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• W1988673487 abstract "TRPC5 forms Ca2+-permeable nonselective cation channels important for neurite outgrowth and growth cone morphology of hippocampal neurons. Here we studied the activation of mouse TRPC5 expressed in Chinese hamster ovary and human embryonic kidney 293 cells by agonist stimulation of several receptors that couple to the phosphoinositide signaling cascade and the role of calmodulin (CaM) on the activation. We showed that exogenous application of 10 μm CaM through patch pipette accelerated the agonist-induced channel activation by 2.8-fold, with the time constant for half-activation reduced from 4.25 ± 0.4 to 1.56 ± 0.85 min. We identified a novel CaM-binding site located at the C terminus of TRPC5, 95 amino acids downstream from the previously determined common CaM/IP3R-binding (CIRB) domain for all TRPC proteins. Deletion of the novel CaM-binding site attenuated the acceleration in channel activation induced by CaM. However, disruption of the CIRB domain from TRPC5 rendered the channel irresponsive to agonist stimulation without affecting the cell surface expression of the channel protein. Furthermore, we showed that high (>5 μm) intracellular free Ca2+ inhibited the current density without affecting the time course of TRPC5 activation by receptor agonists. These results demonstrated that intracellular Ca2+ has dual and opposite effects on the activation of TRPC5. The novel CaM-binding site is important for the Ca2+/CaM-mediated facilitation, whereas the CIRB domain is critical for the overall response of receptor-induced TRPC5 channel activation. TRPC5 forms Ca2+-permeable nonselective cation channels important for neurite outgrowth and growth cone morphology of hippocampal neurons. Here we studied the activation of mouse TRPC5 expressed in Chinese hamster ovary and human embryonic kidney 293 cells by agonist stimulation of several receptors that couple to the phosphoinositide signaling cascade and the role of calmodulin (CaM) on the activation. We showed that exogenous application of 10 μm CaM through patch pipette accelerated the agonist-induced channel activation by 2.8-fold, with the time constant for half-activation reduced from 4.25 ± 0.4 to 1.56 ± 0.85 min. We identified a novel CaM-binding site located at the C terminus of TRPC5, 95 amino acids downstream from the previously determined common CaM/IP3R-binding (CIRB) domain for all TRPC proteins. Deletion of the novel CaM-binding site attenuated the acceleration in channel activation induced by CaM. However, disruption of the CIRB domain from TRPC5 rendered the channel irresponsive to agonist stimulation without affecting the cell surface expression of the channel protein. Furthermore, we showed that high (>5 μm) intracellular free Ca2+ inhibited the current density without affecting the time course of TRPC5 activation by receptor agonists. These results demonstrated that intracellular Ca2+ has dual and opposite effects on the activation of TRPC5. The novel CaM-binding site is important for the Ca2+/CaM-mediated facilitation, whereas the CIRB domain is critical for the overall response of receptor-induced TRPC5 channel activation. In 1994, we demonstrated (1Vaca L. Sinkins W.G. Hu Y. Kunze D.L. Schilling W.P. Am. J. Physiol. 1994; 267: 1501-1505Crossref PubMed Google Scholar, 2Hu Y. Vaca L. Zhu X. Birnbaumer L. Kunze D.L. Schilling W.P. Biochem. Biophys. Res. Commun. 1994; 201: 1050-1056Crossref PubMed Scopus (91) Google Scholar) that the transient receptor potential (Trp) 1The abbreviations used are: trp, transient receptor potential; CaM, calmodulin; Bk, bradykinin; Bk2R, bradykinin receptor type 2; CBII, second CaM binding; CHO, Chinese hamster ovary; CIRB, CaM/IP3R binding; H1R, histamine receptor type 1; HEK, human embryonic kidney; IP3R, inositol 1,4,5-trisphosphate receptor; MBP, maltose-binding protein; mTRPC5, mouse canonical transient receptor potential 5; TRPL, TRP-like; W7, N-aminohexyl-5-chloro-1-naphthalenesulfonamide; TFP, trifluoperazine dimaleate; RT, reverse transcription; HEDTA, N-(2-hydroxyethyl)ethylenediaminetriacetic acid. 1The abbreviations used are: trp, transient receptor potential; CaM, calmodulin; Bk, bradykinin; Bk2R, bradykinin