The Transient Receptor Potential Protein Homologue TRP6 Is the Essential Component of Vascular a 1 -Adrenoceptor–Activated Ca 2 1 -Permeable Cation Channel

—The Drosophila transient receptor potential protein (TRP) and its mammalian homologues are thought to be Ca 2 1 -permeable cation channels activated by G protein (G q/11 )–coupled receptors and are regarded as an interesting molecular model for the Ca 2 1 entry mechanisms associated with stimulated phosphoinositide turnover and store depletion. However, there is little unequivocal evidence linking mammalian TRPs with particular native functions. In this study, we have found that heterologous expression of murine TRP6 in HEK293 cells reproduces almost exactly the essential biophysical and pharmacological properties of a 1 -adrenoceptor–activated nonselective cation channels ( a 1 -AR–NSCC) previously identified in rabbit portal vein smooth muscle. Such properties include activation by diacylglycerol; S-shaped current-voltage relationship; high divalent cation permeability; unitary conductance of 25 to 30 pS and augmentation by flufenamate and Ca 2 1 ; and blockade by Cd 2 1 , La 3 1 , Gd 3 1 , SK&F96365, and amiloride. Reverse transcriptase–polymerase chain reaction and confocal laser scanning microscopy using TRP6-specific primers and antisera revealed that the level of TRP6 mRNA expression was remarkably high in both murine and rabbit portal vein smooth muscles as compared with other TRP subtypes, and the immunoreactivity to TRP6 protein was localized near the sarcolemmal region of single rabbit portal vein myocytes. Furthermore, treatment of primary cultured portal vein myocytes with TRP6 antisense oligonucleotides resulted in marked inhibition of TRP6 protein immunoreactivity as well as selective suppression of a 1 -adrenoceptor–activated, store depletion–independent cation current and Ba 2 1 influx. These results strongly indicate that TRP6 is the essential component of the a 1 -AR–NSCC, which may serve as a store depletion–independent Ca 2 1 entry pathway during increased sympathetic activity. ( Circ Res . 2001;88:325-332.)

T he ␣ 1 -adrenoceptor (␣ 1 -AR) is distributed widely in the vascular system and plays a central role in control of systemic blood pressure via sympathetic nerves. Stimulation of the ␣ 1 -AR leads to activation of G protein (G q/11 )-coupled phospholipase C␤ (PLC␤), which catalyzes formation from phosphoinositide of 2 major metabolites, inositol 1,4,5triphosphate (IP 3 ) and diacylglycerol (DAG), thereby causing a release of stored Ca 2ϩ and an accompanying sustained Ca 2ϩ entry. 1 The ␣ 1 -AR-activated nonselective cation channel (␣ 1 -AR-NSCC) is thought to contribute to this Ca 2ϩ entry in both direct and indirect ways, since it is activated by DAG and allows preferential movement of divalent cations and secondarily evokes Ca 2ϩ entry through the voltage-dependent pathway by depolarizing the membrane. [2][3][4] Despite this potential physiological importance, no clues elucidating the molecular entity of ␣ 1 -AR-NSCC have been obtained so far.
The transient receptor potential (trp) gene and its closest relative trpl (trp-like) were originally identified in investigation of abnormal visual transduction of Drosophila melanogaster and were later shown to encode Ca 2ϩ -entry channels that open during activation of the rhodopsin/G protein/PLC/ IP 3 signaling cascade. 5 Subsequently, their 7 mammalian homologous genes (trp1 to trp7) have been cloned, in the hope of elucidating the molecular counterparts of native receptor-operated Ca 2ϩ entry channels (ROCCs) in mammals (including those activated by store depletion; G protein; or second messengers such as IP 3 , DAG, arachidonic acid, and Ca 2ϩ ) on stimulation of G protein-coupled receptors (GPCRs) or tyrosine kinase-coupled receptors (RTKs). 6 -11 Although functional expression of these mammalian trpencoding proteins (TRP homologues) in the heterologous system demonstrated the appearance of phosphoinositide turnover-linked Ca 2ϩ -permeable cation conductance (channel activity or Ca 2ϩ fluorescence increase), it remains unclear how they correspond to particular ROCCs in the native system, except for some studies implicating TRP1, TRP3, TRP4, and TRP5 in store-operated or capacitative Ca 2ϩ entry. 6 -10,12,13 In this study, we have obtained the first clear evidence that a mammalian TRP homologue, TRP6, 14 the human isoform of which was previously shown to act as a DAG-activatable cation channel rather than the store depletion-operated Ca 2ϩ channel (SOC), 10,15 is likely to be the molecular identification of the ␣ 1 -AR-NSCC that has also been reported to be activated by DAG in rabbit portal vein smooth muscle. 16 To examine this, we made a detailed comparison between recombinantly expressed TRP6 protein and the ␣ 1 -AR-NSCC using molecular and electrophysiological techniques, and examined the expression of TRP6 mRNA and the functional significance of TRP6 proteins in portal vein smooth muscle, employing the reverse transcriptase-polymerase chain reaction (RT-PCR), immunocytochemistry and antisense strategy.

