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J. Biol. Chem., Vol. 277, Issue 16, 13569-13577, April 19, 2002

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Activation of TRPV4 Channels (hVRL-2/mTRP12) by Phorbol Derivatives^*

Hiroyuki Watanabe, John B. Davis^§, Darren Smart^§, Jeff C. Jerman^§, Graham D. Smith^§, Phil Hayes^§, Joris Vriens, William Cairns^§, Ullrich Wissenbach^¶, Jean Prenen, Veit Flockerzi^¶, Guy Droogmans, Christopher D. Benham^§, and Bernd Nilius

From the  Department of Physiology, Campus Gasthuisberg, KU Leuven, B-3000 Leuven, Belgium, ^§ Neurology CEDD and Discovery Research, GlaxoSmithKline, CM195AW Harlow, United Kingdom, and the ^¶ Institut für Pharmakologie und Toxikologie, Universität des Saarlandes, D-66427 Homburg (Saar), Germany

Received for publication, January 3, 2002, and in revised form, January 28, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We have studied activation by phorbol derivatives of TRPV4 channels, the human VRL-2, and murine TRP12 channels, which are highly homologous to the human VR-OAC, and the human and murine OTRPC4 channel. 4-Phorbol 12,13-didecanoate (4-PDD) induced an increase in intracellular Ca²⁺ concentration, [Ca²⁺]_i, in 1321N1 cells stably transfected with human VRL-2 (hVRL-2.1321N1) or HEK-293 cells transiently transfected with murine TRP12, but not in nontransfected or mock-transfected cells. Concomitantly with the increase in [Ca²⁺]_i, 4-PDD activated an outwardly rectifying cation channel with an Eisenman IV permeation sequence for monovalent cations that is Ca²⁺-permeable with P_Ca/P_Na = 5.8. Phorbol 12-myristate 13-acetate also induced an increase in [Ca²⁺]_i but was ~50 times less effective than 4-PDD. EC₅₀ for Ca²⁺ increase and current activation was nearly identical (pEC₅₀ ~ 6.7). Similar effects were observed in freshly isolated mouse aorta endothelial cells which express TRP12 endogenously. By using 4-PDD as a tool to stimulate TRP12, we showed that activation of this channel is modulated by [Ca²⁺]_i; an increase in [Ca²⁺]_i inhibits the channel with an IC₅₀ of 406 nM. Ruthenium Red at a concentration of 1 µM completely blocks inward currents at 80 mV but has a smaller effect on outward currents likely indicating a voltage dependent channel block. We concluded that the phorbol derivatives activate TRPV4 (VR-OAC, VRL-2, OTRPC4, TRP12) independently from protein kinase C, in a manner consistent with direct agonist gating of the channel.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The TRPV subfamily of transient receptor potential cation channels (for a unified nomenclature, see Ref. 1) consists of members with completely different mechanisms of gating. Proteins of this subfamily contain typically three to six ankyrin repeats in the N terminus and six transmembrane segments with a pore region between segments 5 and 6. The first identified nonmammalian member of this subfamily, the Caenorhabditis elegans OSM-9 channel, is activated by changes in osmolarity (2). The second protein identified in the TRPV family is the mammalian vanilloid receptor channel VR1, which is activated by vanilloid compounds such as capsaicin, pepper, hot chili, moderate heat, or protons (3). Unlike VR1, another close relative of this channel, VRL-1, is constitutively activated by growth factors (4) or by noxious heat (5). Other members of this family are mechano-sensitive, such as the stretch-inactivated channel SIC, which seems to be gated by cell shrinkage (6). In contrast, the highly Ca²⁺-selective ECaC1 (CaT2) and ECaC2 (CaT1) channels are down-regulated by intracellular Ca²⁺ and have been reported to be gated by depletion of intracellular Ca²⁺ stores (7).

We have studied, in the present report, the gating properties of yet another cation channel with a moderate Ca²⁺ permeability, TRPV4 (also known as OTRPC4, VR-OAC, or TRP12), which senses changes in cell volume (8-11) by an unknown mechanism, and show that the human VRL-2 channel (identical to VR-OAC) and the murine TRP12 (identical to OTRPC4) can also be gated by binding of an agonist; phorbol esters activate both channels in astrocytoma cells 1321N1 stably transfected with VRL-2 (hVRL-2.1321N1 cells) and in HEK-293 cells transiently transfected with TRP12. In addition, we show that these phorbol esters activate a current in mouse aorta endothelial cells, which endogenously express TRP12 (8), which is identical to that in cells expressing recombinant TRP12. These novel findings may catalyze the search for an endogenous activator of this Ca²⁺-permeable TRPV channel and provide a reliable tool to study functional effects of TRPV stimulation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Culture

hVRL-2 Stable Cell Line (hVRL-2.1321N1)-- At the amino acid level, VRL-2 is 100% identical to hOTRPC4 (GenBank^TM accession number AF258465) and differs in only one amino acid with the human hVROAC (GenBank^TM accession number AF263523). 1321N1 astrocytoma cells, stably transfected with VRL-2, were grown in Dulbecco's modified Eagle's medium containing 10% (v/v) human serum, 2 mM L-glutamine, 2 units/ml penicillin, and 2 mg/ml streptomycin at 37 °C in a humidity controlled incubator with 10% CO₂. Quantitative reverse transcriptase-PCR analysis suggested that there was no expression of VRL-2 in wild type 1321N1 cells.

