Differential Regulation of TRPV1, TRPV3, and TRPV4 Sensitivity through a Conserved Binding Site on the Ankyrin Repeat Domain^*

Cloning of Expression Vectors

cDNA fragments encoding ARDs (human TRPV3-ARD residues 115–367 and chicken TRPV4-ARD residues 132–383) and full-length protein (human TRPV3 and chicken TRPV4) were cloned into the NdeI and NotI sites of pET21-C6H (23) and pFastBac-CFLAG (15) vectors, respectively. Baculovirus stocks were generated and used to infect Sf21 cells as described in the Bac-to-Bac manual (Invitrogen). Full-length TRPVs in pcDNA3 were provided by Michael Caterina (Johns Hopkins School of Medicine; rat TRPV2), David Clapham (Harvard Medical School; human TRPV3) and Stefan Heller (Stanford University; chicken TRPV4). All mutants were generated by mutagenesis, and all clones were verified by DNA sequencing.

Expression and Purification of TRPV ARDs

The ARDs were expressed in Escherichia coli BL21(DE3) by induction with 0.4 mm isopropyl-β-d-thiogalactopyranoside overnight at room temperature after the cells reached A₆₀₀ = 0.6. Cells were resuspended in lysis buffer (20 mm Tris-HCl (pH 8.0), 300 mm NaCl, 20 mm imidazole (pH 8.0), and 1 mm phenylmethylsulfonyl fluoride) with 0.1% Triton X-100, 0.2 mg/ml lysozyme, 50 μg/ml RNase A, and 25 μg/ml DNase I and lysed by sonication. The cleared lysate was loaded onto nickel-nitrilotriacetic acid (Qiagen) and eluted by a step gradient containing 50, 100, 150, and 200 mm imidazole (pH 8) in lysis buffer. Ten mm EDTA (pH 8.0) and 1 mm dithiothreitol (DTT) were added after elution. The fractions containing TRPV3-ARD or TRPV4-ARD were pooled and further purified on Q or SP Sepharose FF (GE Healthcare), respectively, in 20 mm Tris (pH 8.0), 5 mm DTT using a linear gradient of 0–0.4 M NaCl. Size exclusion chromatography on a Superdex 75 column (GE Healthcare) in 10 mm Tris-HCl (pH 8.0), 200 mm NaCl, and 1 mm DTT was used for further purification of TRPV3-ARDs, whereas the TRPV4-ARDs were dialyzed in 20 mm Tris-HCl (pH 8.0), 300 mm NaCl, 10% glycerol, and 1 mm DTT. All proteins were concentrated to >7 mg/ml in a Vivaspin centrifugal filter (10,000 molecular weight cut off; Sartorius AG, Goettingen, Germany), flash frozen, and stored at −80 °C. TRPV1-ARD, TRPV2-ARD, TRPV5-ARD, and TRPV6-ARD were purified as described previously (15, 23, 25).

ATP- and CaM-Agarose Pulldown Assays

All assays were carried out at 4 °C as described previously (25). The ATP-agarose assays were performed in the absence of divalent ions except otherwise noted, in binding buffer (10 mm Tris-HCl (pH 7.5), 50 mm NaCl, 1 mm DTT, and 0.15% n-decyl-β-d-maltopyranoside; except 150 mm NaCl was used for TRPV4-ARD mutant analyses to preserve protein solubility). For ATP competition assays, competing compounds were added to reaction mixtures prior to the agarose slurry. All nucleotides used where sodium salts diluted from 0.5 m stocks adjusted to pH 7 with NaOH. The CaM-agarose assays were performed in binding buffer supplemented with 2 mm CaCl₂ or 5 mm EGTA (pH 7.5). In each load lane, the volumes loaded corresponded to 2 μg of protein. Gels were quantified using ImageJ (26), and shown are the average ± S.D. for at least three independent experiments.

Insect and Mammalian Cell Culture and Full-length TRPV Protein Expression

Sf21 insect cells were maintained in Hink's TNM-FH (Mediatech, Manassas, VA), supplemented with 10% fetal bovine serum, 0.1% pluronic F-68, and 10 μg/ml gentamycin. Cells at 5 × 10⁵ cells/ml were adhered to glass coverslips in medium without pluronic F-68 and infected with baculovirus. HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, GlutaMAX (Invitrogen), 100 units/ml penicillin and 100 μg/ml streptomycin. Cells were co-transfected with pNEGFP and pcDNA3 containing the appropriate full-length TRPV using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer directions.