receptor type 2; CBII, second CaM binding; CHO, Chinese hamster ovary; CIRB, CaM/IP3R binding; H1R, histamine receptor type 1; HEK, human embryonic kidney; IP3R, inositol 1,4,5-trisphosphate receptor; MBP, maltose-binding protein; mTRPC5, mouse canonical transient receptor potential 5; TRPL, TRP-like; W7, N-aminohexyl-5-chloro-1-naphthalenesulfonamide; TFP, trifluoperazine dimaleate; RT, reverse transcription; HEDTA, N-(2-hydroxyethyl)ethylenediaminetriacetic acid. gene and its homologue, Trp-like (Trpl), from Drosophila melanogaster encoded calcium-permeable cationic channels activated either by store depletion or by stimulation of Gq/11-coupled receptors. These initial findings prompted the search for mammalian homologues, leading to the identification of seven TRP genes with different degrees of sequence similarity to the original insect Trp gene (3Zhu X. Jiang M. Peyton M. Boulay G. Hurst R. Stefani E. Birnbaumer L. Cell. 1996; 85: 661-671Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar). These genes are now designated TRP-Canonical or TRPC, symbolizing their close similarity to the original Drosophila Trp. Many recently discovered cation channels are found to share some limited homology with the TRPCs. These include TRPVs (similar to the vanilloid receptor), TRPMs (named after the first identified member, melastatin), and TRPPs (named after PKD2 for polycystic kidney disease), etc. Together, there are at least 28 non-allelic TRP genes in the mammalian genome. The TRP channels serve diverse functions in many tissues from somatosensory to cardiovascular systems (4Clapham D.E. Runnels L.W. Strubing C. Nat. Rev. Neurosci. 2001; 2: 387-396Crossref PubMed Scopus (947) Google Scholar).TRPC5 is a member of the TRPC family of Ca2+-permeable nonselective cationic channels. It has drawn attention recently because of its role in modulating hippocampal growth cone motility and neurite elongation in the mammalian brain (5Greka A. Navarro B. Oancea E. Duggan A. Clapham D.E. Nat. Neurosci. 2003; 6: 837-845Crossref PubMed Scopus (310) Google Scholar). The TRPC5 channel activity is induced upon stimulation of the phosphoinositide signaling cascade by receptors that stimulate phospholipase C; however, the exact mechanism of channel activation remains controversial (6Plant T.D. Schaefer M. Cell Calcium. 2003; 33: 441-450Crossref PubMed Scopus (135) Google Scholar). The activation of TRPC5 is dependent on the presence of Ca2+ at both the extracellular and the intracellular sides of the plasma membrane (7Jung S. Muhle A. Schaefer M. Strotmann R. Schultz G. Plant T.D. J. Biol. Chem. 2003; 278: 3562-3571Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 8Okada T. Inoue R. Yamazaki K. Maeda A. Kurosaki T. Yamakuni T. Tanaka I. Shimizu S. Ikenaka K. Imoto K. Mori Y. J. Biol. Chem. 1999; 274: 27359-27370Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 9Schaefer M. Plant T.D. Obukhov A.G. Hofmann T. Gudermann T. Schultz G. J. Biol. Chem. 2000; 275: 17517-17526Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar, 10Zeng F. Xu S.Z. Jackson P.K. McHugh D. Kumar B. Fountain S.J. Beech D.J. J. Physiol. (Lond.). 2004; 559: 739-750Crossref Scopus (114) Google Scholar). Although the extracellular effect of Ca2+ has been shown to be mediated by the acidic residues, Glu543, Glu595, and Glu598, located at the putative pore loop of the TRPC5 protein (7Jung S. Muhle A. Schaefer M. Strotmann R. Schultz G. Plant T.D. J. Biol. Chem. 2003; 278: 3562-3571Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar), the mechanism for the intracellular Ca2+ dependence of TRPC5 remains to be elucidated.Calmodulin (CaM) is a common intracellular mediator of many Ca2+-dependent regulations. All TRPC proteins possess a C-terminal CaM-binding domain that also interacts with an N-terminal sequence of the inositol 1,4,5-trisphosphate receptor (IP3R) (6Plant T.D. Schaefer M. Cell Calcium. 2003; 33: 441-450Crossref PubMed Scopus (135) Google Scholar). We have demonstrated that IP3R and CaM compete with each other for binding to the common CaM/IP3R-binding (CIRB) site of the TRPC. In functional studies, the TRPC-binding region of the IP3R activated and Ca2+/CaM inhibited the activation of TRPC3 and TRPC4. Moreover, TRPC channels were activated by removing or inactivating CaM from excised inside-out membrane patches, indicating that displacement of the inhibitory CaM from the common CIRB site is sufficient for the activation of TRPC channels (11Zhang Z. Tang J. Tikunova S. Johnson J.D. Chen Z. Qin N. Dietrich A. Stefani E. Birnbaumer L. Zhu M.X. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3168-3173Crossref PubMed Scopus (204) Google Scholar). Consistent with this, we have shown that CaM increased the delay between the release of Ca2+ from internal storage compartments and the activation of Ca2+ influx via endogenous TRPC1 channels in Chinese hamster ovary (CHO) cells, which were subjected to the regulation by IP3R and CaM in a similar fashion as the exogenously expressed TRPC3 and TRPC4 in human embryonic kidney (HEK) 293 cells (12Vaca L. Sampieri A. J. Biol. Chem. 2002; 277: 42178-42187Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Additional C-terminal CaM-binding domains outside of the CIRB sites have been found on TRPC proteins (6Plant T.D. Schaefer M. Cell Calcium. 2003; 33: 441-450Crossref PubMed Scopus (135) Google Scholar, 13Singh B.B. Liu X. Tang J. Zhu M.X. Ambudkar I.S. Mol. Cell. 2002; 9: 739-750Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Unlike the CIRB sites, these sites are not conserved among all TRPC channels, and they do not bind to IP3Rs. The second CaM-binding (CBII) site of TRPC1 has been shown to be involved in the slow Ca2+-induced channel inactivation (13Singh B.B. Liu X. Tang J. Zhu M.X. Ambudkar I.S. Mol. Cell. 2002; 9: 739-750Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar).In the present study we have identified the CBII site from mouse TRPC5 (mTRPC5). We have explored the specific roles of the CIRB and CBII sites in the modulation of channel activity after activation of receptors that stimulate the phosphoinositide signaling cascade by selectively disrupting each CaM-binding site from mTRPC5 and heterologous expression of the wild type and mutant channels in CHO and HEK293 cells. We show for the first time that intracellular application of CaM accelerated the activation of mTRPC5 by receptor agonists. Although mutations in the CIRB site rendered the channel inactive, deletion of the CBII site attenuated the Ca2+/CaM-induced acceleration of receptor-evoked mTRPC5 activation.EXPERIMENTAL PROCEDURESReagents and Solutions—All salts were of analytical grade purchased from Sigma. GTP and ATP were also obtained from Sigma. Bovine brain calmodulin, bradykinin, human thrombin, the CaM inhibitors N-aminohexyl-5-chloro-1-naphthalenesulfonamide (W7) and trifluoperazine dimaleate (TFP), and histamine were purchased from Calbiochem. The pipette (intracellular) solution contained (mm) the following: 130 potassium aspartate, 5 KCl, 10 EGTA or HEDTA, 7.39 (0.4 μm free, with EGTA), 5.07 (5 μm free with HEDTA), or 6.73 (10 μm free with HEDTA) CaCl2, 1 MgCl2, 1 ATP, 2 GTP, 10 HEPES, pH 7.2. Free pipette Ca2+ concentrations were calculated using the MaxChelator program (see below). The extracellular (bath) solution contained (mm) the following: 130 NaCl, 5 KCl, 1 CaCl2, 1.7 Mg2SO4, 5 glucose, 10 HEPES, pH 7.2.DNA Constructs and Mutagenesis—Complementary DNA for the open reading frame of mTRPC5 was subcloned in pIRESneo (Clontech, Mountain View, CA) vector. Mutageneses were performed using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Mutations were confirmed by DNA sequencing. Constructs for maltose-binding protein (MBP) fusion proteins that contained different fragments of mTRPC5 were made in the pAGA3 vector as described previously (14Tang J. Lin Y. Zhang Z. Tikunova S. Birnbaumer L. Zhu M.X. J. Biol. Chem. 2001; 276: 21303-21310Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). cDNA for the guinea pig histamine type 1 receptor (H1R) in pcDNA3 was kindly provided by Dr. Michael Schaefer (Charité-Universitätsmedizin Berlin) and that for the human type 2 bradykinin receptor (Bk2R) was a generous gift from Dr. William W. Schilling (Case Western Reserve University).Analysis of the Expression of mTRPC5 mRNA by RT-PCR—Total RNA was extracted from wild type cells and cells transfected with mTRPC5 and the mTRPC5 mutants using the TRIzol reagent (Invitrogen). Approximately 500 