Recombinant Expression, Electrophysiology, and Fluorescence Measurements
HEK293 cells were transfected with one of the recombinant plasmids pCI-neo-mTRP6, -mTRP3, or -mTRP7 17,18 and were used for electrophysiological experiments within 48 to 72 hours. For antisense experiments, myocytes enzymatically dissociated from the rabbit portal vein smooth muscle 19 were maintained in a short-term culture (3 to 4 days) in a laminin (20 g/mL)coated dish containing DMEM supplemented with 2% FBS plus antibiotics and TRP antisense or sense oligonucleotides (5 mol/L) ( Table 1). Cells were then reseeded on coverslips and used within 12 to 24 hours for electrophysiological and fluorescent measurements.
Ba 2ϩ fluorescence was measured using a dual-excitation wavelength spectrofluorometer (CAM 230, Nihon Bunko). Fura-2-loaded cells (incubated with 2 mol/L fura-2-acetoxymethyl ester for 30 to 45 minutes) were alternately illuminated by UV lights (340 and 380 nm, 100 Hz), and the emitted fluorescence was collected after filtering at 510 nm (Ϯ30 nm). The extent of Ba 2ϩ influx was assessed as the ratio of fluorescence intensity at 340 and 380 nm excitation. All experiments were performed at 24°C to 26°C.
All data are expressed as meanϮSEM. Student t test and 1-way ANOVA were used for single-and multiple-comparison statistical analyses, respectively.

RT-PCR
Total RNA was extracted from the whole rabbit and murine portal veins or isolated smooth muscles, and first-strand cDNA generated from 1 g of total RNA was subjected to PCR amplification using TRP homologue-specific primers. The PCR protocol was as follows: 10 cycles of 30 seconds at 94°C, 30 seconds at 94°C, 30 seconds at 64°C, and 1 minute at 68°C, followed by 30 cycles of 30 seconds at 94°C, 30 seconds at 60°C, and 1 minute at 68°C. PCR products were identified by hybridization with 32 P-5Јend-labeled synthetic oligonucleotide probes. For the PCR primers and oligonucleotide probes used, see the supplementary information (available at http://www.circresaha.org).
An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.

Murine TRP6 Is a DAG-Activated Channel
Human embryonic kidney 293 (HEK293) cells transiently expressing murine TRP6 protein (mTRP6) exhibited virtually no spontaneous currents under normal conditions (current density at Ϫ60 mV, 0.15Ϯ0.1 pA/pF; nϭ31). Exogenously applied ATP and CCh dose-dependently produced inward currents in these cells (ED 50 , 4.5 and 9.1 mol/L, respectively; nϭ5 to 6), whereas no discernible currents were activated by these agonists in control cells transfected with the empty vector (15 of 15 cells). The ATP and CCh-induced inward currents in mTRP6-expressing cells (hereafter designated as mTRP6 currents) were strongly inhibited by pretreatment with suramin (100 mol/L; by 93Ϯ3%, nϭ5) and atropine (1 mol/L; by 91Ϯ5%, nϭ5), respectively, thus suggesting that the endogenous P 2Y and muscarinic receptors in HEK293 cells mediate their activation.