Transient Expression of mTRP12-- We used the recombinant bicistronic expression plasmid pdiTRP12, which carries the entire protein coding region for TRP12 and the green fluorescent protein, GFP (8), for transient transfection. Human embryonic kidney cells, HEK-293, were grown in Dulbecco's modified Eagle's medium containing 10% (v/v) human serum, 2 mM L-glutamine, 2 units/ml penicillin, and 2 mg/ml streptomycin at 37 °C in a humidity-controlled incubator with 10% CO₂. HEK-293 cells, transiently transfected with the above described vector, were visually identified in the patch clamp set up. GFP was excited at a wavelength between 425 and 475 nm. The emitted light was passed via a 495 nm dichroic mirror through a 500 nm long-pass filter. The TRP12-expressing cells were identified by their green fluorescence. Nontransfected cells from the same batch were used as controls.

Endothelial Cells-- The "primary explant technique" was used to study freshly isolated endothelial cells from mouse aorta. This method is described in detail elsewhere (12, 13). These cells express TRP12 as previously shown with Northern blot analysis (8). As a reference we used the endothelial cell line EA.hyb926 (14), which does not respond to TRP12 activation. These cells were cultured as described elsewhere (15, 16).

Solutions

For electrophysiological measurements, the standard extracellular solution contained (in mM): 150 NaCl, 6 KCl, 1 MgCl₂, 1.5 or 5 CaCl₂, 10 glucose, 10 HEPES, buffered at pH 7.4 with NaOH. The osmolality of this solution, as measured with a vapor pressure osmometer (Wescor 5500, Schlag, Gladbach, Germany), was 320 ± 5 mosmol. To compare volume-activated currents with 4-PDD activated currents, we used a solution containing (in mM): 110 NaCl, 1 MgCl₂, 1 CaCl₂, 10 glucose, 10 HEPES, 95 mannitol, buffered pH 7.4 with NaOH. The normal hypotonic stimulation (HTS)¹ was done by superfusion of the cells with this solution but without mannitol (240 mosmol, 25% reduction of osmolarity). Pipette solution was composed of (in mM): 20 CsCl, 100 cesium aspartate, 1 MgCl₂, 4 Na₂ATP, 0.1 EGTA, 10 HEPES, pH adjusted to 7.2 with CsOH. If indicated, [Ca²⁺]_i was buffered in the presence of 10 mM BAPTA to 1 nM or respective intracellular concentrations. 4-Phorbol 12,13-didecanoate (4-PDD), a non-PKC-activating phorbol ester, and phorbol 12-myristate 13-acetate (PMA, both from Sigma) were applied at concentrations between 10 nM and 30 µM from 30 mM stock solutions in ethanol. Ruthenium Red (Fluka) was used at a concentration of 1 µM. Resiniferatoxin (RTX, Sigma) and capsaicin (Sigma) were used in concentrations of 100, 500, and 1000 nM from a stock solution of 1 mM in ethanol. Capsazepine (Sigma) was used at a concentration of 10 µM. 12-HPETE (12-hydroperoxyeicosatetraenoic acid, Sigma) was used at a concentration of 10 µM in the pipette solution. Extra- and intracellular solutions contained Cs⁺ salts to inhibit K⁺ currents, which are present in a few, but not all, HEK cells. For changes in acid load, we have omitted NaOH, which resulted in a pH of the HEPES-buffered solution of 5.5. All experiments were performed at room temperature (20-23 °C).

Electrophysiological Recordings

Whole-cell membrane currents were monitored with an EPC-9 (HEKA Elektronik, Lambrecht, Germany, 8-Pole Bessel filter 2.9 kHz) using ruptured patches. Patch electrodes had a DC resistance between 2 and 4 M. An Ag-AgCl wire was used as a reference electrode. Capacitance and access resistances were monitored continuously. Between 50 and 70% of the series resistance was electronically compensated to minimize voltage errors. If not mentioned otherwise, we have applied one of two ramp protocols. (a) A protocol consisting of a voltage step to 100 mV for 20 ms followed by a 180-ms linear ramp to +100 mV, sampling interval: 0.1 ms. This protocol was repeated every 5 or 10 s. (b) A ramp protocol from 150 to +100 mV, lasting 400 ms and repeated every 5 s, sampling interval 0.8 ms. Time courses of the whole-cell current and current densities were obtained by averaging the current in a narrow window around 80 mV during the voltage ramp protocol.

Measurement of Intracellular Ca²⁺ Concentrations Using the FLIPR^TM

Intracellular Ca²⁺ concentrations were monitored using FLIPR^TM (Molecular Devices, Wokingham, UK) as described previously (17). Briefly, hVRL-2.1321N1 cells, seeded into 96-well plates (25,000 cells per well), were incubated with culture medium containing the cytoplasmic Ca²⁺ indicator, Fluo-3 (4 µM; Teflabs, Austin, TX) at 25 °C for 120 min. The cells were then washed four times with Tyrode medium containing 0.1% bovine serum albumin, before being incubated for 30 min at 25 °C with either Tyrode alone (control) or Tyrode containing various antagonists. The plates were then placed into a FLIPR^TM to monitor cell fluorescence (_ex = 488 nm, _EM = 540 nm) (18) before and after the addition of various agonists. Responses were measured as peak minus basal fluorescence intensity and where appropriate expressed as a percentage of a maximum carbachol-induced response. Data are expressed as means ± S.E. unless otherwise stated. Curve fitting and parameter estimation were carried out using GraphPad Prism 3.00 (GraphPad Software Inc., San Diego, CA). Statistical comparisons were made where appropriate using Student's t test.