ng of total RNA was used as the template for RT-PCR, which was carried out using the ONEStep system with Superscript II (Invitrogen) following the manufacturer's protocols. The reaction was performed on a TC-512 thermal cycler (TECHNE, Cambridge, UK) using 30 cycles of the following PCR protocol: 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s. The primers used for mTRPC5 were 5′-CTATGAGACCAGAGCTATTGATG (forward) and 5′-CTACCAGGGAGATGACGTTGTATG (reverse), which amplify a 220-bp-long product. For glyceraldehyde-3-phosphate dehydrogenase, the primers used were 5′-GACATCAAGAAGCTGGTGAAGC (forward) and 5′-TACTCCTTGGAGGCCATGTAG (reverse), which amplify a 236-bp-long product. The PCR products were sequenced to identify the products, run on a 2% agarose gel, and stained with ethidium bromide. The gel was analyzed with a Typhoon 8600 Imager (Amersham Biosciences).CaM Binding Assay—Fragments of mTRPC5 fused to the C terminus of MBP were prepared by in vitro synthesis using the transcription- and translation-coupled rabbit reticulocyte lysates in the presence of [35S]Met and [35S]Cys as described previously (11Zhang Z. Tang J. Tikunova S. Johnson J.D. Chen Z. Qin N. Dietrich A. Stefani E. Birnbaumer L. Zhu M.X. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3168-3173Crossref PubMed Scopus (204) Google Scholar, 14Tang J. Lin Y. Zhang Z. Tikunova S. Birnbaumer L. Zhu M.X. J. Biol. Chem. 2001; 276: 21303-21310Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). The 35S-labeled proteins were incubated with CaM-Sepharose at room temperature for 30 min in a binding solution that contained 120 mm KCl, 1 mm CaCl2, 0.5% Lubrol, 20 mm Tris-HCl, pH 7.5. After several washes, bound proteins were separated by SDS-PAGE and then revealed by x-ray autoradiography as described (11Zhang Z. Tang J. Tikunova S. Johnson J.D. Chen Z. Qin N. Dietrich A. Stefani E. Birnbaumer L. Zhu M.X. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3168-3173Crossref PubMed Scopus (204) Google Scholar). For studying Ca2+ dependence of mTRPC5-CaM binding, 10 mm EGTA or HEDTA and the desired concentrations of CaCl2 (calculated using the MaxChelator program (www.stanford.edu/∼cpatton/maxc.html)) were included in the binding solution, and the apparent affinity (K1/2) for Ca2+ was determined as described (14Tang J. Lin Y. Zhang Z. Tikunova S. Birnbaumer L. Zhu M.X. J. Biol. Chem. 2001; 276: 21303-21310Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar).Cell Surface Biotinylation Assay—Transfection, biotinylation, and streptavidin precipitation were performed as described previously (15Wang C. Hu H.Z. Colton C.K. Wood J.D. Zhu M.X. J. Biol. Chem. 2004; 279: 37423-37430Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Immunoblotting was performed using anti-mTRPC5 antibodies (Alomone Labs, Jerusalem, Israel). Sample loading for the crude cell lysate and streptavidin precipitated portion represents 12 and 355 μg, respectively, of total proteins in cell lysates. Identical exposure time was used to reveal the chemiluminescent signals for the mTRPC5 proteins in crude lysates and streptavidin-precipitated samples.Fluorescence-based Membrane Potential Measurements—HEK293 cells were cotransfected with mTRPC5 and H1R in wells of a 96-well plate as described previously (16Hu H.Z. Gu Q. Wang C. Colton C.K. Tang J. Kinoshita-Kawada M. Lee L.Y. Wood J.D. Zhu M.X. J. Biol. Chem. 2004; 279: 35741-35748Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar). One day after the transfection, cells were washed once with Hanks' balanced salt solution and then incubated for 30 min with 80 μl of FLIPR membrane potential dye (Molecular Devices, Sunnyvale, CA) diluted in the Hanks' solution. Changes in membrane potential were measured at 32 °C using a fluid handling integrated fluorescence plate reader, FlexStation (Molecular Devices). Histamine was diluted in Hanks' solution at 300 μm, and 40 μl were delivered to the sample plate by the integrated robotic 8-channel pipettor at 20 s after readings began. Samples were excited at 530 nm, and emission of 565 nm was collected from the bottom of the plate at 0.67 Hz.Stable Cell Lines—HEK293 and CHO cells were transfected with bicistronic plasmids containing the wild type mTRPC5 or the mutants