interplay between calmodulin and phosphatidylinositol 4,5

Interplay between Calmodulin and Phosphatidylinositol4,5-Bisphosphate in Ca2�-induced Inactivation of TransientReceptor Potential Vanilloid 6 Channels*

Received for publication, August 9, 2012, and in revised form, January 4, 2013 Published, JBC Papers in Press, January 8, 2013, DOI 10.1074/jbc.M112.409482

Chike Cao, Eleonora Zakharian1, Istvan Borbiro, and Tibor Rohacs2

From the Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey, New Jersey MedicalSchool, Newark, New Jersey 07103

Background: A large number of calmodulin-binding sites have been proposed in TRPV6.Results:Wehave identified the site that is responsible for inhibition of TRPV6 by calmodulin in excised inside-out patch clampexperiments.Conclusion: Calmodulin and PI(4,5)P2 antagonistically regulate TRPV6, but not through direct competition.Significance: This study provides mechanistic insight into Ca2�-induced inactivation of TRPV6.

The epithelial Ca2� channel transient receptor potentialvanilloid 6 (TRPV6) undergoes Ca2�-induced inactivation thatprotects the cell from toxic Ca2� overload and may also limitintestinal Ca2� transport. To dissect the roles of individual sig-naling pathways in this phenomenon, we studied the effects ofCa2�, calmodulin (CaM), and phosphatidylinositol 4,5-bispho-sphate (PI(4,5)P2) in excised inside-out patches. The activity ofTRPV6 strictly depended on the presence of PI(4,5)P2, andCa2�-CaM inhibited the channel at physiologically relevantconcentrations. Ca2� alone also inhibited TRPV6 at high con-centrations (IC50 � �20 �M). A double mutation in the distalC-terminal CaM-binding site of TRPV6 (W695A/R699E)essentially eliminated inhibition by CaM in excised patches. Inwhole cell patch clamp experiments, this mutation reduced butdid not eliminate Ca2�-induced inactivation. Providing excessPI(4,5)P2 reduced the inhibition by CaM in excised patches andin planar lipid bilayers, but PI(4,5)P2 did not inhibit binding ofCaM to the C terminus of the channel. Overall, our data show acomplex interplay between CaM and PI(4,5)P2 and show thatCa2�, CaM, and the depletion of PI(4,5)P2 all contribute to inac-tivation of TRPV6.

The epithelial Ca2� channel TRPV6 is a member of the tran-sient receptor potential (TRP)3 superfamily of ion channels.TRPV6 is a Ca2�-selective inwardly rectifying channelexpressed in the apical membrane of duodenal epithelial cells,where it is thought to be responsible for Ca2� entry from the

lumen of the intestine (1). TRPV6 expression in the duodenumis regulated at the transcription level by active vitamin D. Cal-cium entering through this channel is pumped out on the baso-lateral side of the cell by the plasma membrane Ca2�-ATPase,leading to vectorial transport of Ca2� from the lumen of theintestine to the intersitium then to the blood. TRPV6 is alsoexpressed in several other tissues, including the placenta andepididymal epithelium. Consistent with the latter, male TRPV6mice carrying the D541A mutation that renders the channelnonfunctional show severely reduced fertility (2). TRPV5, aclose homologue of TRPV6, is expressed in the kidney, where itplays an important role in Ca2� reabsorption in the distal con-voluted tubule (1). Both these channels are constitutively activeandundergoCa2�-induced inactivation (3), which is thought toprotect cells from toxic Ca2� levels, and it was also proposed tolimit intestinal Ca2� transport (4).

The membrane phospholipid phosphatidylinositol 4,5-bis-phophate (PI(4,5)P2) commonly known as PIP2, is a generalregulator of many ion channels (5–7), including TRP channels(8, 9). The activity of TRPV5 and TRPV6 depends on the pres-ence of PI(4,5)P2 (10–13). It was proposed that Ca2�-inducedinactivation of TRPV6 proceeds through Ca2� influx activatingphospholipase C and depletion of PI(4,5)P2 (4).The ubiquitous Ca2� sensor calmodulin (CaM) has also been

proposed to play a role in the Ca2�-induced inactivation ofTRPV6 (14, 15) and TRPV5 (16). A distal C-terminal bindingsite was implicated in this phenomenon both for TRPV6 (14,15) and for TRPV5 (16). Removal of that binding site, however,only resulted in a partial inhibition of Ca2�-induced inactiva-tion in whole cell patch clamp experiments. A large number ofadditional binding sites in the C and N termini, as well as in theintertransmembrane loops, have also been described (17–19),and it is unclear whether any of those sites contribute to thephysiological effects of CaM. All studies so far relied on thewhole cell patch clamp technique to study the effects of CaMon TRPV6 or TRPV5, where the concentration of CaM can-not be controlled, nor can the effects of Ca2� and CaM bedifferentiated.

* This work was supported, in whole or in part, by National Institutes of HealthGrants NS055159 and GM093290 (to T. R.) and GM098052 (to E. Z.). Thiswork was also supported by a grant from the University of Medicine andDentistry of New Jersey Foundation (to T. R.).

1 Present address: Dept. of Cancer Biology & Pharmacology, University of Illi-nois College of Medicine Peoria, IL 61605.

2 To whom correspondence should be addressed. Tel.: 973-972-4464; Fax:973-972-7950; E-mail: [email protected].

3 The abbreviations used are: TRP, transient receptor potential; TRPV6, TRPvanilloid 6; HEDTA, N-(2Hydroxyethyl)ethylenediamine-N,N�,N�-triaceticacid; hTRPV6, human TRP vanilloid 6; PI(4,5)P2, phosphatidylinositol 4,5-bisphophate; AASt, arachydonyl and stearyl side chains; MBP, maltose-binding protein.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 8, pp. 5278 –5290, February 22, 2013© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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A recent study proposed that CaM and phosphoinositidesbind to overlapping binding sites in a large number of TRPchannels and that these signaling molecules generally affectTRP channels by physically displacing each other from theirrespective binding sites (20). Again this study was based onwhole cell patch clamp experiments, and the direct effects ofCaM and PI(4,5)P2 were not tested in excised patches.Here we have reconstituted and characterized the effect of

CaM in excised inside-out patches on TRPV6. This techniqueallows the dissection of the effects of Ca2� and CaM and canunequivocally identify the site responsible for the inhibitoryeffect of CaM. This technique is also largely devoid of second-ary effects through various cellular components, which arepresent in whole cell patch clamp experiments, and thus allowsus to examine the effects of phosphoinositides and CaM inisolation.We found that CaM inhibited TRPV6 in excised patches in a

concentration-dependent manner in the presence of 3 �M

Ca2�. CaM in the absence of Ca2� did not inhibit TRPV6,whereas Ca2� alone at higher concentrations also inhibitedTRPV6 activity (IC50 � �20 �M). We also found that the full-length protein and the purified C terminus, but not the N ter-minus, of TRPV6 binds to CaM in the presence of Ca2�. CaMbinding to the full-length proteinwas eliminated by a combinedmutation of Trp-695 and Arg-699 residues in the distal C-ter-minal CaM-binding site of TRPV6. In excised patches, thismutant was essentially not inhibited by CaM; in whole cellpatch clamp experiments, Ca2�-induced inactivation wasreduced but not eliminated. The inhibitory effect of CaM onwild-type TRPV6 in excised patches and planar lipid bilayerswas reduced by higher PI(4,5)P2 concentrations, but we havenot observed direct competition between PI(4,5)P2 and CaM inbiochemical binding experiments. Our data show complex reg-ulation of TRPV6 activity by the interplay between PI(4,5)P2,Ca2�, and CaM.

