopus (928) Google Scholar). These channels are monovalent cation channels that are permeable to sodium or potassium. However, the molecular structure of the mammalian calcium-permeable mechanosensitive channel remains obscure. One of characteristics of this channel is its relation to the cytoskeleton. The current of MscL and TREK are increased or unaffected by the disruption of actin microfilaments (5Hamill O.P. Martinac B. Physiol. Rev. 2001; 81: 687-740Crossref Scopus (928) Google Scholar), whereas the current of most calcium-permeable mechanosensitive channels is abolished by this manipulation, which has been recognized in neurons (6Cornet M. Delpire E. Gilles R. Pfluegers Arch. Eur. J. Physiol. 1987; 410: 223-225Crossref PubMed Scopus (28) Google Scholar, 7Small D.L. Morris C.E. Am. J. Physiol. 1994; 267: C598-C606Crossref PubMed Google Scholar), leukocytes (8Downey G.P. Grinstein S Sue-A-Quan A. Czabam B. Chan C.K. J. Cell. Physiol. 1995; 163: 96-104Crossref PubMed Scopus (61) Google Scholar), the gallbladder (9Foskett J.K. Spring K.R. Am. J. Physiol. 1985; 248: C27-C36Crossref PubMed Google Scholar), hepatocytes (10Khalbuss W.E. Wondergem R. Hepatology. 1991; 13: 962-969Crossref PubMed Scopus (17) Google Scholar), renal epithelia (11Linshaw M.A. Fogwl C.A. Downey G.P. Koo E.W. Gotlieb A.I. Am. J. Physiol. 1992; 262: F144-F150PubMed Google Scholar), and muscle (12Katz B. J. Physiol. 1950; 111: 261-282Crossref PubMed Scopus (151) Google Scholar, 13Gucharay F. Sachs F. J. Physiol. 1984; 352: 685-701Crossref PubMed Scopus (718) Google Scholar). Therefore, the relationship of the calcium-permeable ion channels to the cytoskeleton might differ from that of MscL and TREK. On the other hand, the molecular structure of calcium-permeable mechanosensitive ion channels was not clarified. Great progress in understanding the molecular candidate was made with the cloning and identification of the vanilloid receptor (TRPV1), 1The abbreviations used are: TRPVtransient receptor potential vanilloidCHOChinese hamster ovaryGABACγ-aminobutyric acid (variant C)HEK293human embryonic kidney 203MAP7microfilament-associated protein 7RTreverse transcriptaseNi-NTAnickel-nitrilotriacetic acidPBSphosphate-buffered saline. an ion channel of the transient receptor potential (TRP) ion channel family, which has been proposed to function at the transduction step in the nociceptive pathway (14Caterina M.J. Rosen T.A. Tominaga T. Brake A.J. Julius D. Nature. 1999; 398: 436-441Crossref PubMed Scopus (1253) Google Scholar). Although mice lacking TRPV1 show a marked behavioral response to noxious heat, capsaicin, and acid, they do not show the difference in mechanical nociception (15Caterina M.J. Leffler A. Malmberg A.B. Martin W.J. Trafton J. Petersen-Zeitz K.R. Koltzenburg M. Basbaum A.I. Julius D. Science. 2000; 288: 306-312Crossref PubMed Scopus (2914) Google Scholar). Mechanical nociception was suggested to be processed by other related genes. TRPV4 (SAC1 (16Suzuki M. Ishibashi K. Imai M. J. Am. Soc. Nephrol. 1999; 10: 44Google Scholar), TRP12 (17Wissenbach U. Bodding M. Freichel M. Flockerzi V. FEBS Lett. 2000; 485: 127-134Crossref PubMed Scopus (257) Google Scholar), OTRPC4 (18Strotmann R. Harteneck C. Nunnenmacher K. Schultz G. Plant T.D. Nat. Cell Biol. 2000; 2: 695-702Crossref PubMed Scopus (796) Google Scholar), VR-OAC (19Liedtke W. Choe Y. Marti-Renom M.A. Bell A.M. Denis C.S. Sali A. Hudspeth A.J. Friedman J.M. Heller S. Cell. 2000; 103: 525-535Abstract Full Text Full Text PDF PubMed Scopus (1078) Google Scholar), or VRl2 (20Delany L.S. Hurle M. Facer P. Alnadaf T. Plumpton C. Kinghorn I. See C.G. Costigan M. Anand P. Woolf C.J. Crowther D. Sanseau P. Tate S.N. Physiol. Genomics. 