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(Circulation Research. 2001;88:325.)
© 2001 American Heart Association, Inc.

Cellular Biology

The Transient Receptor Potential Protein Homologue TRP6 Is the Essential Component of Vascular ₁-Adrenoceptor–Activated Ca²⁺-Permeable Cation Channel

Presented in part at the 73rd annual meeting of the Japanese Pharmacological Society, Yokohama, Japan, March 24, 2000, and published in abstract form (Jpn J Pharmacol. 2000;82:83P).

Ryuji Inoue, Takaharu Okada, Hitoshi Onoue, Yuji Hara, Shunichi Shimizu, Shinji Naitoh, Yushi Ito, Yasuo Mori

From the Department of Pharmacology (R.I., H.O., Y.I.), Graduate School of Medical Sciences, Kyushu University, Fukuoka; Laboratory of Humoral Information (T.O., Y.H., S.S., Y.M.), National Institute for Physiological Sciences, Okazaki, Japan; Department of Pathophysiology School of Pharmaceutical Sciences (S.S.), Showa University, Tokyo; and Tissue and Histopathology Section (S.N.), Division at Scientific Data Registry, Atomic Bomb Disease Institute, Nagasaki University School of Medicine, Japan.

Correspondence to Ryuji Inoue, Department of Pharmacology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582. E-mail inouery{at}pharmaco.med.kyushu-u.ac.jp

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Abstract—The Drosophila transient receptor potential protein (TRP) and its mammalian homologues are thought to be Ca²⁺-permeable cation channels activated by G protein (G_q/11)–coupled receptors and are regarded as an interesting molecular model for the Ca²⁺ 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 ₁-adrenoceptor–activated nonselective cation channels (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 Ca²⁺; and blockade by Cd²⁺, La³⁺, Gd³⁺, 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 ₁-adrenoceptor–activated, store depletion–independent cation current and Ba²⁺ influx. These results strongly indicate that TRP6 is the essential component of the ₁-AR–NSCC, which may serve as a store depletion–independent Ca²⁺ entry pathway during increased sympathetic activity.

Key Words: receptor-operated Ca²⁺ channel • transient receptor potential protein • ₁-adrenoceptor

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
The 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 Cß (PLCß), which catalyzes formation from phosphoinositide of 2 major metabolites, inositol 1,4,5-triphosphate (IP₃) and diacylglycerol (DAG), thereby causing a release of stored Ca²⁺ and an accompanying sustained Ca²⁺ entry.¹ The ₁-AR–activated nonselective cation channel (₁-AR–NSCC) is thought to contribute to this Ca²⁺ entry in both direct and indirect ways, since it is activated by DAG and allows preferential movement of divalent cations and secondarily evokes Ca²⁺ 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 Ca²⁺-entry channels that open during activation of the rhodopsin/G protein/PLC/IP₃ 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 Ca²⁺ entry channels (ROCCs) in mammals (including those activated by store depletion; G protein; or second messengers such as IP₃, DAG, arachidonic acid, and Ca²⁺) on stimulation of G protein–coupled receptors (GPCRs) or tyrosine kinase–coupled receptors (RTKs).^{6 7 8 9 10 11} Although functional expression of these mammalian trp-encoding proteins (TRP homologues) in the heterologous system demonstrated the appearance of phosphoinositide turnover–linked Ca²⁺-permeable cation conductance (channel activity or Ca²⁺ 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 Ca²⁺ entry.^{6 7 8 9 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 Ca²⁺ 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.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
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 muscle¹⁹ were maintained in a short-term culture (3 to 4 days) in a laminin (20 µg/mL)–coated dish containing DMEM supplemented with 2% FBS plus antibiotics and TRP antisense or sense oligonucleotides (5 µmol/L) (Table 1). Cells were then reseeded on coverslips and used within 12 to 24 hours for electrophysiological and fluorescent measurements.