EXPERIMENTAL PROCEDURES

Reagents—Natural long acyl chain PI(4,5)P2, purified fromporcine brain, mainly containing arachydonyl and stearyl sidechains (AASt) (Avanti Polar Lipids), was dissolved in water (1mM stock), followed by 5 min of sonication; then it was ali-quoted and stored at�80 °C.Working solutions were prepareddaily by dilution of stock aliquots followed by sonication for 5min. DiC8 PI(4,5)P2 (Cayman Chemical) was dissolved in water(2.5 mM), aliquoted, and stored at �80 °C. Working solutionswere diluted from the stock on the day of the experiments. ATP(as Na2ATP; Sigma-Aldrich) was dissolved in the perfusionsolution to the final concentration of 2 mM on the day of exper-iments. After the addition of 2 mM MgCl2, the pH values of thesolutions were adjusted to 7.4 before use. Calmodulin purifiedfrom bovine testes, purity of �98% (Sigma-Aldrich) was dis-solved as a 100 �M stock in Cl�-free bath solution (see below),aliquoted, stored at�80 °C, and diluted in theworking solutionon the day of the experiments.Molecular Biology and Expression Vectors—For mammalian

expression, the coding region of humanTRPV6 (hTRPV6) sub-cloned into pCMV-tag3A (Stratagene) was used (21). Thisresulted in a c-Myc epitope tag on the N terminus, and we used

this tag for TRPV6 detection. For oocyte expression, thehTRPV6 was subcloned into the pGEMSH vector. ThepGEMSH-TRPV6 cDNA was linearized and purified usingthe QIAquick PCR purification kit (Qiagen). cRNA of hTRPV6was in vitro transcribed using the mMESSAGE mMACHINEkit (Ambion). For bacterial expression, cDNA fragmentsencoding hTRPV6 wild-type C terminus (residues 579–725),truncated C terminus (residues 579–694), and the wild-type Nterminus (residues 1–326) were amplified by PCR and sub-cloned into pMAL-c4x (New England Biolabs) using therestriction enzymes XbaI and HindIII. The cloned gene wasinserted downstream from the malE gene of Escherichia coli,which encoded the maltose-binding protein (MBP). Thisresulted in the expression of MBP fusion proteins. All of theconstructs were confirmed by DNA sequencing. Mutationswere introduced with the QuikChange site-directed mutagen-esis kit (Stratagene).Xenopus Oocyte Electrophysiology—The oocytes were

extracted from mature female Xenopus laevis frogs (XenopusExpress) and digested with 0.2 mg/ml collagenase (Sigma) inOR2 solution (82.5mMNaCl, 2mMKCl, 1mMMgCl2, and 5mM

HEPES, pH 7.4) for �16 h at 18 °C. Defolliculated oocytes wereselected and then maintained in OR2 solution plus 1.8 mM

CaCl2 and 1% penicillin/streptomycin (Mediatech) at 18 °C.cRNA (20 ng) wasmicroinjected into each oocyte using a nano-liter injector system (World Precision Instruments). The exper-iments were performed 72 h after injection.Excised inside-outmacropatch experiments were performed

with borosilicate glass pipettes (World Precision Instruments)of 0.8–1.7 megaohm resistance. The electrode pipette solutioncontained 96 mM LiCl, 1 mM EGTA, and 5 mM HEPES, pH 7.4.For the measurements shown in Fig. 2, the electrode pipettewas first filled half with Cl�-free bath solution containing 93mMpotassiumgluconate, 5mMHEDTA, 5mMHEPES, with thepH adjusted to 7.4; and the other half of the pipette was filledwith Cl�-containing pipette solution (22). After establishinggiagohm resistance seals on devitellinizedXenopus oocytes, thecurrents were measured using an Axopath 200B amplifier(Molecular Devices). For the measurement in Fig. 2, we used aramp protocol from �100 to � 100 mV (0.25 mV/ms), imme-diately preceded by a 100-ms step to �100 mV. The protocolwas applied every second; holding potential was 0 mV. For allother measurements, we used a ramp protocol from �103to �100 mV, performed once a second, immediately precededby a 100-ms step to �103 mV. The perfusion solution con-tained 93 mM potassium gluconate, 5 mM HEDTA, 5 mM

HEPES, with the pH adjusted to 7.4. To obtain various freeCa2� concentrations, we added the appropriate amount ofCa2� in the gluconate and/or HEDTA solution as calculated bythe Maxchelator (WinMaxC32 2.50, Stanford University) pro-gram (23). Ca2� at 1, 3, and 10 �M was buffered with HEDTA,and Ca2� at 30 and 100 �M was not buffered with HEDTA. Ithas been previously shown that gluconate is aweakCa2� buffer,with a Kd of �20 mM (22). Thus in the presence of 5 mM

HEDTA in the 93 mM gluconate solution, 1.24 mM Ca2� wasadded to obtain 1 �M free Ca2�, 2.5 mM to obtain 3 �M freeCa2�, and 3.9 mM to obtain 10 �M free Ca2�. In the 93 mM

gluconate solutionwithoutHEDTA, 174�MCa2�was added to

Calmodulin Regulation of TRPV6 in Excised Patches

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obtain 30�M free Ca2� and 579�M to obtain 100�M free Ca2�.For these measurements, the bath was connected with theground electrode through an agar bridge.Mammalian Electrophysiology—Whole cell patch clamp

experiments were performed as described earlier (4, 10).Briefly, human embryonic kidney (HEK293) cells were trans-fected with either the wild-type ormutant TRPV6 andGFP as atransfection marker, using the Effectene transfection reagent.Recordings were performed 36–72 h post-transfection onHEK293 cells in an extracellular solution containing 137 mM