2001; 4: 165-174Crossref PubMed Scopus (201) Google Scholar, 21Suzuki M. Ohki G. Mochizuki T. Somlo S. Ishibashi K. Imai M. FEBS Lett. 2002; 517: 219-224Crossref PubMed Scopus (8) Google Scholar) is similar to TRPV1 and is reported to be a swelling-activated cation channel, a candidate for the mammalian mechano-gated calcium-permeable cation channel. The mice lacking this gene show a defect in the high-threshold mechanosensation, pressure, but maintain the low-threshold innocuous touch sensation (22Suzuki M. Mizuno A. Kodaira K. Imai M. J. Biol. Chem. 2003; 278: 22664-22668Abstract Full Text Full Text PDF PubMed Scopus (586) Google Scholar). This result is supported in rat by using antisense methodology (23Alessandri-Haber N. Yeh J.J. Boyd A.E. Parada C.A. Chen X. Reichling D.B. Levine J.D. Neuron. 2003; 31: 497-511Abstract Full Text Full Text PDF Scopus (360) Google Scholar) or is proposed in a Drosophila mutant (24Tracey W.D. Wilson R.I. Laurent G. Benzer S. Cell. 2003; 113: 261-273Abstract Full Text Full Text PDF PubMed Scopus (537) Google Scholar). transient receptor potential vanilloid Chinese hamster ovary γ-aminobutyric acid (variant C) human embryonic kidney 203 microfilament-associated protein 7 reverse transcriptase nickel-nitrilotriacetic acid phosphate-buffered saline. We had expressed TRPV4 in CHO cells but found a poor activity by swelling. We also found that TRPV4 was significantly activated by an inflation of the cell (22Suzuki M. Mizuno A. Kodaira K. Imai M. J. Biol. Chem. 2003; 278: 22664-22668Abstract Full Text Full Text PDF PubMed Scopus (586) Google Scholar). We next expressed an N-terminal ankyrin or a C-terminal hydrophilic deletion mutant of TRPV4, resulting in a lack of activation. These findings brought us to a hypothesis that more proteins are needed for TRPV4 to be expressed on membrane and to be sensitive to cell deformity only when transfected in these cells. We, therefore, performed a binding study to search for a protein to reconstitute better mechanosensitive expression of TRPV4. Yeast Two-hybrid Screens—Approximately two million clones were screened from a mouse kidney cDNA library constructed in the Gal4 activation domain carrying vector pACTII (Clontech). The library was screened with bait that encoded the predicted intracellular domain of the mouse TRPV4 cloned in the Gal4 DNA-binding domain vector pAS2-1 (Clontech). The plasmids were transformed into yeast strain Y190, and transformants were selected on triple dropout media (Leu/His/Trp that contained 30 mm 3-amino-triazole) and assayed for β-galactosidase activity. Positive clones were cotransformed into yeast with either the bait vector or the pACTII vector backbone to confirm interactions. The activity of β-galactosidase was measured using the chlorophenol red-β-d-galactopyranoside assay according to the manufacturer's protocol (Clontech). pACTII vectors with higher activity were recloned into DH5α and amplified for identification. Antibodies—A TRPV4-specific antibody (anti-TRPV4) was raised in a rabbit against a C terminus, as reported elsewhere (21Suzuki M. Ohki G. Mochizuki T. Somlo S. Ishibashi K. Imai M. FEBS Lett. 2002; 517: 219-224Crossref PubMed Scopus (8) Google Scholar, 22Suzuki M. Mizuno A. Kodaira K. Imai M. J. Biol. Chem. 2003; 278: 22664-22668Abstract Full Text Full Text PDF PubMed Scopus (586) Google Scholar). A MAP7-specific rabbit polyclonal antibody was raised against a synthetic peptide corresponding to eight C- or N-terminal amino acids (C-terminal TQQTAEVI and N-terminal MDQAKSAE). For coprecipitation and localization, an N-terminal antibody was used. In Vitro Cell-free Binding Assay—The STP3™ (Novagen) was used to generate 35Smethionine-labeled MAP7 protein. TRPV4-His5 fusion proteins were expressed in HEK293 cells and recovered on Ni-NTA beads. The beads were incubated with lysate that contained a 35S-labeled MAP7 protein for 1 h at 45 °C and then washed three times with a buffer (50 mm NaH2PO4, 300 mm NaCl, and 20 mm imidazole). The elution was boiled in sample buffer (2% SDS, 