View this table: [in this window] [in a new window]   Table 1. Sequences of Antisense and Sense Oligonucleotides Used

Whole-cell and single-channel current recordings and data analyses were performed as described elsewhere.^{18 19} Test solutions were topically applied using the so-called "Y-tube" fast solution exchange device. Bath solution contained (in mmol/L) Na⁺ 135, K⁺ 5, Mg²⁺ 1.2, Ca²⁺ 2, Cl^- 151.4, glucose 5, and HEPES 10. Cs⁺ internal solution for whole-cell recording contained (in mmol/L) Cs⁺ 140, Mg²⁺ 2, Cl^- 24, aspartate 120, Na₂ATP 2, phosphocreatine 5, BAPTA 10, Ca²⁺ 4, glucose 10, and HEPES 10. In the experiments shown in Figures 3A and 3B, 1 mmol/L EGTA instead of 10 mmol/L BAPTA/4 mmol/L Ca²⁺ was added in this solution. Pipette solution for cell-attached recording contained (in mmol/L) Na⁺ 140, Ca²⁺ 1, Mg²⁺ 1.2, tetraethylammonium 10, Cl^- 154.4, glucose 5, HEPES 10, and 100 µmol/L ATP or carbachol (CCh).

View larger version (33K): [in this window] [in a new window]   Figure 3. Potentiation of mTRP6 and 1-AR–NSCC currents by externally added Ca2+ under conventional whole-cell clamp. A and B, Actual records at -60 mV of mTRP6 (A) and 1-AR–NSCC (B) under poorly [Ca2+]i-buffering conditions (1 mmol/L EGTA). Ca2+ (1 mmol/L) added in the bath immediately increased both current noise and amplitude (arrows), which was then followed by a slower increase and decrease. C, Immediate increase of 1-AR–NSCC under strongly [Ca2+]i-buffering conditions (10 mmol/L BAPTA). D and E, Summary of immediate (D) and slow peak (E) increases of mTRP6 and 1-AR–NSCC induced by external Ca2+ (n=5 to 11). *Statistically significant difference (P<0.05) with unpaired t test. F, Mean current-variance plots of mTRP6 evaluated from the same cell by noise analysis in the absence (x and dashed curve) and presence ( and solid curve) of 1 mmol/L Ca2+ in the bath. Curves are drawn with parabolic fitting. EGTA (1 mmol/L) was present in Ca2+-free bath solution. Phe indicates phenylephrine.

Ba²⁺ fluorescence was measured using a dual-excitation wavelength spectrofluorometer (CAM 230, Nihon Bunko). Fura-2–loaded cells (incubated with 2 µmol/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 (±30 nm). The extent of Ba²⁺ 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 mean±SEM. 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 µg 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 ³²P-5'end-labeled synthetic oligonucleotide probes. For the PCR primers and oligonucleotide probes used, see the supplementary information (available at http://www.circresaha.org).

Immunocytochemistry
Anti-mouse TRP6 rabbit antiserum was raised against the C-terminal sequence, LIRKLGERLSLEPKLEESRR, and used for immunostaining of portal vein myocytes adherent on coverslips. The protocol used was the following: fixation in 4% paraformaldehyde, 20 minutes; permeabilization in 0.2% Triton/PBS, 15 minutes; preincubation in 10% normal goat serum/PBS, 1 hour; incubation in 1000-fold diluted TRP6 antiserum, 1 hour; washing in 1% normal goat serum/PBS, 1 hour; and incubation in FITC-labeled anti-rabbit goat antiserum, 1 hour. Immunostained cells were observed under a confocal laser scanning microscope (LSM 510, Zeiss; krypton/argon; excitation, 488 nm; emission, 505 nm) with an optical section of 0.8 to 1.1 µm.