NaCl, 5 mM KCl, 10 mM glucose, 10 mM HEPES, with the pHadjusted to 7.4, to which 1 mM MgCl2, 2 or 10 mM CaCl2, or 2mM EGTA was added, depending on the experimental condi-tions (see further details in the figure legends). Borosilicateglass pipettes (Sutter Instruments) of 2–4-megaohm resistancewere filled with a solution containing 135 mM potassium-glu-conate, 5mMKCl, 5mMEGTA, 1mMMgCl2, 2mMNa2ATP, 10mM HEPES, with the pH adjusted to 7.2. The cells were kept inextracellular solution containing 1 mM Mg2� but no Ca2� for20 min before measurements. After formation of gigaohmresistance seals, whole cell configuration was established, andcurrents were measured using an Axopatch 200B amplifier(MolecularDevices). The datawere collected and analyzedwiththe pCLAMP 9.0 software (Molecular Devices). All of the mea-surements were performed at room temperature (20–25 °C).Planar Lipid Bilayer Experiments—The TRPV6 protein was

purified from HEK293 cells with anti-Myc beads as describedearlier (12). Planar lipid bilayers were formed from a solution ofsynthetic 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine and1-palmitoyl-2-oleoyl-glycero-3-phosphoethanolamine; bothfrom Avanti Polar Lipids, in a 3:1 ratio in n-decane (Sigma-Aldrich). The solutionwas used to paint a bilayer in an apertureof �150-�m diameter in a Delrin cup (Warner Instruments)between symmetric solutions of 150 mM KCl, 0.02 mM MgCl2,and 20 mM HEPES (pH 7.4) at 22 °C. Bilayer capacitances werein the range of 50–75 picofarad. After the bilayers were formed,0.2�l of theTRPV6micellar solution (0.02�g/ml)was added tothe cis compartment with gentle stirring. Currents wererecorded with an Axopatch 200B amplifier. The data were col-lected and analyzed with the pCLAMP 9.0 software (MolecularDevices).Expression and Purification of MBP Fusions from Bacteria—

Asingle colony ofE. coliBL21 (DE3) transformedwith theMBPfusion constructs was inoculated into 30ml of LBmedium con-taining 150 �g/ml ampicillin and 20mM glucose and incubatedwith 250 rpm shaking at 37 °C overnight. The culture was usedto inoculate 500 ml of LB containing 150 �g/ml ampicillin and20 mM glucose. When A600 reached to �0.5, isopropyl-�-D-thiogalactopyranoside (Roche Applied Science) at 0.5 mM wasadded, and the culture was shaken continuously at 37 °C for anadditional 3 h, or at 28 °C for 16 h. The cells were harvested bycentrifugation at 5000 � g for 20 min and stored at �20 °Cbefore use. A cell pellet from 50 ml of the culture was resus-pended in 25 ml of column buffer containing 20 mM Tris, pH7.4, 500 mM NaCl, 4 mM EDTA, and 15% glycerol with theaddition of 1mMof the protease inhibitor PMSF, 20mM �-mer-captoethanol, and 2 �g/ml lysozyme. The cells were then lysedby sonication twice, each time for 1 min followed by 1-min

incubations on ice. The lysate was centrifuged at 18,000 � g for45 min. The supernatant was transferred to a fresh 50-ml cen-trifuge tube with 1 ml of amylase resin, freshly washed with0.1% SDS, and equilibrated with column buffer. Ethanol (5%)was added to increase the binding efficiency. After 1 h of incu-bation at 4 °C with gentle shaking, the beads were washed threetimes with 10 ml of column buffer and one time with 10 ml ofcolumn buffer in the presence of 1 �M maltose. MBP fusionproteins were then elutedwith column buffer in the presence of20 �M maltose and stored at 4 °C for use in a week. Proteinconcentrations were determined using the Bio-Rad proteinassay. To detect the protein purity, freshly eluted proteins wereelectrophoretically separated on 10% SDS-PAGE gel (Bio-Rad)using Tris-glycine SDS buffer (Bio-Rad) at a constant voltage of194 volts. The protein bands were visualized by staining withCoomassie G-250 (Bio-Rad).Expression and Preparation of Full-length TRPV6 Protein

fromHEK293Cells—HEK293 cellsweremaintained inminimalessential medium with 10% fetal bovine serum (HyClone) and1% penicillin/streptomycin (Mediatech) in a humidified, 5%CO2 incubator at 37 °C. Wild-type or mutant hTRPV6 inpCMVtag3A vector was used for full-length TRPV6 proteinexpression. The cells were transfected using the Effectenetransfection reagent (Qiagen) with 1.7 �g of DNA in each100-mm culture dish. The cells were washed and collected inPBS�40 h after transfection and stored at�80 °C before use. Acell pellet collected from three transfected 100-mm dishes wasresuspended in 5ml of NCB buffer containing 500mMNaCl, 50mM NaH2PO4, 20 mM HEPES, and 10% glycerol, pH 7.5, withthe addition of 1 mM of the protease inhibitor PMSF, 1 mM

CaCl2, and 5mM �-mercaptoethanol. Then the cells were lysedby the freeze-thawingmethod and centrifuged at 40,000� g for2.5 h, separating cytosolic proteins from membrane proteins.The pellet (crudemembrane fraction) was then resuspended in1 ml of NCB buffer containing half tablet of the protease inhib-itor mixture (Roche Applied Science), 15 �l of prote-CEASETM-50 (G-Biosciences), 1% Nonidet P-40 (RocheApplied Science), and 0.1% Triton X-100 (Sigma) overnight at4 °C and cleared by centrifugation. The supernatant (solubi-lized crude membrane fractions) was stored at 4 °C before use.CaM-Sepharose Pulldown Assay—For the CaM pulldown

assay with MBP fusion proteins, 60 �l of CaM-Sepharose 4B(GEHealthcare) slurry was used that was prewashed with CaMbinding buffer (50 mM Tris, pH 7.5, 250 mM NaCl, 0.2% TritonX-100) in the presence of 2 mM EGTA and then equilibrated inthe CaM binding buffer with various Ca2� concentrations. 80ng of freshly purified MBP fusion proteins was incubated withthe beads in 300 �l of CaM binding buffer at 4 °C for 1 h. Afterseveral washings, the bound fusion proteinswere elutedwith 80�l of 2� SDS sample buffer and boiled for 10 min. 20 �l of thebound proteins was fractionated with 10% SDS-PAGE gels andtransferred onto PVDF membranes (Bio-Rad) in an IDEA Sci-entific (Minneapolis,MN) cell filledwith blotting buffer (25mM

Tris, 192mM glycine, and 20%methanol) at 12 volts for 40min.The membranes were blocked in 5% nonfat dry milk (Bio-Rad)in 1� Tris-buffered saline/Tween 20 and probed with HRP-conjugated anti-MBP monoclonal antibody (1:3500; New Eng-land Biolabs) overnight at 4 °C. After thorough washings, pro-




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tein signals were detected using Supersignal West PicoChemiluminescent Substrate (Thermo Scientific) with Pre-miumAutoradiography films (Denville Scientific). For studyingCa2� dependence of hTRPV6 and CaM interaction, 2 mM

EGTA or the desired concentrations of CaCl2 were included inthe CaM binding buffer. For detecting competition betweenCa2�-CaM and PI(4,5)P2, 80 ng of MBP fusion proteins werefirst incubated with the desired concentrations of PI(4,5)P2overnight at 4 °C with gentle shaking before loading onto theCaM-Sepharose beads.For the CaMpulldown assay with theMyc-tagged full-length

TRPV6 protein, 100 �l of freshly prepared, solubilized crudemembrane fractions were loaded on 100 �l of CaM-Sepharoseslurry equilibrated with NCB buffer in the presence of 20 �M

Ca2�. After incubation on ice for 2.5 h or at 4 °C for overnightwith gentle shaking, the beadswerewashed four times in 1ml ofNCB buffer containing 1% Nonidet P-40 and 0.1% TritonX-100. The bound proteins were eluted with 60 �l of 2� SDSsample buffer and boiled for 10 min. 30 �l of each sample wasfractionated by 10% SDS-PAGE and transferred onto PVDFmembranes in an IDEA Scientific cell filled with blotting buffer(25mMTris, 192mM glycine, 0.03% SDS, and 10%methanol) at22 volts for 1.5 h. Membranes were blocked with 1.2% BSA(Sigma-Aldrich) in 1� Tris-buffered saline/Tween 20 andprobed with monoclonal anti-c-Myc antibody (1:3000; Sigma-Aldrich) overnight at 4 °C. After thorough washings, the blotswere incubated in HRP-conjugated goat/anti-mouse IgG(1:5000; PerkinElmer Life Sciences), followed bywashings. Pro-tein signals were detected using chemiluminescent reagents.

On the Western blots, most of the full-length TRPV6 proteinwas detected at molecular masses higher than the predictedmolecularmass of themonomer (83.2 kDa), representing eitherthe tetramers, or other oligomers, consistentwith earlier results(24). No signal was observed at any molecular mass using theanti-Myc antibody using HEK cells not transfected with Myc-tagged TRPV6.Statistics—The mean � S.E. is shown in the figures. Statisti-

cal significance was calculated using t tests.

RESULTS

We have studied the effects of Ca2� and CaM on the activityof the human TRPV6 in large excised inside-out patches fromXenopus oocytes expressing this channel (Fig. 1). The oocyteshave a large endogenous Ca2�-activated Cl� current; thus weused a gluconate-based Cl�-free intracellular solution to avoidcontamination of TRPV6 currents with the Cl� current whenCa2� was applied. TRPV6 currents were measured at �103mV, and we also monitored the Cl� current at �100 mV inmost measurements (10). Fig. 1A shows that in noninjectedoocytes, application of 3 �M Ca2� to the inner surface of thepatch membrane induced a large apparent outward current at�100 mV, which corresponds to the inward Cl� current, andessentially no current at �103 mV.In TRPV6-expressing oocytes (Fig. 1, B and D), a large

inwardly rectifying TRPV6 current was detected at �103 mV,and only minimal current was detected at �100 mV in theabsence of Ca2�, consistent with TRPV6 being an inwardly rec-tifying channel. Upon excision, TRPV6 activity decreased (run-

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FIGURE 1. Ca2�-CaM inhibits TRPV6 activity in excised inside-out macropatches. Measurements on TRPV6-expressing and noninjected oocytes wereperformed as described under “Experimental Procedures.” Currents are shown at �103 mV (lower traces, Li� current through TRPV6) and at �100 mV (uppertraces, Cl� current through the endogenous Ca2�-activated Cl� channels). A, representative current traces in response to 3 �M Ca2� in a noninjected oocyte.B, representative trace for a TRPV6-expressing oocyte, the applications of 25 �M diC8 PI(4,5)P2, 3 �M Ca2�, 0.5 �M CaM in the presence of 3 �M Ca2�, and 0.5 �M

CaM alone are indicated by the horizontal lines. C, summary of the effects of Ca2�, Ca2�-CaM, and CaM in the absence of Ca2� (n � 6). D, representative tracesfor the effect Ca2�-CaM in a patch where rundown of TRPV6 currents was very slow. E, summary of the time course of inhibition without application ofexogenous PI(4,5)P2 (endo PIP2) and on currents stimulated by diC8 PI(4,5)P2 from five experiments.




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down) to a variable extent; Fig. 1B shows an example of a typicalfast rundown, which is due to the dephosphorylation ofPI(4,5)P2 by lipid phosphatases in the patch membrane (12). Insome patches rundown was much slower, probably because ofthe lower phosphatase activity in the patch membrane. Fig. 1Dshows an example, where essentially no rundown was observedfor �100 s; thus the effects of Ca2� and CaM could be testedwithout the application of exogenous PI(4,5)P2. When weapplied 3 �M Ca2�, a large current appeared at �100 mV, cor-responding to the Ca2� activated Cl� current. At �103 mV, asmall inhibition of TRPV6 was observed. When CaM wasapplied in the presence of 3 �M Ca2�, we observed an almostcomplete inhibition and partial recovery of current activityuponwashout of CaM, followed by rundown of channel activity(Fig. 1D). Fig. 1E summarizes five measurements, where run-down was sufficiently slow to be able to quantify the inhibitionby CaM.Rundown in most patches was fast, however, making it diffi-

cult to test the effects of CaM.To induce stable channel activity,we reactivated TRPV6 in most experiments using the watersoluble diC8 PI(4,5)P2. When applied in the presence of 25 �M

diC8 PI(4,5)P2, 0.5 �M CaM induced an almost complete inhi-bition of TRPV6 activity in the presence of 3 �MCa2� (Fig. 1, Band D). No inhibition was observed by CaM in the absence ofCa2� (Fig. 1, B andC). It is noteworthy that the Ca2�-inducedCl� current was also partially inhibited by CaM (Fig. 1, Band D).Ca2� at 3 �M fully saturates CaM, and this concentration is

probably higher than maximal bulk cytoplasmic Ca2� concen-trations even in stimulated cells. Close to the pore of Ca2�-permeable ion channels, however, local Ca2� concentrationscan reach hundreds of micromolars (25). Thus we testedwhether Ca2� alone exerts substantial inhibition on TRPV6 athigher concentrations. Fig. 2 shows the concentration depend-ence of TRPV6 inhibition by Ca2� applied to excised patches.TRPV6 was almost fully inhibited by 100 �M Ca2�, and half-maximal inhibition was observed at �20 �M. These measure-ments were performed using symmetrical Cl�-free solutions,to avoid any contamination by the Cl� currents, (see “Experi-mental Procedures” for further details). Inhibition by Ca2�

alone was almost instantaneous as opposed to the relatively

slowly developing inhibition by CaM. Similarly currents recov-ered immediately upon returning to Ca2�-free perfusion solu-tion, but the effect of CaM washed out much more slowly.Overall, these data show that the effects of Ca2�-CaM and thatof Ca2� alone have different characteristics and can be dis-sected in excised inside-out patches.In some patches, such as the measurement shown in Fig. 2,

current amplitudes evoked by consecutive applications of diC8PI(4,5)P2 decreased over time. We have noticed this earlierboth with TRPV6 (10) and other PI(4,5)P2-sensitive ion chan-nels such as Kir2.1 (26) and TRPM8 (11). This PI(4,5)P2-inde-pendent rundown is quite variable; it is not seen in all patches,its mechanism is unknown, and we have not investigated itfurther. To avoid overestimating the effect of Ca2� and otherinhibitory compounds caused by this decrease, we alwaysincluded a control period before testing the next concentrationof either Ca2� or Ca2�-CaM (see below).