10% glycerol, and 62 mm Tris (pH 6.8)), and bound proteins were resolved in SDS-PAGE. Immunoprecipitation and Affinity Purification (Pull-down Assay)— Renal extracts were by in 300 mm mm 1 mm mm and were by using to protein The and the were by that used and To the TRPV4, its and mouse MAP7 cDNA were recloned to or which with at the C-terminal or with at the N-terminal For the pull-down extracts of HEK293 cells a were by in mm NaH2PO4, 300 mm NaCl, mm 20 (pH and Ni-NTA beads were used to the complex, which was washed in mm NaH2PO4, 300 mm NaCl, and 20 mm (pH and in mm NaH2PO4, 300 mm NaCl, mm and 20 (pH and were using the antibody The membrane protein of the cells was as of transfected CHO cells were washed three times with (pH and the cells were by at for proteins were then with 1 in (pH at for 30 according to the manufacturer's The cells were washed three times in and then at 4 °C for 1 h in (pH that contained mm and Cell extracts were incubated with of at 4 °C The proteins were by with or were from a and in cells were in and then by with for was according to the manufacturer's protocol MAP7 was using a MAP7 N-terminal antibody and using TRPV4 was in an and using amino was using a according to the manufacturer's and were and a was constructed transcriptase was according to the manufacturer's protocol with of as a The of 30 of at °C for 30 °C for 30 and °C for 30 were to MAP7 from the to the of the number of MAP7 is which is to or TRPV4 has been reported as number and MAP7 were to and transfected to CHO cells with as the of the expression. were according to elsewhere M. J. K. G. Imai M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). were at with an To for cell the current was by of the cell The contained mm NaCl, mm and mm The was by the of The contained a of mm mm and 1 mm (pH of Binding to the C- or N-terminal intracellular domain of TRPV4 (the and the amino as bait in a yeast of an mouse kidney cDNA we to the and clones to the C β-galactosidase activity. and that eight of the clones binding to the C terminus, which had β-galactosidase were These the and membrane was and was found to be MAP7 to of TRPV4 and confirm the yeast we the of TRPV4 with MAP7 using in and in For in TRPV4 was expressed as a fusion protein with and MAP7 was and with by in and TRPV4-His5 fusion proteins on beads were incubated with 35Smethionine-labeled MAP7 protein. MAP7 bound to TRPV4-His5 but not to which that they in We an to TRPV4 and MAP7 in and recognized of and in the kidney. Renal extracts were using and then with The MAP7 was with but not with or with an of the antibody the MAP7 and TRPV4 in and in was also by using a pull-down assay in which MAP7 was with the protein and expressed in HEK293 cells. The binding of TRPV4 on MAP7 was also using deletion acids of amino acid of TRPV4 were for the amino acids were found to be for the binding of MAP7 The amino acid alignment of expression of MAP7 was using The of MAP7 in was similar to that of TRPV4, in the and but was also in the and the is not This of TRPV4 is similar to that reported by (17Wissenbach U. Bodding M. Freichel M. Flockerzi V. FEBS Lett. 2000; 485: 127-134Crossref PubMed Scopus (257) Google Scholar, R. Harteneck C. Nunnenmacher K. Schultz G. Plant T.D. Nat. Cell Biol. 2000; 2: 695-702Crossref PubMed Scopus (796) Google Scholar, W. Choe Y. Marti-Renom M.A. Bell A.M. Denis C.S. Sali A. Hudspeth A.J. Friedman J.M. Heller S. Cell. 2000; 103: 525-535Abstract Full Text Full Text PDF PubMed Scopus (1078) Google Scholar, L.S. Hurle M. Facer P. Alnadaf T. Plumpton C. Kinghorn I. See C.G. Costigan M. Anand P. Woolf C.J. Crowther D. Sanseau P. Tate S.N. Physiol. Genomics. 