Electrophysiology

Insect cells were tested 44–48 h postinfection and HEK293 cells were tested 20–25 h post-transfection under continuous perfusion using a multichamber perfusion apparatus for agonist application. 2-APB and thymol were dissolved in dimethyl sulfoxide and 4α-phorbol 12,13-didecanoate (4αPDD) in ethanol prior to dilution in bath solution. Currents were recorded and analyzed as described (15). Data are presented as mean ± S.E. The intracellular/pipette solution contained 140 mm NaMethanesulfonate, 10 mm HEPES, and either (4 mm NaCl and 10 mm EGTA) for EGTA conditions or (0.6 mm MgCl₂ and 10 mm BAPTA, resulting in 0.4 mm free Mg²⁺ according to MaxChelator (27)) for BAPTA conditions. The BAPTA conditions were very similar to those used in Ref. 21. The pH was adjusted to 7.2 with NaOH, and the final osmolarity was ∼315 mOsm. As indicated, the intracellular solution was supplemented with 4 mm ATP (sodium salt) or ATPγS (lithium salt) from 0.5 m stocks (pH adjusted to ∼7 with NaOH). In EGTA conditions, all ATP should be free ATP, whereas in BAPTA conditions, the presence of 0.6 mm MgCl₂ results in 0.001 mm free Mg²⁺, 0.58 mm Mg-ATP, and 3.42 mm free ATP (27). For CaM depletion experiments, the intracellular solution was supplemented with 2 μg/ml CaM85 or an isotype-matched control antibody (Invitrogen). The extracellular/perfusion solution was 150 mm NaCl, 5 mm KCl, 1 mm MgCl₂, 2 mm CaCl₂, 10 mm HEPES, and 10 mm d-glucose (pH adjusted to 7.4 with NaOH; ∼315 mOsm), except for TRPV3 dose-response experiments and TRPV4 voltage step experiments in insect cells, where the extracellular solution was 150 mm NaGluconate, 10 mm NaCl, 2 mm CaCl₂, 10 mm HEPES, and 10 mm d-glucose (pH adjusted to 7.2 with NaOH; ∼ 315 mOsm), which produced more stable seals with less leak current at high agonist concentrations.

Data Analysis

EC₅₀ values were calculated by fitting the average normalized current at −100 mV for a range of agonist concentrations to the Hill Equation, I(S) = 1 − (Kⁿ/(Kⁿ + Sⁿ)), where I is the current, K is the EC₅₀, S is the agonist concentration, and n is the Hill coefficient. Tail currents from voltage step experiments in HEK293 cells used for the determination of TRPV4 V½ were measured during the first millisecond of a step to a voltage of −160 mV and normalized to the maximum current. Average tail currents were fit to a modified Boltzmann function: G(V) = G_max − (G_max − G_min)/(1 + exp(zF/RT*(V − V½))), where z is the valence of the gating charge and F/RT is 25 mV⁻¹. Statistical analyses were performed using a two-tailed t test, with p < 0.05 being considered statistically significant. Data are presented as mean ± S.E.

TRPV3-ARD and TRPV4-ARD Bind ATP and Ca²⁺-CaM

To determine whether the ATP/CaM-binding site on the TRPV1-ARD is conserved in other TRPV channels, the ARDs from all six TRPV channels common to warm-blooded vertebrates were tested for ATP binding in pulldown assays (Fig. 1). As previously observed (15, 25), the TRPV1-ARD bound ATP-agarose, while the TRPV2-, TRPV5- and TRPV6-ARDs did not. Both TRPV3-ARD and TRPV4-ARD were precipitated by ATP-agarose (Fig. 1A), suggesting that the TRPV1-ARD ATP-binding site is conserved in TRPV3 and TRPV4. Furthermore, the three ARDs that interact with ATP, the TRPV1-, TRPV3- and TRPV4-ARDs, were also precipitated with CaM-agarose in the presence of Ca²⁺, and this interaction was eliminated in the presence of EGTA, a Ca²⁺-chelator (Fig. 1A). As previously determined, the TRPV2-, TRPV5-, and TRPV6-ARDs interacted either very weakly or not at all with CaM-agarose (Fig. 1B) (15, 25).