Combined Ca²⁺ and Patch Clamp Measurements-- For [Ca²⁺]_i measurements combined with patch clamp, single cells were loaded with 100 µM Fura-II-potassium salt. The dye was excited alternately at wavelengths of 360 and 390 nm through a filter wheel rotating at 2 cycles/second. The fluorescence was measured at 510 nm and corrected for autofluorescence, measured from the cell-free background. Apparent free [Ca²⁺] was calculated from the fluorescence ratio R by [Ca²⁺]_i = K_eff (R  R₀)/(R₁  R), where K_eff is the effective binding constant, R₀ the fluorescence ratio at zero Ca²⁺, and R₁ that at high Ca²⁺. These calibration constants were determined experimentally for the given set-up and the actual experimental conditions used.

Data Analysis-- Electrophysiological data were analyzed using the WinASCD software (G. Droogmans, Leuven, Belgium). Pooled data are given as means ± S.E. from n cells. Significance was tested using Student's paired t test (p < 0.05 are marked with an asterisk). Dose-response curves were fitted with the following equation,

(Eq. 1)

where E_max is the maximal effect, c the agonist concentration, and EC₅₀ the concentration of half-maximal response. Inhibitory effects were described by,

(Eq. 2)

with IC₅₀ as the half-maximal blocking concentration. The permeability of monovalent cations was obtained from the shift in the reversal potentials after complete substitution of extracellular divalent cations (1 nM free [Ca²⁺]_i buffered by 5 mM EGTA and nominal Ca²⁺- and Mg²⁺-free extracellular solution) and was calculated by the following equation,

(Eq. 3)

where V_rev is the measured shift in reversal potential.

Permeability of the divalent cations Ca²⁺ and Ba²⁺ relative to Na⁺ was calculated from the reversal potential measured with 30 mM of the respective cation in the extracellular solution.

(Eq. 4)

P_X represents the permeability of the divalent cation, [X]_e its extracellular concentration,  is P_Cs/P_Na obtained from Equation 3, [Na⁺]_e, [Na⁺]_i, and [Cs⁺]_e, [Cs⁺]_i are the extra- and intracellular concentrations for Na⁺ and Cs⁺, respectively, and V_rev is the reversal potential (19, 20).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

4-PDD Activates Ca²⁺ Entry in hVRL-2-transfected 1321N1 Cells-- In hVRL-2.1321N1 cells capsaicin and RTX were without effect, while PMA elicited a Ca²⁺ response with a slow onset reaching a maintained phase in ~130 s (Fig. 1A). The phorbol ester 4-PDD, which does not activate PKC, also elicited a Ca²⁺ response, which was typified by an initially rapid, and later slower, onset that reached a maintained phase in ~40 s (Fig. 1A). These responses were not observed in the absence of extracellular Ca²⁺, and all four ligands were inactive in wild type and mock-transfected 1321N1 cells (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   4-PDD is an agonist of hVRL-2. A, [Ca2+]i (as fluorescent intensity) was monitored using Fluo-3AM in hVRL-2.1321N1 cells before and after the addition of 4-PDD (, 1 µM), PMA (, 3 µM), RTX (, 100 nM), or capsaicin (, 100 nM). Data shown are representative traces, typical of at least n = 5. B, [Ca2+]i was monitored using Fluo-3AM in hVRL-2.1321N1 cells before and after the addition of 4-PDD (100 pM to 10 µM) or PMA (100 pM to 10 µM). Responses were measured as peak increase in fluorescence minus basal, expressed relative to a maximum carbachol response, evoked by activation of endogenous muscarinic receptors, and are given as means ± S.E., where n = 5.

In hVRL-2.1321N1 cells the 4-PDD- and PMA-induced Ca²⁺ responses were concentration-dependent, with pEC₅₀ values of 6.73 ± 0.03 and 5.86 ± 0.10 (n = 5), respectively, although PMA was only a partial agonist (E_max = 65%) compared with 4-PDD (Fig. 1B).

Ruthenium Red (1 µM) abolished both the PMA (3 µM)- and 4-PDD (1 µM)-induced responses in hVRL-2.1321N1 cells, whereas capsazepine (10 µM) was without effect (data not shown).

Activation of hVRL-2 in Stably Transfected 1321N1 Cells-- Fig. 2 shows examples of combined current and Ca²⁺ measurements in the control human astrocytoma cell line 1321N1 and in hVRL-2.1321N1 cells. The control cell line showed background currents, which were outwardly rectifying with densities of 0.4 ± 0.2 pA/pF at 80 mV and ± 1.8 ± 0.4 pA/pF at +80 mV and a reversal potential of 7 ± 2 mV (n = 7). After application of 1 µM 4-PDD neither [Ca²⁺]_i nor the current was increased (Fig. 2, A-C). If the stable cell line expressing hVRL-2 was used, application of 4-PDD increased [Ca²⁺]_i, inward and outward currents, and shifted the reversal potential of the activated current toward more positive potentials (Fig. 2, D-I). The increase in [Ca²⁺]_i was absent in Ca²⁺-free bath solutions (n = 7, data not shown). As shown later, monovalent ion current could be measured in nominally Ca²⁺-free solutions, indicating that 4-PDD still functioned as an agonist in these conditions.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Stimulating effects of 4-PDD application on astrocytoma 1321N1 cells stably expressing hVRL-2 (D-I) and nontransfected control astrocytoma 1321N1 cells (A-C). A, Ca2+ measurements in a voltage-clamped 1321N1 astrocytoma cell, which does not express VRL-2. Holding potential is 0 mV. Application of 1 µM 4-PDD does not induce any increase in [Ca2+]i. B, time course of whole cell currents obtained from linear voltage ramps. Currents were measured at 80 and +80 mV. C, IV curves obtained from voltage ramps from 100 to +100 mV measured at the times indicated in B. Note that no current is activated during application of 4-PDD. D, same protocol as in A. Note that 0.1 µM 4-PDD induced an increase in [Ca2+]i. E, application of 0.1 µM 4-PDD activates a current. Time course was measured as in B. F, IV curves show activation of an outwardly rectifying current by 4-PDD. Data are from the time indicated in E. G, 1 µM 4-PDD activates a large increase in [Ca2+]i. The same protocol as in A was used. H, current activation by 1 µM 4-PDD concomitant with the increase in [Ca2+]i. I, IV curves of current activated by 1 µM 4-PDD in both inward and outward direction.