It has recently been reported that the human TRP6 channels expressed in CHO-K1 cells with the G q/11 -coupled H 1 histamine receptor are activated by DAG through a mechanism independent of protein kinase C (PKC). 15 We therefore tested whether this applies to mTRP6 recombinantly expressed in HEK293 cells at the whole-cell current level. As summarized in Figure 1A, (1) significant suppression of mTRP6 current activation occurred in the presence of the PLC inhibitor U73122 (10 mol/L) and with intracellular perfusion of GDP␤S (100 mol/L), but not by the PKC inhibitor calphostin C (1 mol/L) or intracellular perfusion of the IP 3 receptor inhibitor heparin (1 mg/mL); (2) inward currents having a similar nature to mTRP6 current were activated by bath-applied membrane-permeable analogues of DAG, 1-oleoyl-2-acetyl-sn-glycerol (OAG, 100 mol/L) and 1,2-dioctanoyl-sn-glycerol (100 mol/L; data not shown), and the DAG lipase inhibitor RHC80267 (100 mol/L) or intracellular perfusion of GTP␥S; and (3) the PKC activator phorbol 12,13-dibutyrate (up to 1 mol/L), photolytic release of IP 3 , and depletion of internal Ca 2ϩ stores by thapsigargin (2 mol/L) were almost ineffective at activating inward currents. These results collectively suggest the primary significance of DAG mediated through G protein-coupled PLC stimulation and largely exclude the involvement of PKC or IP 3 -mediated store depletion in activating mTRP6 currents, thus being consistent with the conclusions obtained for human TRP6. 15 The activation profile of mTRP6 described above is strongly reminiscent of a native second messenger-activated cation channel, ie, the ␣ 1 -AR-NSCC, in the rabbit portal vein smooth muscle. 15,16 Thus, to determine a possible molecular correspondence between TRP6 and the native ␣ 1 -AR-NSCC, we made a detailed comparison of their biophysical and pharmacological properties in terms of patch-clamp technique (see below).

mTRP6 Shows a Voltage Dependence Similar to That of ␣ 1 -AR-NSCC
The current-voltage (I-V) relationship of mTRP6 showed a marked voltage-dependent inhibition at strongly negative potentials (ϽϪ40 mV; Figure 1B). This inhibition, which is also observed for mTRP5 20 but not for other TRP subtypes, is unlikely due to ion permeation blockade by divalent cations such as Ca 2ϩ and Mg 2ϩ , as the degree of inhibition was not appreciably affected by adding 2 mmol/L Ca 2ϩ and 1.2 mmol/L Mg 2ϩ in divalent cation-free bath solution (in mmol/L, Ca 2ϩ 0, Mg 2ϩ 0, and mmol/L Na ϩ 140). At potentials slightly positive to the reversal potential (E rev ) of mTRP6 (0 to 30 mV), there is a range in which little current flows in the outward direction, whereas at more positive potentials (Ͼ30 mV), a prominent outward rectification is seen ( Figure 1B). A very similar I-V relationship (S shape and outward rectification) was also obtained for the ␣ 1 -AR-NSCC current recorded under the same experimental conditions ( Figure 1C; see also References 19 and 21).
The mTRP6 current is cationic, as it was completely abolished when all external cations were substituted by large impermeant cations such as N-methyl-D-glucamine but was not affected on total anion substitution with benzenesulfonate. E rev of the mTRP6 current was also close to 0 mV under nearphysiological conditions ( Figure 1B). The value of E rev was significantly shifted toward more positive potentials, when divalent cations such as Ca 2ϩ , Ba 2ϩ , and Sr 2ϩ were the sole charge-carrying cations in the bath (by 29.7Ϯ3.2, 25.8Ϯ3.2, and 16.5Ϯ9.9 mV, respectively [nϭ5], at 100 mmol/L; for actual I-V see the dotted curve in Figure 1B). The relative permeabilities of mTRP6 determined from such E rev measurements under biionic conditions 22 (see also supplementary information available at http://www.circresaha.org) were P Na :P Ca :P Ba :P Sr :P Rb :P K : P Cs :P Li :P Mn ϭ1.0:4.54:3.52:1.94:1.12:1.06:1.0:0.77:0.58. These results strongly suggest that mTRP6 is several times more permeable to Ca 2ϩ and Ba 2ϩ than to monovalent cations such as Na ϩ (Eisenman sequence III).
Single-channel activities accounting for the reversal potential and voltage dependence of mTRP6 currents (hereafter designated as mTRP6 channels) were recorded from cellattached patches of mTRP6-expressing HEK293 cells (19 of 45 patches) but not from those of the empty vector-expressing cells (0 of 38 patches). As displayed in Figure 1D, the polarity of mTRP6 channels reversed at Ϸ0 mV, and their openings became less frequent on hyperpolarization (Figures 1E and  1F). The slope conductance of mTRP6 channels calculated from the inward portion of I-V relationship gave a unitary conductance of Ϸ28 pS on average, under normal ionic conditions ( Figure 1E).