2001; 4: 165-174Crossref PubMed Scopus (201) Google Scholar). We next the of MAP7 and TRPV4 in of mouse was suggested by results in in cells in the the membrane of renal and the membrane of cells was in and renal cells The in in the renal was an by was marked in the other as which may be a that MAP7 not to TRPV4 that MAP7 may a function in this of TRPV4 by the coexpression we cells the MAP7 was in or cells as as in but not in CHO cells To investigate the that MAP7 the of TRPV4, we expressed TRPV4 and MAP7 in CHO cells. cells with the TRPV4 antibody or MAP7 are shown with the are two cells in of TRPV4 was expressed in the cell on the in of whereas TRPV4 and MAP7 were in the cell shown on the in of The expression the TRPV4 by surface cell on the in of TRPV4 with MAP7 cell on the in resulted in a in the of TRPV4. TRPV4, with was brought toward the membrane surface in the To surface we membrane protein by is a that to membrane surface amino acids and was as a with membrane protein TRPV4 is expressed on the membrane when is transfected TRPV4 colocalized with MAP7 an in the TRPV4 of the membrane MAP7 bound to TRPV4 and TRPV4 surface expression of TRPV4 with to the expression of TRPV4 toward the membrane we that MAP7 mechanical transmission actin microfilaments. The of the activity of TRPV4 was to investigate its functional and to actin by The of the was from 300 to and then to The of TRPV4 with MAP7 and TRPV4 were not However, the current by 300 to was The current by TRPV4 with MAP7 sensitive to the A of the activated but by in TRPV4 or TRPV4 with MAP7 that the two molecules induced a The activated current was by H. J. G. B. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The current of TRPV4 deletion with MAP7 the results of protein binding study by To the of cytoskeletal the current of TRPV4 with MAP7 was with in The and abolished the whereas the microtubule-freezing and not Therefore, the activation of TRPV4 with MAP7 by actin microfilaments but not We the swelling-activated current in or CHO and HEK293 cells in the The current was measured in a from 300 to and then to The current of TRPV4 in HEK293 was significantly activated by and by In the was not significantly in CHO cells of MAP7 and the was cloned as a protein associated with FEBS Lett. PubMed Scopus Google Scholar), a study suggested its to with actin microfilaments T. C. J. Biol. Chem. 1984; Full Text PDF PubMed Google Scholar). which is to D. J. Biol. Chem. Scholar), is a that is expressed in cells. MAP7 may a in cells and may to N. J.M. G. P. D. 1998; PubMed Scopus Google Scholar). The has been to microfilament channels as is an of in the cytoskeleton in P. F. Nature. 1999; PubMed Scopus Google Scholar, H. K. N. J. Nature. 1999; PubMed Scopus Google Scholar). Although the of MAP7 and TRPV4 in the cell was suggested by results we not intracellular of the two molecules in MAP7 is in FEBS Lett. PubMed Scopus Google Scholar), the of cell surface or the of molecules in the The results of of pull-down and findings that MAP7 is an associated protein of TRPV4. MAP7 in the whereas TRPV4 is to be more TRPV4 and to other This is the with which is more P. F. Nature. 1999; PubMed Scopus Google Scholar, H. K. N. J. Nature. 1999; PubMed Scopus Google Scholar). The coexpression of MAP7 and TRPV4 resulted in the surface expression of TRPV4 This is also the with The coexpression of and their at the cell surface P. F. Nature. 1999; PubMed Scopus Google Scholar, H. K. N. J. Nature. 1999; PubMed Scopus Google Scholar). The surface expression that we was suggested by However, these results not the of two On the other hand, membrane expression was by of MAP7 and TRPV4 and the membrane using This is more as has been suggested in an with channel H. G. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). MAP7 with the C of TRPV4. pull-down assay and functional study suggested the of amino acids However, the of the binding domain was not predicted by the and is We to search for the by using in a of MAP7 and is on E. E. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). The of MAP7 was in the activation of TRPV4 by the current was significantly when was in the not However, the current of TRPV4 in HEK293 (18Strotmann R. Harteneck C. Nunnenmacher K. Schultz G. Plant T.D. Nat. Cell Biol. 2000; 2: 695-702Crossref PubMed Scopus (796) Google Scholar) or CHO (19Liedtke W. Choe Y. Marti-Renom M.A. Bell A.M. Denis C.S. Sali A. Hudspeth A.J. Friedman J.M. Heller S. Cell. 2000; 103: 525-535Abstract Full Text Full Text PDF PubMed Scopus (1078) Google Scholar) cells was not significantly by the of from the TRPV4 was also activated by a using E. E. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), which is a protein C in HEK293 cells H. J. G. B. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). the of MAP7 or its by is in the mechanism of the remains This mechanism may the TRPV4 response of expressed cells to TRPV4 was more activated in HEK293 cells in CHO possibly HEK293 cells MAP7 The difference in the study is not to the findings of (19Liedtke W. Choe Y. Marti-Renom M.A. Bell A.M. Denis C.S. Sali A. Hudspeth A.J. Friedman J.M. Heller S. Cell. 2000; 103: 525-535Abstract Full Text Full Text PDF PubMed Scopus (1078) Google Scholar), used CHO cells and an activated current of which to a of However, number of used HEK293 which a The of the activation by is by the MAP7 in HEK293 cells. The of functional expression in the cells is also in the activation we (19Liedtke W. Choe Y. Marti-Renom M.A. Bell A.M. Denis C.S. Sali A. Hudspeth A.J. Friedman J.M. Heller S. Cell. 2000; 103: 525-535Abstract Full Text Full Text PDF PubMed Scopus (1078) Google Scholar) activation of TRPV4, was in HEK293 cells (18Strotmann R. Harteneck C. Nunnenmacher K. Schultz G. Plant T.D. Nat. Cell Biol. 2000; 2: 695-702Crossref PubMed Scopus (796) Google Scholar). HEK293 is an cell that was from the TRPV4 is is therefore that HEK293 cells a of the to TRPV4 expression. In CHO cells are cells from ovary which channel This hypothesis also results the of the which a swelling-activated cation current B. G. Physiol. Rev. 2001; Scopus Google Scholar), contained TRPV4 but not MAP7 that a difference in was in the of with molecules of the may the mechanosensitive calcium-permeable cation channel The mechanosensitive molecular be with the channel complex, and an ion channel, which is to In the of the activity of the channel increases R. M. Nature. 2002; PubMed Scopus Google Scholar). MAP7 TRPV4 to resulting in an in activity at the the of to membrane surface the mechanosensitive may require the channel and the to a However, TRPV4 with MAP7 not in the channel. We a channel on TRPV4 and CHO cells. Although we were to a cation channel, was not similar to the channels is This result is with that in cells (18Strotmann R. Harteneck C. Nunnenmacher K. Schultz G. Plant T.D. Nat. Cell Biol. 2000; 2: 695-702Crossref PubMed Scopus (796) Google Scholar). The of TRPV4 has been suggested in (22Suzuki M. Mizuno A. Kodaira K. Imai M. J. Biol. Chem. 2003; 278: 22664-22668Abstract Full Text Full Text PDF PubMed Scopus (586) Google Scholar, N. Yeh J.J. Boyd A.E. Parada C.A. Chen X. Reichling D.B. Levine J.D. Neuron. 2003; 31: 497-511Abstract Full Text Full Text PDF Scopus (360) Google Scholar) and in of the mechanism the activation of TRPV4 were that of the H. H. W. K. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar) or H. J. G. B. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, H. J. J. G. T. B. Nature. 2003; Scopus Google Scholar) results in the activation of the TRPV4 channel. be the transduction of cell in B. 1991; PubMed Scopus Google Scholar). Therefore, deformity in the cell may the to actin and then TRPV4 MAP7 in cells. This should be in is the to an associated protein of TRPV4. with TRPV4 by an in the membrane expression. MAP7 is a protein that is associated with cytoskeletal this may the mechanism of to cell We the for the of the and we also Y. and Y. for