Data obtained from all cells are shown in Fig. 3. Half-maximal increase of [Ca²⁺]_i and the current measured at 80 mV (to avoid an eventual contribution of Ca²⁺-activated Cl currents at positive potentials) occurred at pEC₅₀ of 6.71 ± 0.04 and 6.86 ± 0.11 (n between 6 and 17), respectively (Fig. 3, A and B). The reversal potential shifted by ~5 and 15 mV toward positive potentials after application of 0.1 and 1 µM 4-PDD, respectively (Fig. 3C). Obviously, 4-PDD activated a Ca²⁺ influx and an outwardly rectifying current in hVRL-2-expressing, but not in hVRL-2-lacking cells.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Concentration response relationship of the elevation of [Ca2+]i and the increase of the VRL-2 current by 4-PDD. A, dependence of the elevation of [Ca2+]i at a holding potential of 0 mV on 4-PDD. Data were fitted to Equation 1 (n indicates the number of cells). A half-maximal activation was achieved at pEC50 = 6.71. The same experimental protocol as shown in Fig. 2 was used. All data are given as means ± S.E. B, maximal increase in current at 80 mV during application of 4-PDD. Data were obtained from simultaneous [Ca2+]i  current measurements. Note that [Ca2+]i is not buffered. The pipette solution contains 0.1 mM EGTA. pEC50 is 6.86. C, application of 4-PDD induced a shift of reversal potential Vrev toward more positive potentials. This shift also depends on the 4-PDD concentration.

Current Activation in TRP12-transfected HEK-293 Cells-- TRP12 is a channel cloned from mouse kidney and is ~95% identical to human VRL-2 (mouse mTRP12; GenBank^TM accession number CAC20703), suggesting that it is the mouse orthologue of VRL-2. Fig. 4 shows combined current and Ca²⁺ measurements in nontransfected HEK cells and in HEK cells transiently transfected with murine TRP12. Background currents in the control cell line showed weak outward rectification, current densities of 3 ± 1 pA/pF at 80 mV and 8 ± 5 pA/pF at +80 mV, and a reversal potential of 5 ± 2 mV (n = 8). The background current in TRP12-expressing cells was 5 ± 3 pA/pF at 80 mV and 13 ± 5 pA/pF at +80 mV (n = 8). The reversal potential of this current was measured at 6 ± 2 mV (n = 8). Application of 1 µM 4-PDD induced a small increase in [Ca²⁺]_i in nontransfected cells, activated an outwardly rectifying current, and shifted the reversal potential of the net current toward more positive potentials (Fig. 4, A-C). These effects were significantly enhanced in TRP12-transfected cells (Fig. 4, D-F). Application of 1 µM 4-PDD increased [Ca²⁺]_i to 0.08 ± 0.02 µM in nontransfected cells (n = 8) and to 0.43 ± 0.1 µM in TRP12-transfected cells (n = 8).

View larger version (37K): [in this window] [in a new window]   Fig. 4.   Elevation of [Ca2+]i and current activation by 4-PDD in a nontransfected HEK cell and in a HEK cell expressing mTRP12. A, application of 1 µM 4-PDD induced in a nontransfected HEK cell a very small increase in [Ca2+]i. Cells were clamped at 0 mV. Experimental protocol was the same as in Fig. 2. Note that the HEK cells used in these experiments expressed endogenous VRL-2 as shown in reverse transcriptase-PCR experiments. B, currents measured before and during application of 4-PDD, same cell as in A. C, current-voltage relationships obtained from voltage ramps measured at the times indicated in B. D, in TRP12-transfected HEK cells a much larger increase in [Ca2+]i can be observed after application of 4-PDD. The same protocol as in A was used. E, current activation from the same cells as shown in D, which closely correlated to the increase in [Ca2+]i. Note again that in these cells pipette solution only contains 0.1 mM EGTA. Note that in this experiment [Ca2+]i is buffered at 1 nM, whereas in A-G, [Ca2+]i is not buffered. IV curves were measured at the times indicated in E. Note the increase in inward and outward currents and the shift of the reversal potential. F, stimulation of TRP12 by cell swelling (HTS, solid line above the traces). On top of the HTS stimulus, 1 µM 4-PDD was applied (dotted line). The cell was clamped at 0 mV. The same ramp protocol was used as described in Fig. 2. Current was measured at +80 and 80 mV. Note that application of NMDG+ completely inhibits the inward current. G, IV curves measured at the times indicated in G. Note the increase in inward and outward currents and the shift of the reversal potential.