An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
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 -60 mV, 0.15±0.1 pA/pF; n=31). Exogenously applied ATP and CCh dose-dependently produced inward currents in these cells (ED₅₀, 4.5 and 9.1 µmol/L, respectively; n=5 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 µmol/L; by 93±3%, n=5) and atropine (1 µmol/L; by 91±5%, n=5), 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 H₁ 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 µmol/L) and with intracellular perfusion of GDPßS (100 µmol/L), but not by the PKC inhibitor calphostin C (1 µmol/L) or intracellular perfusion of the IP₃ 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 µmol/L) and 1,2-dioctanoyl-sn-glycerol (100 µmol/L; data not shown), and the DAG lipase inhibitor RHC80267 (100 µmol/L) or intracellular perfusion of GTPS; and (3) the PKC activator phorbol 12,13-dibutyrate (up to 1 µmol/L), photolytic release of IP₃, and depletion of internal Ca²⁺ stores by thapsigargin (2 µmol/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 IP₃-mediated store depletion in activating mTRP6 currents, thus being consistent with the conclusions obtained for human TRP6.¹⁵

View larger version (39K): [in this window] [in a new window]   Figure 1. Activation profile, voltage-dependent gating, and ionic selectivity of mTRP6. A, mTRP6 current density at -60 mV. Results are shown for (in µmol/L) ATP 100, CCh 100, OAG (left inset) 100, RHC80267 (RHC) 100, U73122 10, calphostin C (calc C) 1, thapsigargin (TG) 2, with CCh (GTP, heparin, or GDPßS), GTP 100 µmol/L, heparin 1 mg/mL, or GDPßS 300 µmol/L included in the pipette. Right inset, GTPS (100 µmol/L) was included in the pipette. Numbers of experiments are given in parentheses. *P<0.05, ANOVA and pooled-variance t test. B and C, I-V relationships of mTRP6 under 2 biionic conditions and that of 1-AR–NSCC from the rabbit portal vein (RPV) under conventional whole-cell clamp. D, mTRP6 channel activities at 4 different potentials from the same cell-attached patch. Resting membrane potential of HEK293 cell was almost nulled by excess K+ (128 mmol/L) in the bath. E and F, I-V and open probability-vs-voltage relationships of single mTRP6 channel (n=11 to 12). Solid line indicates best linear fit of data points; mp, membrane potential; and Vp, pipette potential.

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 (<-40 mV; Figure 1B). This inhibition, which is also observed for mTRP5²⁰ but not for other TRP subtypes, is unlikely due to ion permeation blockade by divalent cations such as Ca²⁺ and Mg²⁺, as the degree of inhibition was not appreciably affected by adding 2 mmol/L Ca²⁺ and 1.2 mmol/L Mg²⁺ in divalent cation-free bath solution (in mmol/L, Ca²⁺ 0, Mg²⁺ 0, and mmol/L Na⁺ 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 (>30 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^{19 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 near-physiological conditions (Figure 1B). The value of E_rev was significantly shifted toward more positive potentials, when divalent cations such as Ca²⁺, Ba²⁺, and Sr²⁺ were the sole charge-carrying cations in the bath (by 29.7±3.2, 25.8±3.2, and 16.5±9.9 mV, respectively [n=5], 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 conditions²² (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=1.0:4.54:3.52:1.94:1.12:1.06:1.0:0.77:0.58. These results strongly suggest that mTRP6 is several times more permeable to Ca²⁺ and Ba²⁺ than to monovalent cations such as Na⁺ (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 cell-attached 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 0 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 28 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 Ca²⁺ and Ba²⁺ relative to Na⁺ (E_rev of ₁-AR–NSCCs in Ba²⁺-rich external solution [26.4 mV with 89 mmol/L Ba²⁺]²³ is comparable with that of mTRP6 current [25.8 mV with 100 mmol/L Ba²⁺; 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 25 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 µmol/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 µmol/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 75/85% identity/similarity to mTRP6.¹⁸ We also tested another known blocker of the ₁-AR–NSCC current, Cd²⁺.¹⁹ As illustrated and summarized in Figures 2A, 2D, and 2F, the concentration-inhibition curves for Cd²⁺ blockade of mTRP6 and ₁-AR–NSCC currents gave similar IC₅₀ values (253 and 213 µmol/L, respectively) and the same Hill coefficient (1.2). In addition, other commonly used cation channel blockers such as Gd³⁺, La³⁺, amiloride, and SK&F96365 also inhibited mTRP6 and ₁-AR–NSCC currents with similar IC₅₀ values (Table 2).