Multiple regions have been suggested in the TRPV6 proteinto bindCaM. Fig. 3A shows the seven different putative bindingsites identified in various articles (14–19). To evaluate whichregion is responsible for the inhibition by CaM, we haveexpressed and purified the C- and N-terminal cytoplasmicregions of TRPV6 in bacteria, using a MBP tag. We measuredthe binding of the isolated cytoplasmic domains to CaM-sep-harose beads and detected the fragments with anti-MBP anti-bodies. Fig. 3B shows that the cytoplasmic C terminus (residues579–725) showed a strong binding to CaM beads, whereas theN terminus (residues 1–326) showed no binding. Binding of theC terminus depended on Ca2�. We focused then on the verydistal C-terminal region in the human TRPV6 (14) (15), whichwas also demonstrated in the closely related TRPV5 to bindCaM (16). Removing the distal part of the C terminus (�694–725) eliminated CaM binding (Fig. 3B).We have also deleted this region from the full-length TRPV6,

expressed the mutant and wild-type protein in mammaliancells, and measured binding to CaM. Fig. 3C shows that thefull-length wild-type TRPV6 displayed robust binding to CaM,but the truncated (�694–725) TRPV6 did not bind CaM at all.The equivalent of this region in TRPV5 was further analyzed ina study by de Groot et al. (16), who identified individual aminoacids responsible for CaM binding in TRPV5 with NMR spec-

FIGURE 2. Concentration dependence of the Ca2� inhibition of TRPV6 activity in excised inside-out macropatches. A, representative trace for theapplication of 3, 30, and 100 �M free Ca2� on wild-type TRPV6 activity induced by 25 �M diC8 PI(4,5)P2. This experiment was performed in symmetrical Cl�-freeconditions, as described under “Experimental Procedures”; traces at �100 and �100 mV are shown. Note the absence of the Cl� current. B, summary of thedata (n � 5–18). The IC50 for Ca2� inhibition is 20.8 �M, and the Hill coefficient is 1.01.




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troscopy. We have introduced into the human TRPV6 theequivalent mutations to the two residues that had the mostdramatic effect in TRPV5 (Fig. 4A). We then tested the effectsof this doublemutation on CaMbinding. Fig. 4B shows that theW695A/R699E double mutant of the C terminus of TRPV6failed to bind CaM. The same mutations also eliminated bind-ing of the full-length TRPV6 to CaM beads (Fig. 4C). Fig. 4 (Dand E) shows that 0.5 �M CaM did not inhibit the W695A/R699E either in the presence or absence of 3�MCa2� in excisedpatches.Next we compared the effects of CaM in excised patches on

the mutant and wild-type channels in more detail. Fig. 5 showsthat the wild-type channel is inhibited in a dose-dependentmanner by CaM in the presence of 3 �M Ca2�. In our hands 50nM CaM still almost completely inhibited TRPV6 activity, eventhough its effect developed much more slowly than those ofhigher concentrations (Fig. 5,A,C, andD). It was estimated thatfree Ca2�-CaM concentration in cells reaches up to 45 nM (27);thus the inhibition we observe in excised patches is likely to bephysiologically relevant. When the same concentrations ofCaM were tested on the W695A/R699E double mutant, weobserved essentially no inhibition at lower CaM concentra-

tions, andmuch slower and smaller effects than in thewild-typechannel at higher CaM concentrations (Fig. 5, B and C). Thesedata indicate that the inhibitory effect of CaM in the physiolog-ical range is almost exclusively due to binding to the distalC-terminal region. At supraphysiological CaM concentrations,other binding sites or nonspecific effects by CaMmay also con-tribute to inhibition.

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References 1 IAALYDNLEAAMVLME TRPV6 17 2 VRALLARRASVSARATGTAFRR TRPV5 18 3 QTPVKELVSLKWKRYGRPYFC TRPV5 18 4 LVEVPDIFRMGVTRFFGQTILGGPFHVLI TRPV5, TRPV6 18 5 ELWRAQIVATTVMLERKLPR TRPV5 18,19 6 RQRIQRYAQAFHTRGSE TRPV6 17 7 ANWERLRQGTLRRDL TRPV5, TRPV6

C

14,15,16,18

FIGURE 3. CaM binds to TRPV6 via a distal C-terminal binding site. A, pro-posed CaM-binding sites in TRPV5 and TRPV6. The amino acid sequencescorrespond to the equivalent fragments in the human TRPV6. B, CaM-Sephar-ose pulldown assay using MBP fusion proteins of TRPV6 wild-type cytoplas-mic C terminus (amino acids 579 –725), truncated C terminus without thedistal region (�694 –725), and wild-type cytoplasmic N terminus (amino acids1–326) in the presence of various Ca2� concentrations or 2 mM EGTA. Boundproteins were detected by Western blot analysis using anti-MBP antibody.The images are representative of five or six experiments. C, CaM bindingassays were performed using the crude membrane fractions from HEK293cells expressing full-length wild-type Myc-hTRPV6 or mutant Myc-hTRPV6�694 –725 in the presence of 20 �M Ca2�. Bound proteins eluted fromthe CaM beads were detected by Western blot analysis using an anti-Mycantibody. The images are representative of eight experiments.

FIGURE 4. Two highly conserved amino acid residues in the distal C ter-minus of TRPV6 are responsible for interacting with CaM. A, sequencealignment shows that residues Trp-695 and Arg-699 in hTRPV6 are fully con-served among all studied TRPV5 and TRPV6 species (16). B, CaM-Sepharosepulldown assays were performed in the presence of 100 �M Ca2� on theisolated C terminus of wild-type and mutant TRPV6. The image is represent-ative of five experiments. C, CaM-Sepharose pulldown assays were performedin the presence of 20 �M Ca2� for full-length, wild-type, and three mutatedTRPV6 proteins transiently expressed in HEK293 cells. Input proteins for load-ing control and bound proteins eluted from the CaM beads are detected byWestern blot using an anti-Myc antibody. The images are representative ofthree experiments. D, representative trace for the effect of Ca2�, Ca2�-CaM,and CaM on double mutant W695A/R699E (TRPV6-WR) channel activity stim-ulated by 25 �M diC8 PI(4,5)P2 in excised inside-out macropatches. E, sum-mary data for D normalized to the current evoked by diC8 PI(4,5)P2 (n � 5).




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We also compared the effects of CaMonTRPV6when chan-nel activity was maintained by MgATP, which serves as a sub-strate for endogenous lipid kinases in the patch membrane toallow resynthesis of PI(4,5)P2 (12). Fig. 6 shows that whenMgATP is applied to excised patches after current rundown, itactivated TRPV6with slower kinetics than diC8 PI(4,5)P2. CaM(0.2 �M) inhibited the activity of the wild-type TRPV6 stimu-lated by either PI(4,5)P2 or MgATP (Fig. 6, A and C), and theW695A/R699E double mutant was not inhibited or only mini-mally inhibited in either case (Fig. 6, B and D).It was proposed for many TRP channels mainly based on

biochemical binding experiments that phosphoinositides affecttheir activity by competing with CaM (20). Because TRPV6activity depends on PI(4,5)P2, and CaM inhibits it, we testedwhether this model can apply to this channel. First, we mea-sured the effects of CaM in the presence of different concentra-tions and forms of PI(4,5)P2. Fig. 7 (A and B) shows that 0.2 �M

CaM inhibited TRPV6 slightly but significantly less in the pres-ence of 100 �M diC8 PI(4,5)P2 than in the presence of 25 �M

diC8 PI(4,5)P2. The W695A/R699E double mutant was essen-tially not inhibited in the presence of either concentration of

DiC8 PI(4,5)P2 (Fig. 7, C and D). We also tested the effects ofCaM in the presence of the long chain natural AASt PI(4,5)P2(Fig. 7,E and F). This compound accumulates in themembrane;thus higher effective membrane concentrations can be reachedthan with the diC8 analogue (28). The effect of AASt PI(4,5)P2took longer time to develop, and even on washout, channelactivity increased further for a while, consistent with our earlierreport (12). This is likely due to the slow incorporation of thelipidmicelles and lateral diffusion to reach the channel. Becausethis compound accumulates in the membrane, we used a two-pulse protocol to test whether CaM inhibits the channel less inthe presence of higher PI(4,5)P2 concentrations. Fig. 7E showsthat current activity was significantly increased upon the sec-ond application of AASt PI(4,5)P2 to the same patch, indicatinga higher concentration of the lipid in the membrane. CaMinhibited current activity significantly less after the secondapplication of AASt PI(4,5)P2 than after the first. ExcessPI(4,5)P2 also reduced the inhibition by CaM in planar lipidbilayers (Fig. 8, A and B).Our data show so far that PI(4,5)P2 can functionally compete

with CaM both in excised patches and in planar lipid bilayers.

FIGURE 5. Concentration dependence of the effect of CaM on TRPV6 in excised inside-out macropatches. A and B, representative traces for the effects ofdifferent concentrations of CaM in the presence of 3 �M Ca2� on wild-type TRPV6 (TRPV6) and double mutant W695A/R699E (TRPV6-WR) activated by 25 �M

diC8 PI(4,5)P2. C, summary data, normalized to the current evoked by 25 �M diC8 PI(4,5)P2 (n � 5– 8). D, T1⁄2 values for the different concentrations of CaM.




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This can happen either through competition for the same oroverlapping binding site or via allosteric effects. To differenti-ate between these two possibilities, we tested whether PI(4,5)P2competes with CaM for binding to the C terminus of TRPV6.Fig. 8 (C and D) shows that the C terminus of TRPV6 showedvery similar binding to CaM beads in the absence or the pres-ence of 20 and 100 �M diC8 PI(4,5)P2. This measurement indi-cates that PI(4,5)P2 does not directly compete with CaM for thesame binding site.To assess the role of CaM in Ca2�-induced inactivation, we

have performed whole cell patch clamp experiments in mam-malian cells expressing wild-type and W695A/R699E doublemutant TRPV6 channels. Fig. 9 (A–C) shows measurements inwhich monovalent currents through TRPV6 were measured,interspersed with applications of 2 mM Ca2�. In wild-typechannels, monovalent currents decreased, on average, by 60%after the first and�70% after second application of 2mMCa2�.In the doublemutant, only�20% inactivation was observed, onaverage, after the first application of Ca2�, with a �40%decrease after the second. These data show that CaM isinvolved in Ca2�-induced inactivation, but it is probably notthe only factor.We have also measured Ca2�-induced inactivation in a dif-

ferent protocol often used by several laboratories (3, 14, 15),where Ca2� influx was initiated with a 3-s voltage step from�70 to �100 mV in the presence of 10 mM Ca2�. Fig. 9 (D–F)shows that in this protocol, a quickly inactivating phase that iscomplete after �50 ms is followed by a slower phase through-out the 3-s voltage pulse. The fast phase was not differentbetween the wild-type and the W695A/R699E mutant; how-

ever, inactivation of the mutant channel was significantlysmaller in the slow phase (Fig. 9G), in accordance with earlierresults using different mutations in this region of TRPV6 (14,15) or equivalent mutations in TRPV5 (16).

DISCUSSION

TRPV6, similarly to many other Ca2� permeable channels,undergoes Ca2�-induced inactivation. Given that this channelis constitutively active, this process is especially important forprotecting the cell from toxic Ca2� overload. To understandthe roles of individual signaling molecules, we have usedexcised inside-out patch clamp measurements to study theeffects of CaM, PI(4,5)P2 and Ca2� on TRPV6 channels. Usingthis technique, we fully control the concentration of CaM andavoid the effects of cellular components present in whole cellpatch clamp experiments, and we can dissect the direct effectsof Ca2� from those mediated by CaM.We found that CaMapplied to the intracellular surface of the

patch membrane reproducibly inhibited TRPV6 channel activ-ity. In the absence of Ca2�, channel activitywas not inhibited byCaM. This is consistent with earlier reports showing that CaMbinding to TRPV6 requires Ca2� (15). For most CaM experi-ments, we used 3 �M Ca2�, which by itself only caused a smallinhibition. CaM is amajor intracellular protein, its total cellularconcentration is though to be �10 �M, but most of it binds toother proteins, and the maximal free concentration of Ca2�-CaM was estimated to be 45 nM at saturating Ca2� concentra-tions (3 �M or above) (27). CaM inhibited TRPV6 activity atconcentrations as low as 50 nM in excised patches, suggestingthat the effect of CaM on TRPV6 is physiologically relevant.

FIGURE 6. Ca2�-CaM inhibits TRPV6 activity stimulated by MgATP in excised inside-out patches. A and B, representative traces for the application of Ca2�

(3 �M) and Ca2�-CaM (3 �M Ca2� and 0.2 �M CaM) on wild-type TRPV6 and the W695A/R699E double mutant TRPV6 (TRPV6-WR) activity, respectively. Channelactivity was stimulated by 25 �M diC8 PI(4,5)P2 and 2 mM MgATP (2 mM NaATP and 2 mM Mg2�). C and D, summary of the data for six or seven experiments.




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Ca2� itself without the application of CaM also inhibitedTRPV6 activity in excised patches, but substantial inhibitionrequired very high concentrations (EC50 � �20 �M). CaMassociates with many different ion channels in the absence ofCa2� (29). Can the effect of Ca2� be due to CaM preassociatedtoTRPV6?We think this is unlikely for the following reasons: 1)Our data and earlier reports (15) show that CaM associationwith the channel requires Ca2�. 2) The CaM1234 mutant thatcannot bind Ca2� is often used to study the effects of preasso-ciated CaM. It was shown that this dominant negative CaM didnot bind TRPV6 and had no effect on its Ca2�-induced inacti-vation (15). 3) Binding of Ca2� to CaM is though to saturate�3�M, and in our experiments, we needed Ca2� concentrations ashigh as 100 �M for full inhibition.

Although global intracellular Ca2� hardly reaches concen-trations over 1 �M, in the subplasmalemmal regions near themouth of an open Ca2� channel, local Ca2� concentrationsmay reach hundreds of micromolars (25). Despite the require-ment for very high Ca2� concentrations, it is possible thatdirect inhibition by Ca2� contributes to Ca2�-induced inacti-vation. The lack of effect of the CaM-binding site mutation onthe first very fast phase of TRPV6 inactivation (Fig. 9,D and E),also described by others (14), is consistent with the idea of localhigh concentrations of Ca2� directly inhibiting the channel.

At the physiologically relevant CaM concentrations, theinhibitory effect of CaMdeveloped quite slowly andwas partial,suggesting that other factors also contribute to Ca2�-inducedinactivation. To be able to address the role of CaM and other

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FIGURE 7. PI(4,5)P2 competes with Ca2�-CaM on TRPV6 activity in excised inside-out patches. A, representative trace for the effect of 3 �M Ca2� andCa2�-CaM (3 �M Ca2� and 0.2 �M CaM) on wild-type TRPV6, in the presence of 25 and 100 �M diC8 PI(4,5)P2. B, summary data for A, normalized to the currentevoked by diC8 PI(4,5)P2 with the corresponding concentration for the wild-type TRPV6 channel. The inhibition by CaM at 40 s is significantly less at 100 �M diC8PI(4,5)P2 than at 25 �M (n � 9, p � 0.0104). C, representative experiment for the double mutant TRPV6 (TRPV6-WR). D, summary for the effect of CaM on theW695A/R699E mutant of TRPV6 (n � 4). E, representative trace for the effect of 3 �M Ca2� and 0.2 �M Ca2�-CaM on wild-type TRPV6 activity, in the presence ofnatural AASt PI(4,5)P2 (10 �M). Because of the micelle form of AASt PI(4,5)P2 in the bath solution, the amount of AASt PI(4,5)P2 incorporated into the patchmembrane was associated with the time of application. F, summary data for E, normalized to the current evoked by AASt PI(4,5)P2. The inhibition by CaM wassignificantly less at the second application of AASt PI(4,5)P2 than at the first one, when measured at 60 s (n � 6, p � 0.0056). Asterisks denote statisticalsignificance.




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signaling pathways, we used the excised inside-out patch tech-nique to unequivocally identify the site responsible for CaM-mediated inhibition. CaM binds to linear �-helical protein seg-ments �20 amino acids long (30). Various CaM-binding siteshave been proposed in TRPV6 in the N- and C-terminal cyto-plasmic domains and in the cytoplasmic loop between the sec-ond and third transmembrane domains (Fig. 3A). Here wefocused on the distal C-terminal CaM-binding region, residues694–716 in the human TRPV6 (14, 15). A recent article foundthat CaM also binds to TRPV5, a close relative of TRPV6, viathe equivalent distal C-terminal site (16). That study also iden-tified individual amino acids that are important in this regionfor CaM binding. Our data show that mutation of two con-served residues (W695A/R699E) in the distal C-terminal regionofTRPV6 eliminatedCaMbinding to the isolatedC terminus ofTRPV6 and to the full-length channel protein. Furthermore,this double mutant showed dramatically reduced inhibition byCa2�-CaM in excised patches. Some residual inhibitionremained at supraphysiological CaM concentrations, whichmay be due to nonspecific interactions of CaM with the chan-nel, or interactions with some of the additional CaM-bindingsites. Overall, these data show that the inhibitory effect of CaMat physiological CaM levels ismediated by the distal CaM-bind-ing site.

An earlier study showed using FRET in live cells that the cyanfluorescent protein-tagged CaM interacts with the full-lengthYFP-tagged TRPV6 in a Ca2�-dependent manner (15). Con-sistent with our results, they also found that the Ca2�-depen-dent FRET between the full-length TRPV6 and CaM was elim-inated by the removal of the distal C-terminal CaM-binding site(15).The activity of TRPV6 requires the presence of PI(4,5)P2 (10,

12). This appears to be a full dependence, because channelactivity invariably showed a complete loss over time in excisedpatches when MgATP or PI(4,5)P2 was not supplied and suffi-cient time has passed in the inside-out configuration. We alsocould not detect any TRPV6 activity in planar lipid bilayerswithout supplying PI(4,5)P2 (12).Without the presence of somePI(4,5)P2, the effects of CaM could not have been studied. Wehave used four different ways to supply PI(4,5)P2 to supportchannel activity: 1) performing the experiment immediatelyafter excision, relying on the endogenous PI(4,5)P2 in the patchmembrane; 2) applying short acyl chain diC8 PI(4,5)P2; 3)applying natural long acyl chain PI(4,5)P2; and 4) applyingMgATP to allow the lipid kinases in the patch to resynthesizePI(4,5)P2. In all of these cases, CaM inhibited TRPV6 activity.Both CaM (31) and PI(4,5)P2 (32) regulate a large number of

TRP channels. Are there any general principles for the interac-

FIGURE 8. PI(4,5)P2 competes with CaM in planar lipid bilayers, but not in biochemical binding experiments. A, planar lipid bilayer measurements wereperformed as described under “Experimental Procedures.” TRPV6 activity was stimulated by 5 �M diC8 PI(4,5)P2. Then 200 nM CaM was applied, and PI(4,5)P2concentration was increased to 10 �M. Clamping potential was �100 mV. The closed state is indicated by a horizontal line on the left side of traces. B, summaryof the bilayer experiments (n � 5 for CaM inhibition and n � 3 for PI(4,5)P2 reactivation). C, CaM-Sepharose pulldown assay was performed using MBP fusionproteins of TRPV6 wild-type C terminus in the absence and the presence of 20 or 100 �M diC8 PI(4,5)P2 as described under “Experimental Procedures.” Free Ca2�

concentration was 100 �M. Bound proteins were detected by SDS-PAGE and Western-blotting using anti-MBP antibodies. D, statistical summary based on fivemeasurements.




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tion of these two regulating molecules? It was proposed forTRPC6 that phosphoinositides, especially PI(3,4,5)P3, regulatechannel activity by disrupting binding to CaM (20). In the samearticle, CaM and phosphoinositides were shown to bind tooverlapping binding sites to isolated C-terminal fragments ofseveral other TRP channels (not including TRPV6), and thisdirect competitionwas proposed to be a generalmechanism forregulation of TRP channels by these two intracellular signalingmolecules. TRPV6 activity fully depends on the presence ofPI(4,5)P2. Does CaM inhibit this channel by displacingPI(4,5)P2 from its binding site? This is quite an attractivehypothesis, because the distal C-terminal CaM-binding site hasa number of positively charged residues, which are invariablyinvolved in interactions of phosphoinositides with proteins,including ion channels (33).Wehave found that higher concen-trations of PI(4,5)P2 could reduce inhibition by CaM in excisedpatches and planar lipid bilayers, which is compatible with this

model. This finding, however, can also be explained by allos-teric effects, for example if PI(4,5)P2 stabilizes the open states,whereas CaM stabilizes the closed state.To explore this idea further, we have tested whether

PI(4,5)P2 can interfere with binding of the isolated C terminusto CaM.We have used the isolated C terminus because it is farless likely than the full-length channel to undergo significantconformational change upon binding of either molecules,which could reduce the binding of the other, without any actualoverlap between their respective binding sites (34). We foundno competition by PI(4,5)P2 in these experiments, which arguesagainst a simple model in which CaM displaces PI(4,5)P2 fromits binding site through direct competition.As mentioned earlier, CaM invariably binds to short �-heli-

cal polypeptide segments. Phosphoinositides may also bind toshort highly positively charged peptides (35), in most cases,however, well defined three-dimensional structures such as the

FIGURE 9. Ca2�-induced inactivation is reduced but not eliminated in the W695A/R699E mutant. Whole cell patch clamp experiments in TRPV6-express-ing HEK cells were performed as described under “Experimental Procedures.” A and B, representative measurements for wild-type and W695A/R699E (WR)TRPV6 channels at constant �60 mV holding potential. At the beginning of the measurements, the cells were kept in a nominally Ca2�-free solution containing1 mM Mg2�. Monovalent currents were evoked by the application of 2 mM EGTA in bivalent free extracellular solution (0 Ca); then Ca2�-induced inactivation wasinduced by the application of a solution containing 2 mM Ca2� and no Mg2� (2 Ca). C, summary of current amplitudes in the first, second, and third applicationsof EGTA. The difference between wild-type and the mutant channel was statistically significant in the second (p � 0.024, n � 11), but not at the third pulse (p �0.058). D and E, Ca2�-induced inactivation of Ca2� currents for wild-type and mutant TRPV6 channels. Ca2� currents were initiated by a 3-s voltage step from�70 to �100 mV in an extracellular solution containing 10 mM Ca2�. The traces shown are averages for six measurements for both groups. F, summary ofinactivation kinetics at 100 ms; the data are normalized to the point 3 ms after the voltage step, to avoid capacitative artifacts. G, summary at 1 and 3 s after the�100 mV voltage step; the data are normalized to the current 100 ms after the voltage step, after the initial fast phase inactivation. The difference betweenwild-type and mutant channel was statistically significant both at 1 s (p � 0.025) and at 3 s (p � 0.0054). Asterisks denote statistical significance. H, model forCa2�-induced inactivation of TRPV6.




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pleckstrin homology domain are responsible for binding, wherethe actual binding residues come together from various parts ofthe linear sequence. Ion channels generally do not have welldefined phosphoinositide-binding domains with homology toother lipid-binding domains. Among ion channels, phospho-inositide interactions are best characterized in inwardly recti-fying K� (Kir) channels. It is thought that the PI(4,5)P2-bindingpocket is formed by a three-dimensional structure where resi-dues contributing to lipid binding come from various parts ofthe C and N terminus (33, 36). This picture was confirmed bythe recent co-crystal structure of Kir2.2 and PI(4,5)P2 (37, 38).Most residues playing significant roles in PI(4,5)P2 interactionsare located in proximal regions, relatively close to the trans-membrane domains. If PI(4,5)P2 interactions in TRP channelsare similar to those in Kir channels, it is quite unlikely that thevery distal C terminus by itself could serve as the sole PI(4,5)P2-binding domain. We found that when the whole distal CaMbinding segment is removed, the channel is still fully functional(data not shown), consistent with earlier findings (14, 15).Because our data show full dependence of channel activity onPI(4,5)P2, it is unlikely that this short segment contributes sig-nificantly to PI(4,5)P2 binding. This, again, also argues againstCaM inhibiting the channel by displacing PI(4,5)P2 from itsbinding site.What is the contribution of CaM inhibition to the overall

Ca2�-induced inactivation of TRPV6 in a cellular context? Toaddress this, we have tested the effect of the doublemutation inthe CaM-binding region on the Ca2�-induced inactivation ofTRPV6 in whole cell patch clamp measurements. We havemeasured the decrease of monovalent currents throughTRPV6, interspersed with applications of 2 mM Ca2�, as wedescribed earlier (4, 10). Here we have found that the doublemutant TRPV6, lacking CaM binding and displaying no inhibi-tion by CaM in excised patches, showed significantly reducedCa2�-induced inactivation compared with wild-type TRPV6.Some inactivation did occur, however, especially after the sec-ond application of Ca2�, where the difference between wild-type and mutant channels was not statistically significant. Inour earlier work, we have shown that inclusion of PI(4,5)P2 inthe patch pipette completely eliminated Ca2�-induced inacti-vation of TRPV6 in this protocol (10). These data suggest thatboth CaM and PI(4,5)P2 depletion is needed for maximalinactivation.We have also measured Ca2�-induced inactivation in a dif-

ferent protocol, where Ca2� influx is initiated by a voltage stepto �100 mV, from a positive voltage that does not allow Ca2�

influx. This protocol allows detection of very fast events at theonset of Ca2� influx. We have found that the first fast phase ofinactivation, which was complete at �50 ms, was not affectedby the CaM-binding site mutation. Because the effects of Ca2�

developed very fast in excised patches, it is quite likely that thisfast phase is caused by the direct effect of Ca2� on the channel.Similar to the protocol measuring monovalent currents, Ca2�-induced inactivation in the second, slower phase was reducedbut not eliminated in the CaM-binding site mutant, pointing tothe involvement of other factors in Ca2�-induced inactivation.

Our excised patch measurements show that excess PI(4,5)P2can reduce the effect of CaM on the channel, and the effect of

PI(4,5)P2 is less in the presence of CaM; in other words there isfunctional competition between these two signalingmolecules.Overall, these data suggest a model in which Ca2� plays a dualrole in inactivation: by binding to CaM it reduces the effective-ness of PI(4,5)P2, and by reducing PI(4,5)P2 levels, it enhancesthe effect of CaM. Overall, it is quite likely that neither CaM,nor PI(4,5)P2 depletion alone is sufficient to induce full inacti-vation of the channel, but the two act together to reduce furtherCa2� influx.

Our data show that although CaM did inhibit TRPV6 at 50nM, that effect developedmuchmore slowly than at higher con-centrations. Thus physiological CaM levels, even when satu-ratedwithCa2�, are suboptimal for inhibiting TRPV6. Channelactivity fully depends on the presence of PI(4,5)P2; thus takingthis lipid away, should fully inhibit the channel by itself. Indeedwhenwe artificially depleted PI(4,5)P2with a rapamycin-induc-ible 5� phosphatase, we saw a �75% inhibition (10) of TRPV6activity. Ca2� influx through TRPV6, however, results in only amoderate reduction in PI(4,5)P2 levels (10) when comparedwith that induced by capsaicin in TRPV1-expressing cells (39).Thus upon Ca2� influx through TRPV6, two suboptimal inhib-itory signals converge and act together to reduce channelactivity.In conclusion, we found that Ca2�-CaM inhibits TRPV6

activity via direct binding to the distal C-terminal region.Increased PI(4,5)P2 levels may override this inhibition, but theeffect of CaM is not due to the displacement of PI(4,5)P2 viadirect competition. During Ca2�-induced inactivation, Ca2�,CaM, and PI(4,5)P2 contribute to reduced channel activity. Fig.9H shows our model for Ca2�-induced inactivation of TRPV6.

Acknowledgment—The clone for the human Myc-tagged TRPV6 wasgenerously provided byDr. T. V.McDonald (Albert EinsteinCollege ofMedicine, New York, NY).

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Chike Cao, Eleonora Zakharian, Istvan Borbiro and Tibor Rohacs-induced Inactivation of Transient Receptor Potential Vanilloid 6 Channels

2+Interplay between Calmodulin and Phosphatidylinositol 4,5-Bisphosphate in Ca

doi: 10.1074/jbc.M112.409482 originally published online January 8, 20132013, 288:5278-5290.J. Biol. Chem.

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