To compare the 4-PDD stimulation of TRP12 with its previously described activation by cell swelling, we applied 4-PDD during perfusion of the cell with a hypotonic solution. As shown in Fig. 4G, volume-activated TRP12 currents were stimulated with a latency and reached a constant level. This behavior has been described already elsewhere (11). Application of 4-PDD further increased the current substantially. The activated current is a cation current, because its inward component disappeared when permeable cations were substituted by NMDG⁺ (Fig. 4G). The current component activated by 4-PDD was 9.1 ± 2.2 times larger than the volume-activated component (n = 6, all measured at 80 mV). Under both conditions, currents are relatively slowly activated. Current activation appeared with a latency of 22 ± 5 s for 4-PDD (n = 8) and 29 ± 4 s for cell swelling (n = 15). Peak currents appeared after 85 ± 26 s for 4-PDD (n = 8) and 76 ± 9 s for HTS (n = 15).

Concomitantly, the phorbol ester enhanced the amplitude of the outwardly rectifying current. Application of the phorbol ester PMA (10 µM) also activated TRP12 (Fig. 5, A and B). However, the maximal effects of PMA were approximately two times smaller than those of 4-PDD, and the IC₅₀ value was 10 times higher than for 4-PDD (Fig. 5C), similar to the relative efficacy seen in VRL-2 (Fig. 1B). Fig. 5 shows the dose-response relationship of the current response during application of 4-PDD and PMA in cells with a pipette solution with [Ca²⁺]_i buffered at 1 nM. From the fit of the current responses at various concentrations of 4-PDD to Equation 1, we obtained a maximal current of 131 pA/pF (80 mV) with a pEC₅₀ of 6.67 (Fig. 5C). The reversal potential shifted toward a more positive potential, reaching a maximal shift of about 10 mV at 1 µM 4-PDD (Fig. 5D).

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Dose-response relationship of the current increase by 4-PDD and PMA in HEK cells transiently expressing mTRP12. A, activation of an outwardly rectifying current after 10 µM PMA. Holding potential is 0 mV. The same protocol as in Fig. 4 was used. B, IV relationship measured at the times indicated in A. C, the increase in current by 4-PDD (open circles) was measured at 80 mV. Data were fitted by Equation 1. Half-maximal activation was obtained at a pEC50 of 6.67, nearly identical to that in VRL-2-transfected cells. Maximal current was 131 pA/pF. In these cells [Ca2+]i was buffered at 1 nM, which results in much larger current as shown in Fig. 4. Dose-response relationship of PMA (open squares) is characterized by a pEC50 of 5.52 and a maximal current at 60 pA/pF. D, shift of the reversal potential depends on the 4-PDD concentration.

The characteristics of the 4-PDD-activated current are similar to those of the current activated by cell swelling in these same cells (11). The relative conductance measured at 80 mV changed in the sequence K⁺:Cs⁺:Na⁺:Li⁺ = 1.34 ± 0.1:1.1 ± 0.11:1:0.68 ± 0.13 (n = 7, Fig. 6C). The permeability sequence of the 4-PDD activated current for various monovalent cations, as determined from shifts in reversal potential upon extracellular cation substitution (Fig. 6, A and B), was Eisenman IV with P_K:P_Cs:P_Na:P_Li = 1.21 ± 0.02:1.10 ± 0.015:1:0.85 ± 0.03 (n = 7, Fig. 6D). The cation channel activated by 4-PDD was also permeable for Ca²⁺ as reported elsewhere (9, 11). Fig. 7 shows an example in which Ca²⁺ is the only charge carrier for inward current after substitution of all extracellular monovalent cations with NMDG⁺. Stimulation of TRP1-expressing HEK cells with 1 µM 4-PDD increased the inward current at 80 mV as well as the outward current at +80 mV (Fig. 7A), but induced only very small effects in nontransfected cells. This increase of the current was transient and decreased in the maintained presence of 4-PDD. Current-voltage relationships, as shown in Fig. 7B, indicate the activation of a strongly outwardly rectifying current with a P_Ca:P_Na ratio of 5.8 ± 0.5, as calculated from Equations 3 and 4. The reversal potential of the current measured in the presence of intracellular Cs⁺ and 30 mM extracellular Ca²⁺ as the only cation was 26.6 ± 1.4 mV. Ba²⁺ was much less permeable than Ca²⁺ with a ratio P_Ba:P_Na of 0.7 ± 0.5 calculated from the reversal potential of 5.4 ± 2.6 mV in the presence of 30 mM Ba²⁺. Increasing [Ca²⁺]_e from 1 to 30 mM shifted the reversal potential by 33 ± 2.5 mV, which is less than the expected 44-mV shift for a Ca²⁺-selective cation channel. Taken together these data indicate that the channel activated by 4-PDD is a Ca²⁺-permeable cation channel as described for TRP12, OTRPC4, and VR-OAC activated by cell swelling.

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Permeation properties of the 4-PDD-activated channel, mTRP12. A, current-voltage relationships obtained after maximal stimulation of TRP12-transfected HEK cells with 1 µM 4-PDD. During stimulation, the extracellular Na+ was completely substituted by NMDG+, Li+, Cs+, or K+. The same experimental protocol as in Fig. 4 was used. B, same cell as in A. The voltage range close to the reversal potential is zoomed to show the shift due to the various Na+ substitutions. Note that K+ is the best permeating cation and Li+ the least permeable. C, relative conductance was calculated from the maximal increase in current after application of 1 µM 4-PDD at 80 mV divided by the current carried by Na+. Ratios were obtained from the same cells (n = 6). D, the relative permeability was measured from the shift in the reversal potentials (B) and calculated by Equation 3. Data are from six cells with a complete set of substitutions as shown in A.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Permeation of Ca2+ though the 4-PDD-activated channel. A, time course of the whole-cell current measured at 80 and +80 mV from linear voltage ramps. Up to the dotted line, TRP12-transfected HEK cells were perfused with normal bath solution. At the time indicated, all monovalent cations were substituted by NMDG+ and 30 mM Ca2+. Therefore, inward currents can only be carried by Ca2+. Application of 1 µM 4-PDD induced a large but transient increase in both inward and outward current. Ca2+ in the pipette solution is buffered at 1 nM. B, current-voltage relationships measured at the times a and b, indicated in A. Note the rightward shift of the IV curve.