These biophysical properties of mTRP6 currents and channels are very similar to those of the ␣ 1 -AR-NSCC, ie, in unique voltage dependence (S shape and outward rectifying I-V), unitary conductance of 25 to 30 pS, 19 and preferential permeation of Ca 2ϩ and Ba 2ϩ relative to Na ϩ (E rev of ␣ 1 -AR-NSCCs in Ba 2ϩ -rich external solution [26.4 mV with 89 mmol/L Ba 2ϩ ] 23 is comparable with that of mTRP6 current [25.8 mV with 100 mmol/L Ba 2ϩ ; this study]).

Similar Pharmacology of mTRP6 and ␣ 1 -AR-NSCC
To further confirm the similarity between mTRP6 and the ␣ 1 -AR-NSCCs, we next investigated the effects of a nonspecific but frequently used cation channel blocker flufenamate 24 -26 on mTRP6 currents. This compound has previously been shown to uniquely "enhance" the ␣ 1 -AR-NSCC current in the rabbit portal vein. 27 Surprisingly, flufenamate (100 mol/L) reversibly enhanced both mTRP6 and ␣ 1 -AR-NSCC current amplitudes to a similar extent (Figures 2A, 2C, and 2E), and this was unaffected by the mode of activation or presence of the cyclooxygenase inhibitor indomethacin (10 mol/L) ( Figure 2E). It seems that this enhancing action results from a subtle difference in the molecular structure of mTRP6 from other TRP subtypes, because the same drug dose-dependently inhibited the currents due to mTRP3 or mTRP7 expressed in HEK293 cells ( Figures 2B and 2E), which exhibit Ϸ75/Ϸ85% identity/similarity to mTRP6. 18 We also tested another known blocker of the ␣ 1 -AR-NSCC current, Cd 2ϩ . 19 As illustrated and summarized in Figures 2A, 2D, and 2F, the concentration-inhibition curves for Cd 2ϩ blockade of mTRP6 and ␣ 1 -AR-NSCC currents gave similar IC 50 values (253 and 213 mol/L, respectively) and the same Hill coefficient (1.2). In addition, other commonly used cation channel blockers such as Gd 3ϩ , La 3ϩ , amiloride, and SK&F96365 also inhibited mTRP6 and ␣ 1 -AR-NSCC currents with similar IC 50 values ( Table 2).
Dependence on external (Ca 2ϩ o ) and internal Ca 2ϩ (Ca 2ϩ i ) is an interesting feature of several native and recombinant TRP and TRP-related channels, 26,28 -31 and a biphasic dependence on [Ca 2ϩ ] o , ie, potentiation and inhibition, is another hallmark to characterize the ␣ 1 -AR-NSCC. 21 We therefore compared the effects of varying [Ca 2ϩ ] o on mTRP6 and ␣ 1 -AR-NSCC currents under the same experimental conditions. As demonstrated in Figures 3A and 3B, when [Ca 2ϩ ] i was poorly buffered, these currents showed a complex dependence on Ca 2ϩ o ; after a sudden jump of [Ca 2ϩ ] o from 0 to 1 mmol/L, the amplitude of both mTRP6 and ␣ 1 -AR-NSCC currents increased immediately (indicated by arrows). This was then followed by a more dramatic slower increase and decrease, although this time course (latency, time to peak, and time to decline) varied considerably between cells examined. In contrast, when [Ca 2ϩ ] i was rigorously buffered by intracellularly applied BAPTA (10 mmol/L) via a large patch pipette (access resistance, 5 to 7 M⍀), only the immediate increase remained ( Figure 3C; not illustrated for mTRP6). This strongly suggests that the slow Ca 2ϩ o -induced increase/decrease may be mediated by a secondary rise in [Ca 2ϩ ] i . The extents of immediate and slow increases were similar between mTRP6 and ␣ 1 -AR-NSCC currents (Figures 3D and

3E
), but the latter may be more susceptible to Ca 2ϩ o -induced immediate increase ( Figure 3D). The immediate Ca 2ϩ oinduced increase was accompanied by a markedly increased current noise (Figures 3A through 3C), which reflects the increased mTRP6 channel or ␣ 1 -AR-NSCC conductance. On average, the unitary conductance of mTRP6 estimated by noise analysis increased from 7.4Ϯ0.7 to 20.0Ϯ1.9 pS (nϭ13) for a [Ca 2ϩ ] o change from 0 to 1 mmol/L ( Figure 3F). Very similar values were also obtained for the ␣ 1 -AR-NSCC in the present study (8.9Ϯ1.1 versus 19.5Ϯ2.1 pS; nϭ7) and by others. 32 These results strongly suggest that the potentiating action of Ca 2ϩ o on mTRP6 is essentially the same as on the ␣ 1 -AR-NSCC.