described in this study followed by an internal ribosome entry site and the gene conferring resistance to neomycin. Cells were maintained in 1 mg/ml G418 for 1 month and later assayed by RT-PCR to confirm the presence of the mRNA for mTRPC5 and aminoglycoside phosphotransferase. Stably transfected cell clones were isolated by progressive dilution and tested for mTRPC5 expression. Cells (both CHO and HEK293) were transfected also with a similar bicistronic plasmid containing the cDNA for Bk2R and the gene conferring resistance to puromycin. Clonal cell lines expressing both mTRPC5 and Bk2R were isolated using the two selection markers, neomycin and puromycin.Whole-cell Measurements of mTRPC5 Currents—The whole-cell configuration of the patch clamp technique was utilized to study mTRPC5-mediated currents as described previously (12Vaca L. Sampieri A. J. Biol. Chem. 2002; 277: 42178-42187Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Briefly, cells were plated on glass coverslips and mounted on the stage of an inverted microscope (Nikon Instruments, Japan). The amplifier used was the Axopatch 200A (Axon Instruments, Union City, CA). Pipette resistance was 10–12 megohms when tested with pipette and bath solutions. Whole-cell resistances were in the range of 1–2 gigohms, and cell capacitance ranged from 10 to 12 picofarads for CHO and 12 to 16 picofarads for HEK293 cells. Cells were held at the holding potential of –80 mV and repetitively stepped to –120 through +60 mV for 500 ms each in a 20-mV increment once every second. Receptor agonists were applied through bath perfusion, and CaM was introduced intracellularly by dialysis through the patch pipette.Data Analysis and Curve Fitting—All data were plotted and fitted using Sigmaplot 8 (SYSTAT, Point Richmond, CA). To obtain activation time constants, the mean outward current was plotted over time for the duration of the experiments under the different experimental conditions indicated in each figure legend. Data were fitted to sigmoidal Equation 1 as follows: f=a/(1+exp(-(x-x0)/b))(Eq. 1) where a indicates the maximum outward current value (top asymptote); b indicates the minimum outward current value (bottom asymptote); X0 indicates time constant for half-activation, and x indicates the time explored for each data point.RESULTSTime Course of the Activation of mTRPC5 by Agonists— Agonist stimulation of CHO cells stably expressing the wild type mTRPC5 channel results in a time-dependent activation of outwardly rectifying currents (Fig. 1). We followed current activation in the presence of agonists for 15 min (only the first 8 min are shown). The current reached a steady-state level after ∼7 min in the continuous presence of the agonists. After this period of time, 30% of the cells showed a small (about 5%) reduction of current amplitude. This phenomenon was not further studied in the present work.Fig. 1A shows a family of currents evoked by voltage pulses from a holding potential of –80 to –120 mV through +60 mV in 20-mV steps. Thrombin (1 unit) induced activation of an outwardly rectifying current with the reversal potential near 0 mV (–6 ± 4 mV, n = 25). The time course of the current-voltage (I-V) relationships is illustrated in Fig. 1B. The gray rectangle in Fig. 1B shows the limit of current induced by the agonist in cells not expressing mTRPC5. Notice that basal current level (before agonist stimulation) in mTRPC5-expressing cells was slightly higher when compared with wild type cells (∼10% more current). Although this current may reflect mTRPC5 channel activity, this was not further explored in the present study.To confirm the expression of mTRPC5, we performed RT-PCR experiments with total RNA obtained from control cells (wild type CHO cells) and cells expressing mTRPC5. The inset in Fig. 1B shows that that only mTRPC5-transfected cells contained the mTRPC5 transcript.The time course of mTRPC5 current activation was independent of the agonist and receptor utilized. Fig. 1 only illustrates the response to thrombin. We have compared the responses to thrombin and bradykinin (Bk) at concentrations that gave the maximal response (1 unit and 100 nm, respectively) in CHO cells stably expressing mTRPC5, and we obtained similar time constant values for half-activation of the outward current of 4.25 ± 0.4 and 4.01 ± 0.7 min, respectively.Calmodulin Accelerates the Time Course of the Activation of mTRPC5—In order to examine how CaM affects the activation of mTRPC5, we introduced CaM into mTRPC5-expressing cells by including the Ca2+-binding protein in the patch pipette. As illustrated in Fig. 2A, addition of CaM (10 μm) to the pipette resulted in a faster activation of mTRPC5 currents. Plotting the activation time courses for the outward currents in the absence (Fig. 2B, gray circles) and presence (black circles) of the exogenously applied CaM yielded activation curves that can be fitted with the sigmoidal equation as described under “Experimental Procedures.” The time constant for half-activation was reduced from 4.25 ± 0.4 min (n = 17) in the absence of exogenous CaM to 1.56 ± 0.85 min with 10 μm CaM in the pipette. This effect was obtained with thrombin and Bk in CHO cells. Similar results were found in HEK293 cells stimulated with Bk (cells expressing mTRPC5 and Bk2R, see under “Experimental Procedures”). Time constants for outward currents in the absence and presence of CaM were comparable across cell lines and agonists tested (Table I). Because inward currents were relatively small compared with the amplitude of outward currents, and because they were difficult to separate from endogenous inward currents (this was especially difficult with HEK293 cells that showed larger inward currents than CHO cells), all the analyses in this study were conducted only on the outward currents.Fig. 2CaM accelerates the agonist-stimulated activation of mTRPC5 currents. A, comparison of the time course of current activation in the presence (black) and absence (gray) of 10 μm CaM in the patch pipette. Current amplitudes at +60 (upward bars) and –120 mV (downward bars) are shown in averages ± S.D. from 17 cells. B, time course of outward current at +60 mV in the presence and absence of CaM. The solid lines represent the fit to a sigmoidal equation (“Experimental Procedures”) to obtain the half-activation time constants.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IEffect of CaM on TRPC5 activation (rate constants in minutes) by two agonists in CHO and HEK293 cellsConditionCHOHEK293Thrombin - CaM4.25 ± 0.4NTaNT indicates not tested.Bradykinin - CaM4.01 ± 0.74.29 ± 0.81Thrombin + CaM1.56 ± 0.85NTBradykinin + CaM1.75 ± 0.621.47 ± 0.74a NT indicates not tested. Open table in a new tab Identification of Two CaM-binding Sites at the C Terminus of mTRPC5—The above results suggest that CaM plays an important role in facilitating the receptor-induced activation of mTRPC5 channels. We have reported previously (14Tang J. Lin Y. Zhang Z. Tikunova S. Birnbaumer L. Zhu M.X. J. Biol. Chem. 2001; 276: 21303-21310Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar) the presence of a conserved common CIRB site at the near C-terminal regions of all TRPC proteins. For mTRPC5, the CIRB site lies in between Glu701 and Lys733 and requires higher Ca2+ concentrations (apparent K1/2 = 44.2 μm) for binding to CaM than the CIRB sites of other TRPC proteins. We also reported that the rest of the mTRPC5 C terminus downstream from the CIRB site (Gly762–Leu975, clone J1170 in Fig. 3A) bound to CaM as well. To narrow down the CBII site of mTRPC5, we have tested a large number of smaller fragments generated from J1170 by using an in vitro pull-down assay. As shown in Fig. 3, the minimal binding site for CaM is confined to a stretch of 27 residues, Pro828–Asn854, 95 amino acids downstream from the CIRB site. The isolated mTRPC5 CBII fragment bound to CaM in a Ca2+-dependent manner (Fig. 3D) with an apparent K for Ca2+ 1/2 of 3.1 ± 0.2 μm (n = 3). This value is lower than the previously determined K1/2 value (44.2 μm) of Ca2+ for CaM binding to the mTRPC5 CIRB site but within the range of those values (1.6–12.9 μm) for the CIRB sites of other TRPC isoforms (14Tang J. Lin Y. Zhang Z. Tikunova S. Birnbaumer L. Zhu M.X. J. Biol. Chem. 2001; 276: 21303-21310Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar).Fig. 3Identification of a second CaM-binding site on the C terminus of mTRPC5. A, diagram of mTRPC5 and its fragments included in the MBP fusion proteins for the in