To further evaluate whether the slow increase of [Ca²⁺]_i is indeed the result of a slow influx of Ca²⁺ during the slow channel activation, we have activated TRP12 by 4-PDD in Ca²⁺-free solutions and then applied extracellular Ca²⁺. Under these conditions, 4-PDD, as expected, induced a delayed activation of an outward current (the inward component is absent because of substitution of all cation by NMDG⁺, Fig. 8, A and C). After activation, 30 mM [Ca²⁺]_e was applied, which induced a nearly instantaneous appearance of an inward current and a very fast increase in [Ca²⁺]_i (Fig. 8B). These data indicate a fast entry of Ca²⁺ when the channel has been pre-activated. In addition, no Ca²⁺ signal was obtained during stimulation with 4-PDD in the absence of [Ca²⁺]_e, indicating the absence of Ca²⁺ release during activation (identical results in five cells).

View larger version (22K): [in this window] [in a new window]   Fig. 8.   Fast Ca2+ entry after pre-activation of TRP12 by 4-PDD. A, TRP12-transfected HEK cells were incubated in a Ca2+-free solution. All extracellular cations have been substituted by NMDG+. The current at the holding potential of 0 mV is shown. At the time indicated, 1 µM 4-PDD was applied and induced a large outward current but no inward current because of the absence of permeable extracellular cations. Reapplication of 30 mM Ca2+ resulted in an instantaneous appearance of an inward current. B, time course of [Ca2+]i after reapplication of Ca2+. Note the fast increase in Ca2+ due to a fast Ca2+ influx through the pre-activated channel (pipette solution: 0.1 mM EGTA as in all simultaneous current/Ca2+ measurements). C, whole cell current measured at 80 and +80 mV from linear voltage ramps. Note the appearance of an inward current after Ca2+ reapplication and the decrease in the outward current. D, current-voltage relationships measured at the times a, b, and c indicated in C. Note the rightward shift of the IV curve.

We have shown that Ca²⁺ entry through VRL-2 was blocked by Ruthenium Red (data not shown). Ruthenium Red at a concentration of 1 µM completely blocked the 4-PDD-activated inward current through TRP12, but was less effective on the outward current (Fig. 9, A-D).

View larger version (23K): [in this window] [in a new window]   Fig. 9.   Block by Ruthenium Red of mTRP12 channels activated by 4-PDD. A, activation of a membrane current in TRP12-transfected HEK cells by application of 1 µM 4-PDD. Experimental protocol is the same as used in the previous figures. Ruthenium Red (RR, 1 µM) induced a rapid decrease of the holding current at 0 mV. After washing out of RR, the 4-PDD-induced current reappears. The spikes in the current recordings are from the 100 to +100 mV voltage ramps applied every 5 s. B, time course of the inward current at 80 mV and the outward current at +80 mV. Note the complete block of the inward current and the partial inhibition of the outward current. C, IV curves measured at the times indicated in B show the voltage-dependent block by RR. D, inhibition of the mTRP12 current by RR at 80 and +80 mV from five cells.

Because several members of the TRPV family are modulated by [Ca²⁺]_i (for a detailed example, see Ref. 21), we have also studied a possible modulation of the 4-PDD-activated TRP12 channel by changes in [Ca²⁺]_i. Fig. 10 gives an example for the response to 1 µM 4-PDD of TRP12-expressing HEK cells that were dialyzed by intracellular solution in which [Ca²⁺]_i was buffered at 1, 200, and 1000 nM. The response was clearly reduced when [Ca²⁺]_i was increased (Fig. 10, A and B). Both inward and outward currents were reduced to the same extent. A concentration response plot of the maximal current at 80 mV evoked by 1 µM 4-PDD showed a half-maximal inactivation at a Ca²⁺ concentration of 406 nM (Fig. 10C). Clearly, TRP12 is inhibited by elevation of [Ca²⁺]_i.

View larger version (27K): [in this window] [in a new window]   Fig. 10.   Inhibition of TRP12 currents by intracellular Ca2+. A, currents were activated by 1 µM 4-PDD (three separate TRP12-transfected HEK cells). [Ca2+]i was buffered at 1, 200, and 1000 nM. Shown are the time courses of the whole-cell current at 80 and +80 mV. Note the decrease in inward and outward current when [Ca2+]i is increased. B, IV relationships from the times indicated by the black circles in A. Clearly, current activation was decreased when [Ca2+]i was elevated. C, from maximal current at 80 mV the concentration response curve was measured. Data points were fit to Equation 2. Assuming a complete block by high [Ca2+]i, half-maximal inhibition was calculated to be 406 nM.