The ATP and Ca²⁺-CaM Binding Site Is Conserved in TRPV3-ARD and TRPV4-ARD

To further characterize the properties of the ATP-binding site on TRPV3 and TRPV4, we tested its specificity in competition assays with other nucleotides. As previously reported with TRPV1 (15), free GTP and ATP most efficiently competed for binding to ATP-agarose for both TRPV3-ARD and TRPV4-ARD (Fig. 1C). Furthermore, Ca²⁺ and Mg²⁺ also reduced binding to ATP-agarose (Fig. 1C). Therefore, the ATP-binding sites on the ankyrin repeats of TRPV3 and TRPV4 have the highest affinity for divalent-free triphosphate nucleotides, with a small preference for purines over pyrimidines, a specificity profile comparable to TRPV1-ARD (15).

The similar nucleotide specificities of TRPV3-ARD, TRPV4-ARD, and TRPV1-ARD strongly suggest that ATP interacts with these domains at a conserved site. The overall sequence conservation of TRPV1, TRPV3, and TRPV4 (supplemental Fig. 1) was mapped onto the structure of TRPV1-ARD bound to ATP (Fig. 2A). The most conserved surface encompasses the ATP-binding site. We used mutagenesis to confirm that the conserved phosphate-binding residues are important for the interaction of TRPV3-ARD and TRPV4-ARD with ATP. Lys¹⁵⁵ and Lys¹⁶⁰ interact with the triphosphate moiety of ATP in the TRPV1-ARD structure and are important for ATP and CaM binding (15). The corresponding lysines, Lys¹⁶⁹ and Lys¹⁷⁴ in TRPV3-ARD and Lys¹⁷⁸ and Lys¹⁸³ in TRPV4-ARD, were mutated to alanine. Arg¹⁸⁸ in TRPV3 and Lys²⁰⁵ in TRPV4, predicted to lie on the opposite face of the ARDs, were also mutated to alanine and used as negative controls. In pulldown assays, the TRPV3-ARD and TRPV4-ARD lysine mutants showed reduced binding to both ATP and CaM compared with the wild type proteins and negative control mutants (Fig. 2B and 2C). In summary, the lysines homologous to Lys¹⁵⁵ and Lys¹⁶⁰ in TRPV1 are also necessary for TRPV3 and TRPV4 interactions with ATP and CaM, indicating that the multiligand binding site previously identified in TRPV1 (15) is conserved in TRPV3 and TRPV4.

TRPV2 Is Insensitive to Intracellular ATP

Electrophysiology experiments demonstrated that intracellular ATP can sensitize TRPV1 and prevent its desensitization to repeated applications of capsaicin (15). Experiments with the K155A and K160A mutants of TRPV1 also indicated that these effects of ATP were through its direct interaction with the TRPV1-ARD. Rat TRPV2 was hypothesized to be a natural negative control; ATP and Ca²⁺-CaM were not expected to affect its sensitivity because its ARD did not bind either. Rat TRPV2 expressed in HEK293 cells responded to 2-APB in whole cell patch clamp recordings as reported previously (10, 28). TRPV2 exhibited similar currents when stimulated with 2-APB in the absence or presence of intracellular ATP (Fig. 3). Furthermore, no significant desensitization or tachyphylaxis was observed in response to repeated 2-APB applications. Therefore, TRPV2 activity was not affected by the presence of intracellular ATP, correlating with the lack of interaction between ATP and the TRPV2-ARD.

We attempted to generate a TRPV2-ARD mutant that could bind ATP and/or CaM. We looked at two mutations: D78N, which neutralizes a negatively charged side chain which maps in close proximity of the phosphate-interaction site, and H165Q, to attempt to restore the adenine-binding pocket (supplemental Fig. 2). Neither of the single mutants bound to ATP- or CaM-agarose in our assays. The D78N/H165Q mutant bound weakly but significantly to ATP, but not CaM. Because the TRPV2-ARD is only 50% identical to the TRPV1-ARD, it is difficult to determine which other sequence differences may be responsible for the differences in biochemical properties.

TRPV4 Is Sensitized by Intracellular ATP

We used whole cell patch clamp electrophysiology to determine the effect of intracellular ATP on the sensitivity of TRPV4 expressed in insect and HEK293 cells. TRPV4 showed constitutive basal activity in both cell types (Fig. 4 and supplemental Fig. 3), similar to previous observations (e.g. Refs. 6, 7). In voltage step experiments in insect cells, TRPV4 currents were significantly increased in the presence of intracellular ATP or the nonhydrolyzable ATP analog ATPγS (Fig. 4A). Furthermore, the K178A mutation, which reduces ATP binding, abolished sensitization by ATP (Fig. 4A).

Similar results were obtained from basal TRPV4 currents in HEK293 cells (Fig. 4B), although the lower constitutive activity in HEK293 cells enabled us to also look at 4αPDD-stimulated activity. Currents observed after perfusion with 4αPDD were also significantly increased by the addition of ATP to the recording solution (Fig. 4B). The effect of ATP was similar in both 4αPDD-stimulated and constitutive conditions (at +100 mV, constitutive currents increased 1.9-fold and 3.0-fold in insect and HEK293 cells, respectively, while 4αPDD-stimulated currents increased 2.4-fold). Furthermore, depleting HEK293 cells of CaM by including a monoclonal anti-CaM antibody in the intracellular solution, as was previously done in TRPV1-expressing cells (15), did not affect the voltage response of unstimulated TRPV4, but did significantly increase inward currents in TRPV4 expressing HEK293 cells treated with 4αPDD (p < 0.05 at V_m ≤ −20 mV).

Similar to TRPV1 (15), this increased current density for TRPV4 in the presence of intracellular ATP appears to be a result of increased whole cell conductance, rather than a shift in the current-voltage relationship. Tail-current analyses from voltage step experiments in TRPV4-expressing HEK293 cells with control, 4 mm ATP, or anti-CaM antibody intracellular solutions (Fig. 4C and supplemental Fig. 3) show that the V½ of TRPV4 is not altered by ATP or CaM with V½ values of 104 ± 36.4, 102 ± 8.3, and 102 ± 13.6 mV for the control, ATP and anti-CaM experiments, respectively. Of note, the anti-CaM antibody increases the steepness of the G/V curve, suggesting that CaM may affect the intersubunit cooperativity of TRPV4. Overall, these results strongly suggest that the previously observed potentiation or desensitization by binding of intracellular ATP or CaM, respectively, to the N-terminal ankyrin repeats of TRPV1 is conserved in TRPV4.

ATP Lowers the Agonist Sensitivity of TRPV3

Similar to previously published reports using mammalian cells (21, 29), TRPV3 expressed in insect cells is sensitized by repeated applications of 2-APB (Fig. 5A). Once sensitized, TRPV3 also showed biphasic currents (Fig. 5A) where the initial outward rectified current (I₁) is followed by an off-response with the appearance of a less rectified, higher amplitude current that is slower to inactivate (I₂), similar to the currents reported in HEK293 cells and primary keratinocytes overexpressing TRPV3 (30). The sensitization of TRPV3 to repeated agonist applications is in contrast to what is observed with TRPV1, which is desensitized by repeated agonist applications (14, 15). Also unlike TRPV1 and TRPV4, intracellular ATP blocked the sensitization of TRPV3 to repeated 2-APB applications (Fig. 5B). The same effect was observed when ATPγS was used, supporting the idea that it is ATP binding, not an ATP hydrolysis-dependent process, that prevents TRPV3 sensitization. There is no significant difference between the currents observed during the first and twelfth 2-APB applications in presence of intracellular ATP or ATPγS. Furthermore, the currents observed on the twelfth 2-APB application with the control cells are significantly larger than in cells with intracellular ATP or ATPγS (Fig. 5B). Additionally, while biphasic currents and off-responses were observed for seven of the nine control cells tested, none of the ATP (0/6) or ATPγS (0/7) cells showed biphasic currents or off-responses.

The sensitization of TRPV3 is dependent on the strength of the intracellular Ca²⁺ buffer. When BAPTA, a more rapid and specific Ca²⁺ buffer, was used in place of EGTA, TRPV3 was pre-sensitized, showing large responses to the first application of 2-APB and little increased sensitivity to subsequent 2-APB applications (21). This behavior could also be reproduced in our insect cell system (Fig. 5, C and D). Also, TRPV3 K169A (one of the ATP/CaM site mutants that no longer bound ATP or CaM) (Fig. 2) showed initial current densities similar to those of wild type TRPV3 in the presence of BAPTA, even when EGTA was used as the Ca²⁺ buffer (Fig. 5). The TRPV3 K169A currents were similar to the I₂ currents observed with sensitized wild type TRPV3, with large amplitudes, little rectification, and slower deactivation after removal of 2-APB. Consistent with a sensitized state, the average current density from the first 2-APB application for TRPV3 K169A was as large as that for wild type TRPV3 either from the twelfth 2-APB application in experiments with EGTA as the Ca²⁺ buffer, or the first 2-APB application when presensitized with BAPTA as the Ca²⁺ buffer (Fig. 5). These results show that disruption of ATP/CaM binding eliminates the sensitivity of TRPV3 to intracellular Ca²⁺ levels and indicate that interaction of ATP and/or Ca²⁺-CaM on the N-terminal ankyrin repeats regulates TRPV3 sensitization.

To further characterize the mechanism by which intracellular ATP regulates TRPV3 sensitivity, dose-response relationships were measured for two different TRPV3 agonists, 2-APB and thymol, in the absence or presence of intracellular ATP (Fig. 6). Dose-response experiments were carried out with BAPTA to presensitize TRPV3 and remove any confounding effects on the dose response from repeated agonist applications. Additionally, the chloride ions in the extracellular solution were replaced with gluconate. Replacing chloride with gluconate lowers the agonist-induced TRPV3 currents, allowing us to also determine the dose response of TRPV3 K169A, which was otherwise difficult to inactivate after the first agonist application (supplemental Fig. 4). Thymol concentrations above 1 mm were toxic and as a result saturated currents could not be recorded in the presence of ATP. The responses of TRPV3 to thymol in the absence or presence of ATP were both normalized to the maximum current density from the experiments without ATP. ATP increases the EC₅₀ of both agonists by ∼3-fold (Fig. 6), indicating that intracellular ATP reduces the sensitivity of TRPV3 to its agonists.

TRPV3 K169A showed an increased sensitivity to 2-APB with an EC₅₀ ∼2-fold lower than wild type TRPV3, and it is unchanged in the presence of ATP (Fig. 6). The changes in sensitivity between K169A and wild type TRPV3 are not due to differences in expression (supplemental Fig. 5) and instead could result from the loss of ATP and CaM binding in K169A. In contrast, the behavior of the TRPV3 R188A control mutant is indistinguishable from wild type either in the presence or absence of intracellular ATP (Fig. 6). These results strongly support the role of ATP binding to the TRPV3-ARD in altering agonist sensitivity.

TRPV3 behaved similarly in HEK293 cells. Intracellular ATP or ATPγS reduced sensitization compared with control (EGTA), whereas intracellular BAPTA caused significantly increased current densities for the first three 2-APB applications (Fig. 7). In HEK293 cells, we could also test the effects of CaM depletion using an anti-CaM monoclonal antibody. Addition of anti-CaM antibody into the intracellular solution led to immediate sensitization and significantly larger current densities for all 2-APB applications, while currents in the presence of an isotype-matched control antibody were indistinguishable from control (Fig. 7B). Furthermore, the 2-APB-induced currents from CaM-depleted cells were similar to those observed with the K169A mutant in insect cells in that they did not inactivate upon agonist removal and showed little rectification (compare Fig. 5C with Fig. 7A). Taken together, the results from HEK293 and insect cells indicate that there is direct role for CaM binding to the conserved ARD site in TRPV3 inactivation and that ATP binding to the same site can maintain TRPV3 in a low sensitivity state. This is in agreement with a previous report that sensitization of TRPV3 results from a loss of CaM binding (21) and further demonstrates a role for the ARD in this CaM-mediated regulatory mechanism.