Dominant TRP6 mRNA and Protein Expression in Portal Vein
The almost identical electrophysiological and pharmacological properties of mTRP6 and ␣ 1 -AR-NSCC described above strongly suggest that the TRP6 protein may be an essential molecular component of ␣ 1 -AR-NSCC. To test this possibility more directly, we examined the expression of TRP6 mRNA and protein in portal vein smooth muscles. As shown in Figure 4A, total RNA was isolated and subjected to reverse transcription combined with PCR amplification and Southern blot hybridization for determination of expressed TRP subtypes in portal vein smooth muscle cells. In the mouse portal vein, TRP6 RNA was abundantly expressed, whereas TRP1, TRP3, and TRP4 RNAs were present at much lesser levels, and TRP5 and TRP7 RNAs were undetectable. Abundance of TRP6 RNA was similarly found in the whole rabbit portal vein and the smooth muscle isolated from it, suggesting that smooth muscle cells are the major expression site for TRP6 RNA in the portal vein. Immunocytochemistry using anti-TRP6 antisera (see supplementary information, available at http://www.circresaha.org) revealed that TRP6 protein is localized near the sarcolemmal region of an acutely dissoci-  ated rabbit portal vein myocyte (Figures 4Bb and 4Bc), whereas no immunoreactivity was detected from the myocytes treated with FITC-labeled secondary antibody alone (data not shown) or with preabsorption of anti-TRP6 antibody by the immunizing peptide (Figure 4Be). These results strongly suggest that TRP6 is the dominant TRP subtype expressed in the portal vein smooth muscle.

TRP6 Functions as the ␣ 1 -AR-Activated Ca 2؉ Entry Channel
Finally, to determine whether the endogenously expressed TRP6 protein really functions as the ␣ 1 -AR-activated channels, we cultured myocytes enzymatically dissociated from the rabbit portal vein with the TRP6 antisense oligonucleotide that was expected to selectively inhibit TRP6 expression (Table 1). Three to 5 days of antisense oligonucleotide treatment almost completely abolished the expression of TRP6 protein in portal vein myocytes ( Figure 5E), whereas in those treated with the sense oligonucleotide, substantial TRP6 immunoreactivity remained ( Figure 5C). Correspondingly, the density of cation current activated by the ␣-AR agonist phenylephrine (100 mol/L) was markedly decreased with the TRP6 antisense oligonucleotide (Figures 6B and 6D) compared with cells treated with the TRP6 sense oligonucleotide ( Figures 6A and 6D) or antisense oligonucleotides for other TRP homologues detected in the portal vein by RT-PCR ( Figure 6D). We also tested the contribution of TRP6 to Ca 2ϩ entry through the ␣ 1 -AR-NSCC by measuring Ba 2ϩ fluorescence (see supplementary information, available at http://www.circresaha.org), because Ba 2ϩ is almost equally as permeable as Ca 2ϩ through the mTPP6 channel ( Figure 1B), whereas it permeates the native SOCs to a lesser extent than Ca 2ϩ . 33 The use of Ba 2ϩ may also be advantageous to measure genuine influx, as it is not extruded or taken up into internal stores by Ca 2ϩ -ATPases. 34 As shown in Figure 6C and and DIC images (B, E, and F) of the myocytes incubated without (A and B) and with either sense (C and D) or antisense (E and F) TRP6-specific oligonucleotides for 5 days. Immunoreactivity in cultured myocytes is rather scattered over the cell, some of which appears to be condensed near the perinuclear region, presumably in the endoplasmic reticulum and/or Golgi apparatus. DIC indicates differential interference contrast. Figure 6. TRP6 antisense oligonucleotide inhibits ␣ 1 -AR-activated cation current and Ba 2ϩ influx in primary cultured portal vein myocytes. A through C, Phenylephrine (Phe)-induced currents (Ϫ60 mV; nystatin-perforated recording) and Ba 2ϩ fluorescence ratio changes in TRP6 antisense or sense oligonucleotide-treated myocytes. Ba 2ϩ (2 mmol/L) was added in the bath after stimulation with 100 mol/L Phe (200 to 250 seconds) or 2 mol/L thapsigargin (TG; 700 to 800 seconds) in Ca 2ϩ -free bath solution (1 mmol/L EGTA added). D, Averaged density of Pheinduced cation current with TRP1, TRP3, and TRP6 antisense (1AS, 3AS, and 6AS) and sense (1S, 3S, and 6S) oligonucleotides. E and F, Phe (100 mol/L)or TG (2 mol/L)-induced Ba 2ϩ fluorescence ratio increase (⌬ratio) and its rate, d(ratio)/dt, with TRP6 antisense (6AS) and sense (6S) oligonucleotide treatment. Probability value are results of ANOVA and pooledvariance t test.
summarized in Figures 6E and 6F, treatment with TRP6 antisense oligonucleotide significantly reduced the rate and peak of Ba 2ϩ fluorescence ratio increase in response to the ␣ 1 -AR activation but did not affect those evoked by store depletion per se (thapsigargin 2 mol/L) (Figures 6E and 6F). These results strongly point to the functional importance of TRP6 protein as a Ca 2ϩ entry pathway independent of SOCs during the ␣ 1 -AR stimulation via sympathetic nerves in this muscle.
Striking similarity between recombinantly expressed mTRP6 and ␣ 1 -AR-NSCC currents, ie, in activation profile ( Figure 1A), unique voltage dependence (S-shaped and outward rectifying I-V), divalent cation permeability (Ca 2ϩ , Ba 2ϩ ϾNa ϩ ), unitary conductance (25Ϸ30 pS), efficacy of organic and inorganic blockers, and augmentation by flufenamate and external Ca 2ϩ , strongly suggests that the TRP6 protein is the essential molecular component of ␣ 1 -AR-NSCC channels in the portal vein smooth muscle. This is further corroborated by the high expression level of TRP6 mRNA, localization of TRP6-specific immunoreactivity near the cell membrane, and marked inhibition of TRP6 protein expression and ␣ 1 -AR-activated cation current and Ba 2ϩ entry by the antisense strategy. Although possible roles of other endogenous TRPs ( Figure 4A) or yet-unidentified accessory regulatory proteins, which may form a heteromultimer with TRP6, cannot be excluded, there is little doubt that TRP6 has central importance in fulfilling the function of ␣ 1 -AR-NSCC in some vascular tissues as a store depletionindependent, receptor-activated Ca 2ϩ entry pathway.
Looking at other native systems, there are groups of nonselective cation channels activated by GPCRs independently of store depletion that show considerable resemblance to the ␣ 1 -AR-NSCC from an electrophysiological point of view. For example, muscarinic cation channels ubiquitously identified in gastrointestinal smooth muscle are of Ϸ25 pS in unitary conductance; severalfold more permeable to Ca 2ϩ and Ba 2ϩ than Na ϩ ; suppressed by hyperpolarization; sensitive to [Ca 2ϩ ] i ; and immediately potentiated by Ca 2ϩ o , although their primary activator is likely to be the activated G i /G o protein. [2][3][4] Some of the 30-pS Ca 2ϩ -activated nonselective cation channels in cardiac and epithelial tissues are also known to be voltage dependent and/or activated by GPCRs. 35,36 Considering that many biologically important signals produced through GPCR or RTK stimulation (Ca 2ϩ , IP 3 , DAG, arachidonic acid, activated G protein, and store depletion signal, etc) are also recognized as key activators/modulators of TRPs, 6 -10,29 -31,37-40 it is quite possible that a much broader range of ROCCs than currently envisaged may be associated with TRPs in some way. Consistent with this idea, the evidence is gradually accumulating that the TRPs are a requisite component of native Ca 2ϩ -permeable cation channels activated by GPCRs, RTKs, and other stimuli. 12,13,41,42