Current Activation in Endothelial Cells Expressing Endogenous TRP12-- To test whether the novel agonist for TRP12 is also effective on native channels, we tested freshly isolated mouse aorta endothelial cells (MAEC). These cells express TRP12 as shown in Northern blots (8). As a reference we used human umbilical vein endothelial cell-derived EA cells, which do not express VRL-2. Stimulation with 1 µM 4-PDD evoked, in both clamped and unclamped MAEC, a Ca²⁺ signal in the presence of 5 mM [Ca²⁺]_e but not in the absence of extracellular Ca²⁺ (Fig. 11). The agonist was, however, ineffective in EA cells. 4-PDD also activated in MAEC, but not in EA cells, an outwardly rectifying cation current and a concomitant shift of the reversal potential toward more positive potentials (Fig. 12). In conclusion, the agonist 4-PDD induced the same responses in cells that express TRP12 endogenously as in VRL-2- or TRP12-transfected cells.

View larger version (19K): [in this window] [in a new window]   Fig. 11.   Increase of [Ca2+]i by 4-PDD in MAEC but not in the EA-926hyb cell line derived from human umbilical vein endothelium. A, 4-PDD does not increase [Ca2+]i in EA-926hyb cells. B, increase in [Ca2+]i in nonclamped MAEC by 1 µM 4-PDD. C, pooled data from clamped (holding potential: 0 mV) and unclamped EA-926hyb and MAEC. MAEC, which express TRP12, respond to 4-PDD by an increase in [Ca2+]i. This increase is accentuated in nonclamped cells, likely due to activation of BKCa in MAEC (12, 43).

View larger version (23K): [in this window] [in a new window]   Fig. 12.   Increase of [Ca2+]i and current by 1 µM 4-PDD in MAEC. A, 4-PDD increases [Ca2+]i in voltage-clamped MAEC. Holding potential: 0 mV. The same protocol as in Figs. 2 and 4 was used (0.1 mM EGTA in the pipette solution). B, current activation by 4-PDD. C, IV curves measured at the times indicated in B. D, increase in current measured at 80 mV in TRP12-expressing MAEC and the reference EA-926hyb cells. E, shift in the reversal potential of the 4-PDD-activated currents toward positive potentials but not in the EA-926hyb reference cells, which do not express VRL-2.

	DISCUSSION

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VRL-2 is identical to the human TRPV channel VR-OAC and apparently represents the human orthologue of the murine TRP12 and OTRPC4 channels (mouse mTRP12, GenBank^TM accession number CAC20703; hOTRPC4, GenBank^TM accession number AF258465; human hVROAC, GenBank^TM accession number AF263523). These channels belong to the TRPV family and are widely expressed in mammals. Originally, these channels were considered as sensors of cell volume, because they can be activated by cell swelling (8-11). These channels are all Ca²⁺-permeable, providing an influx route for extracellular Ca²⁺ and could therefore play an important role in Ca²⁺ signaling during mechanical or osmotic stimulation as well as under conditions coupled to changes of the cell volume. In addition, any stimulation of these channels will change the membrane potential of the stimulated cells. However, the mechanism of activation by cell swelling was clearly different from activation of the best studied volume-regulated anion channel, which likely senses changes in cell volume as a variation of intracellular ionic strength (22, 23). It has been shown that in contrast with volume-regulated anion channels TRP12 cannot be activated by a reduction in intracellular ionic strength or by an intracellular perfusion of TRP12-expressing cells with GTPS (11). We have therefore tested other possibilities of channel activation.

Several members of the TRPV family are agonist-gated channels, which are opened by binding of capsaicin and related compounds (3, 24). However, an endogenous ligand remains to be identified (24). Only recently, the endogenous lipid anandamide, which has some structural similarities with olvanil, has been identified as a possible endogenous agonist of the hVR-1 receptor (17). With a similar approach, using the FLIPR^TM screening technique, we have now also identified a highly selective activator of VRL-2 and its murine orthologue TRP12. Phorbol derivatives stimulate VRL-2 and TRP12. PMA and 4-PDD stimulate both VRL-2 and TRP12, with 4-PDD being ~50 times more potent than PMA. Several lines of evidence demonstrate that 4-PDD can be considered as a VRL-2 and TRP12 activator; (a) in the 1321N1 stable cell line transfected with VRL-2, 4-PDD induces an increase in [Ca²⁺]_i by entry of extracellular Ca²⁺, a concomitant activation of an outwardly rectifying cation current, and a substantial shift in the reversal potential of the net whole-cell current toward more positive potentials. All effects occur with a pEC₅₀ of ~6.8 and are absent in cells that do not express VRL-2; (b) 4-PDD induced the same effect in HEK cells transiently transfected with mTRP12. Current activation occurs with the same pEC₅₀. The 4-PDD-activated channel shows an Eisenman IV permeation profile, which has previously been shown for TRP12 (11) and has similar Ca²⁺ over Na⁺ permeability as reported for OTRPC4 (9). In wild type HEK cells, which express VRL-2 under our conditions, 4-PDD has a small but identical effect as in the cells overexpressing TRP12. (c) In freshly isolated endothelial cells, MAEC, which substantially express endogenous TRP12 (8), 4-PDD induced an identical effect as in the other models used. Because 4-PDD does not activate PKC (25), the stimulating effect is obviously not mediated by PKC activation. Several reports have already identified phorbol esters as direct, PKC-independent channel modulators of N- and L-type Ca²⁺ channels and K_ATP channels (26, 27-31). It is known that phorbol esters bind to a variety of lipophilic receptors (25) to which now, as a novel target, VRL-2/TRP12 should be added. From our data, it seems unlikely that the identified agonist binds to an extracellular site because of the very slow wash-out (Fig. 9). Furthermore, the slow activation may indicate that 4-PDD must enter the cell and may activate the channel by binding to an intracellular site.