View larger version (34K): [in this window] [in a new window]   Figure 2. Effects of cation channel blockers on mTRP6 and 1-AR–NSCC currents under conventional whole-cell clamp with holding potential of -60 mV. A through D, Representative records of the effects of flufenamate and Cd2+ on mTRP6 (A), mTRP7 (B), and 1-AR–NSCC activated by 100 µmol/L phenylephrine (Phe) recorded from rabbit portal vein smooth muscle (RPV) (C and D). E, Potentiating effects of flufenamate (100 µmol/L) under various conditions. The following were used (in µmol/L) to activate mTRP6 currents: CCh 100 or ATP 100 with and without indomethacin (indo) 10, RHC80267 (RHC) 100, or GTPS 100 included in the pipette . Data for 1-AR–NSCC, mTRP3, and mTRP7 currents are also shown. F, Concentration-inhibition curves for mTRP6 (•) and 1-AR–NSCC () currents by Cd2+ (n=5 to 15) in divalent cation-free bath solution. Curves are best nonlinear fits to the Hill equation, 1/{1+ [(Cd2+)/Ki]n}, where (Cd2+), Ki and n denote the concentration of Cd2+ applied, dissociation constant, and Hill coefficient, respectively.

View this table: [in this window] [in a new window]   Table 2. IC50 Values of Inorganic and Organic Blockers on mTRP6 and 1-AR–NSCC

Dependence on external (Ca²⁺_o) and internal Ca²⁺ (Ca²⁺_i) is an interesting feature of several native and recombinant TRP and TRP-related channels,^{26 28 29 30 31} and a biphasic dependence on [Ca²⁺]_o, ie, potentiation and inhibition, is another hallmark to characterize the ₁-AR–NSCC.²¹ We therefore compared the effects of varying [Ca²⁺]_o on mTRP6 and ₁-AR–NSCC currents under the same experimental conditions. As demonstrated in Figures 3A and 3B, when [Ca²⁺]_i was poorly buffered, these currents showed a complex dependence on Ca²⁺_o; after a sudden jump of [Ca²⁺]_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 [Ca²⁺]_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 Ca²⁺_o-induced increase/decrease may be mediated by a secondary rise in [Ca²⁺]_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 Ca²⁺_o-induced immediate increase (Figure 3D). The immediate Ca²⁺_o-induced 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 7.4±0.7 to 20.0±1.9 pS (n=13) for a [Ca²⁺]_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 (8.9±1.1 versus 19.5±2.1 pS; n=7) and by others.³² These results strongly suggest that the potentiating action of Ca²⁺_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 dissociated 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.

View larger version (79K): [in this window] [in a new window]   Figure 4. Dominant expression over other TRP homologues and cellular distribution of TRP6 in the portal vein smooth muscle. A, Autoradiogram of blot hybridization analyses of TRP6 and cyclophilin cDNA fragments amplified by RT-PCR from total RNA of whole mouse brain, mouse portal vein, rabbit portal vein, and smooth muscle isolated from rabbit portal vein (left panel) and of TRP1, TRP3, TRP4, TRP5, and TRP7 cDNA fragments amplified from total RNA of mouse brain and mouse portal vein (right 2 panels). B, Confocal images of TRP6 immunoreactivity in single rabbit portal vein myocytes without or with preabsorption of anti-TRP6 antibody by the immunizing peptide (b and e), and their DIC images (a and d). Inset c is a 2-fold magnification with enhanced contrast from a part boxed in panel b. Anti-TRP6 indicates anti-TRP6 antibody; IP, immunizing peptide; and DIC, differential interference contrast.

TRP6 Functions as the ₁-AR–Activated Ca²⁺ 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 µmol/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 Ca²⁺ entry through the ₁-AR–NSCC by measuring Ba²⁺ fluorescence (see supplementary information, available at http://www.circresaha.org), because Ba²⁺ is almost equally as permeable as Ca²⁺ through the mTPP6 channel (Figure 1B), whereas it permeates the native SOCs to a lesser extent than Ca²⁺.³³ The use of Ba²⁺ may also be advantageous to measure genuine influx, as it is not extruded or taken up into internal stores by Ca²⁺-ATPases.³⁴ As shown in Figure 6C and summarized in Figures 6E and 6F, treatment with TRP6 antisense oligonucleotide significantly reduced the rate and peak of Ba²⁺ fluorescence ratio increase in response to the ₁-AR activation but did not affect those evoked by store depletion per se (thapsigargin 2 µmol/L) (Figures 6E and 6F). These results strongly point to the functional importance of TRP6 protein as a Ca²⁺ entry pathway independent of SOCs during the ₁-AR stimulation via sympathetic nerves in this muscle.

View larger version (119K): [in this window] [in a new window]   Figure 5. TRP6 immunoreactivity in primary cultured portal vein myocytes. Representative TRP6 immunoreactivity (A, C, and E) 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.

View larger version (33K): [in this window] [in a new window]   Figure 6. TRP6 antisense oligonucleotide inhibits 1-AR–activated cation current and Ba2+ influx in primary cultured portal vein myocytes. A through C, Phenylephrine (Phe)–induced currents (-60 mV; nystatin-perforated recording) and Ba2+ fluorescence ratio changes in TRP6 antisense or sense oligonucleotide–treated myocytes. Ba2+ (2 mmol/L) was added in the bath after stimulation with 100 µmol/L Phe (200 to 250 seconds) or 2 µmol/L thapsigargin (TG; 700 to 800 seconds) in Ca2+-free bath solution (1 mmol/L EGTA added). D, Averaged density of Phe-induced cation current with TRP1, TRP3, and TRP6 antisense (1AS, 3AS, and 6AS) and sense (1S, 3S, and 6S) oligonucleotides. E and F, Phe (100 µmol/L)– or TG (2 µmol/L)–induced Ba2+ 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 pooled-variance t test.

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 (Ca²⁺, Ba²⁺>Na⁺), unitary conductance (2530 pS), efficacy of organic and inorganic blockers, and augmentation by flufenamate and external Ca²⁺, 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 Ba²⁺ 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 depletion–independent, receptor-activated Ca²⁺ 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 25 pS in unitary conductance; severalfold more permeable to Ca²⁺ and Ba²⁺ than Na⁺; suppressed by hyperpolarization; sensitive to [Ca²⁺]_i; and immediately potentiated by Ca²⁺_o, although their primary activator is likely to be the activated G_i/G_o protein.^{2 3 4} Some of the 30-pS Ca²⁺-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 (Ca²⁺, IP₃, DAG, arachidonic acid, activated G protein, and store depletion signal, etc) are also recognized as key activators/modulators of TRPs,^{6 7 8 9 10 29 30 31 37 38 39 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 Ca²⁺-permeable cation channels activated by GPCRs, RTKs, and other stimuli.^{12 13 41 42}

	Acknowledgments

 
This work is supported by Grant-in-Aid 12670088 to R.I. from the Japan Society for the Promotion of Sciences. We thank Prof A.F. Brading, University Department of Pharmacology, Oxford University, for English correction of the manuscript; Drs Brian Seed and Gary Yellen for providing the H3-CD8 plasmid; and Hiroshi Fujii, Miyo Ikeda, Emiko Mori, and Kumiko Saito for their expert technical assistance.

	Footnotes

 
Original received July 24, 2000; revision received December 7, 2000; accepted December 8, 2000.

	References

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