vitro binding experiments. S1–S6 indicates the six transmembrane segments. White, gray, and black bars denote no, weak, and strong binding, respectively, to CaM-Sepharose. The names and positions, indicated by the numbers in parentheses, of the fragments are labeled either above or at right. Shaded areas indicate the CIRB and CBII sites. B and C, representative binding results showing the autoradiograms of input 35S-labeled MBP and MBP fusion proteins containing the mTRPC5 fragments and those retained by CaM-Sepharose and glutathione S-transferase bound to glutathione-Sepharose (negative controls). The binding buffer contained 1 mm Ca2+. Input represents 40% of 35S-labeled protein added to the binding assay. D, Ca2+ dependence of the interaction between mTRPC5 CBII and CaM. Ca2+ was buffered by EGTA or HEDTA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Mutations on CaM-binding Sites Alter mTRPC5 Channel Activation—To investigate the function for each of the CaM-binding sites of mTRPC5, we destroyed the CIRB and CBII sites by substituting Arg718, Lys722, and Arg723 with alanines (CIRBm1) and deleting Pro828–Asn854 (ΔCBII), respectively (Fig. 4A). The loss of CaM binding in these mutants was confirmed by in vitro binding studies (Fig. 4B). Receptor-mediated activation of mTRPC5 currents was studied after coexpression of the full-length clones of the mutated mTRPC5 with Bk2R or by stimulation of the endogenous thrombin receptor in CHO. As shown in Fig. 4C, without CaM in the pipette, the activation of mTRPC5ΔCBII was slightly delayed as compared with the wild type mTRPC5. Inclusion of 10 μm CaM in the pipette only weakly accelerated the activation of the mutant channel, indicating that CBII is important for the Ca2+/CaM-mediated facilitation of mTRPC5 activation. After fitting the outward current, the time constants for mTRPC5ΔCBII of 6.04 ± 0.5 (control) and 5.48 ± 0.7 min (with 10 μm CaM) were obtained.Fig. 4Deletion of CBII site attenuated the Ca2+/CaM-induced facilitation of receptor-induced mTRPC5 channel activation. A, diagram showing the amino acid substitutions in mTRPC5-CIRBm1 and the deletion in mTRPC5ΔCBII. B, in vitro binding results showing that the mutations abolished the binding to Ca2+/CaM of the mTRPC5 fragments. Clone names are shown in Fig. 3A. Equivalent regions from the mutants were tested under the same condition as described in Fig. 3. wt, wild type. C, time course of the current activation of wild type mTRPC5 (squares) and the mutants mTRPC5ΔCBII (circles) and mTRPC5-CIRBm1 (triangles) in the presence or absence of 10 μm CaM. Currents were induced by the application of 100 nm Bk. The outward current was measured by a voltage pulse to +60 mV as described under “Experimental Procedures.” D, histamine-induced membrane depolarization in HEK293 cells that coexpressed H1R and mTRPC5 or its mutant. 100 μm histamine was added as indicated. Fluorescence values shown are 1/100,000 of those displayed by the instrument. E, biotinylation experiments illustrating that all mutants reached the plasma membrane similarly to wild type mTRPC5. Actin was used as loading control on each lane. kD illustrates the molecular mass markers (in kilodaltons).View Large Image Figure ViewerDownload Hi-res image Download (PPT)In contrast, the mTRPC5-CIRBm1 mutant failed to respond to the stimulation by thrombin and Bk in both CHO and HEK293 cells. In a fluorescence-based membrane potential assay using the FLIPR membrane potential dye, coexpression of mTRPC5 with H1R in HEK293 cells resulted in a histamine-evoked membrane depolarization (Fig. 4D). Although cells expressing mTRPC5ΔCBII showed a" @default.
• W1988673487 created "2016-06-24" @default.
• W1988673487 creator A5008927356 @default.
• W1988673487 creator A5011922155 @default.
• W1988673487 creator A5012607889 @default.
• W1988673487 creator A5048083035 @default.
• W1988673487 creator A5056672870 @default.
• W1988673487 creator A5077216733 @default.
• W1988673487 creator A5084786673 @default.
• W1988673487 date "2005-09-01" @default.
• W1988673487 modified "2023-10-18" @default.
• W1988673487 title "Calmodulin and Calcium Interplay in the Modulation of TRPC5 Channel Activity" @default.
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