So far, we have not clearly identified an endogenous activator. However, preliminary experiments showed that anandamide (from Sigma, 10 µM, extracellular application, n = 8) is a weakly potent and slow activator of TRP12 (n = 8).² 1-Oleolyl-2-acetyl-sn-glycerol (50-100 µM extracellular, n = 6), 11,12-epoxyeicosatrionic acid (300 nM, intracellular, n = 5), leukotriene D₄ (100 nM intracellular, n = 3, and 100 nM extracellular, n = 5), 500 nM extracellular epoxyeicosatrionic acid (n = 3), 1 µM capsaicin (extracellular, n = 4), 10 µM 12-HPETE (intracellular, n = 5) and pH 5.5 (n = 3), did not induce any activating effect.³ However, 4-PDD can be used as a robust and reliable tool to study several features of TRPV channels and to probe functional effects of the activation of this channel in in vivo systems. As an example, we have already demonstrated the utility of 4-PDD for any kind of permeation studies, including studies on pore mutants. To further characterize properties of VRL-2/Trp2, we have used 4-PDD as a tool to demonstrate inhibition of this channel by elevated [Ca²⁺]_i. Half-maximal inhibition occurred at 406 nM, which is clearly in the physiological range for many activated cells. A similar, but more sensitive, inhibition by intracellular Ca²⁺ has been also described for ECaC channels, which are close relatives of the VR1 and VRL channels (21). We further show that by using 4-PDD as a reliable tool for activation of VRL-2 and TRP12, we could demonstrate block of both channels by Ruthenium Red (RR). This hexavalent cation binds to phospholipids and fatty acids (32), to many Ca²⁺-binding proteins, the mitochondrial Ca²⁺ uniporter and various Ca²⁺ channels (33-35), the ryanodine receptor (36, 37), and has recently also been identified as a selective blocker of the founding member of the TRPV family, VR-1 (38). We have recently shown that the TRPC members, ECaC1 and ECaC2, which are about 30% identical at the amino acid level to VR-1, are blocked in the submicromolar range by RR (39, 40). We show here in addition that block of TRP12 is voltage-dependent as the block decreased at positive potentials. A voltage-dependent block by RR has been shown so far only for a plant vascular Ca²⁺ channel (41). Inward currents through TRP12 are completely blocked by 1 µM RR. Therefore, RR might be usable as a high affinity tool for TRP12 and in general for TRPV-related channels.

For all TRPV4 members (TRP12, OTRPC4, and VR-OAC), it has been shown that an increase in cell volume activated a current and increased [Ca²⁺]_i (8-11). The channels activated by an increase in volume and 4-PDD are identical referring to rectification behavior, Ca²⁺ permeability, and permeability sequence of monovalent cations. A comparison of the current amplitudes shows that the volume-activated TRP12 current has a density at 80 mV of 44 pA/pF (11) and 131 pA/pF after stimulation with 4-PDD (Fig. 5). When we compared, in this series of experiments, the stimulation of TRP12 with 25% hypotonic solution and 1 µM 4-PDD in the same cells, we even found a 9.1 ± 2.2 times larger activation by the agonist. Although difficult to compare, the time course of activation by both methods was not different. This can be explained in the case of 4-PDD by a slow diffusion to an intercellular binding site or in the case of cell swelling by the slow signal transduction in which synthesis of a messenger could be involved. Thus, an increase in cell volume seems to be only a partial activator, and both stimuli might act additively. It is not clear whether cell swelling activates TRP12 via a similar pathway than 4-PDD, but with less efficiency, or via another, independent mechanism. It has also been previously demonstrated that cell swelling increased [Ca²⁺]_i, probably via an arachidonic acid-dependent mechanism (42). Therefore, it is intriguing to speculate that the volume-dependent activation of TRP12 might be due to a messenger, which shares structural similarities with 4-PDD, and might be related to metabolites of arachidonic acid or anandamide.

In conclusion, we show here for the first time that binding of a lipophilic ligand can activate VRL-2 and TRP12. These findings will aid the search for specific endogenous activators and provide a reliable tool for studying functional effects of this novel class of TRPV channels, which were until now only considered as mechano- or volume-activated Ca²⁺-permeable cation channels. In addition, 4-PDD will be a valuable pharmacological tool with which to characterize endogenous swelling-activated currents that may be carried by native VRL-2/TRP12 channels.

	FOOTNOTES

^* This work was supported by the Belgian Federal Government, the Flemish Government and the Onderzoeksraad KU Leuven (GOA 99/07, F.W.O. G.0237.95, F.W.O. G.0214.99, F.W.O. G. 0136.00; Interuniversity Poles of Attraction Program, Prime Ministers Office IUAP Nr.3P4/23), by "Levenslijn" (7.0021.99), and by a grant from the "Alphonse and Jean FortonFonds-Koning Boudewijn Stichting" R7115 B0.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Laboratorium voor Fysiologie, Campus Gasthuisberg, KU Leuven, Herestraat 49, B-3000 Leuven, Belgium. Tel.: 32-16-34-5937; Fax: 32-16-34-5991; E-mail: bernd.nilius@med.kuleuven.ac.be.

Published, JBC Papers in Press, February 4, 2002, DOI 10.1074/jbc.M200062200

² H. Watanabe and B. Nilius, unpublished results.

³ J. Vriens, H. Watanabe, and B. Nilius, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: HTS, hypotonic stimulation; 4-PDD, 4-phorbol 12,13-didecanoate; F, farad; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; RTX, resiniferatoxin; 12-HPETE, 12-hydroperoxyeicosatetraenoic acid; MAEC, mouse aorta endothelial cells; GTPS, guanosine 5'-O-(thiotriphosphate); RR, Ruthenium Red.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES