Functional Role of Vanilloid Transient Receptor Potential 4-Canonical Transient Receptor Potential 1 Complex in Flow-Induced Ca2+ Influx
Objective— The present study is aimed at investigating the interaction of TRPV4 with TRPC1 and the functional role of such an interaction in flow-induced Ca2+ influx. Hemodynamic blood flow is an important physiological factor that modulates vascular tone. One critical early event in this process is a cytosolic Ca2+ ([Ca2+]i) rise in endothelial cells in response to flow.
Methods and Results— With the use of fluorescence resonance energy transfer, coimmunoprecipitation, and subcellular colocalization methods, it was found that TRPC1 interacts physically with TRPV4 to form a complex. In functional studies, flow elicited a transient [Ca2+]i increase in TRPV4-expressing human embryonic kidney (HEK) 293 cells. Coexpression of TRPC1 with TRPV4 markedly prolonged this [Ca2+]i transient; it also enabled this [Ca2+]i transient to be negatively modulated by protein kinase G. Furthermore, this flow-induced [Ca2+]i increase was markedly inhibited by anti–TRPC1-blocking antibody T1E3 and a dominant-negative construct TRPC1Δ567-793 in TRPV4-C1–coexpressing HEK cells and human umbilical vein endothelial cells. T1E3 also inhibited flow-induced vascular dilation in isolated rat small mesenteric artery segments.
Conclusion— This study shows that TRPC1 interacts physically with TRPV4 to form a complex, and this TRPV4-C1 complex may mediate flow-induced Ca2+ influx in vascular endothelial cells. The association of TRPC1 with TRPV4 prolongs the flow-induced [Ca2+]i transient, and it also enables this [Ca2+]i transient to be negatively modulated by protein kinase G. This TRPV4-C1 complex plays a key role in flow-induced endothelial Ca2+ influx.
Hemodynamic blood flow is one of most important physiological factors that control vascular tone.1 Flow shear stress acts on the endothelium to stimulate the release of vasodilators, such as NO and endothelium-derived hyperpolarizing factors, causing endothelium-dependent vascular relaxation.1 In many cases, a key early signal in this flow-induced vascular dilation is Ca2+ influx in endothelial cells in response to flow.2–4 There is intense interest in searching for the molecular identity of the channels that mediate flow-induced Ca2+ influx. Several candidate channels have been proposed. In renal epithelial cells, polycystins 1 and 2 form a channel complex to allow Ca2+ influx in response to flow.5 In vascular endothelial cells, flow may activate P2X4 purinoceptors, which are Ca2+-permeable channels, resulting in vascular dilation.6 Interestingly, several recent studies2,3 demonstrate that TRPV4 channels play a key role in flow-induced endothelial Ca2+ influx and subsequent vascular dilation.
TRP channels are a superfamily of cation channels that can be divided into 7 subfamilies, which include TRPC, TRPV, and 5 others. TRPV4 is a Ca2+-permeable channel in the TRPV subfamily.7 The channel is polymodally activated by hypotonic cell swelling, moderate heat, synthetic phorbol ester 4α-phorbol 12,13-didecanoate (4α-PDD), arachidonic acid, and its metabolites.7 TRPV4 is also involved in flow sensation. Flow shear stress activates TRPV4 in TRPV4-overexpressing HEK 293 cells and native renal epithelial cells.8 More important, there is convincing evidence that TRPV4 is involved in endothelium-dependent vascular dilation to flow.2,3 In rat carotid arteries and the gracilis artery, flow dilation is blocked by TRPV blocker ruthenium red (RuR).3 Furthermore, endothelium-dependent vascular dilation to flow is significantly reduced in TRPV4 knockout (−/−) mice.2
Previous studies have demonstrated an interaction between flow-induced Ca2+ influx and store-operated Ca2+ influx in vascular endothelial cells.9 Presumably, flow-induced Ca2+ influx involves TRPV4. For store-operated Ca2+ influx, several possible candidates could be involved, which include stromal interaction molecule (STIM1), Orai, and TRPC1.10,11 In the present studies, we explored the possible interaction between the flow-sensing TRPV4 and store-operated Ca2+ influx candidate TRPC1. Our results suggest that TRPC1 associates with TRPV4 to form a complex. Such an association alters the kinetics of TRPV4-mediated [Ca2+]i transient in response to flow; more important, it enables the channel complex to be negatively regulated by protein kinase G (PKG). This TRPV4-C1 complex is present in native endothelial cells, where it plays a key role in flow-induced endothelial [Ca2+]i influx and subsequent vascular relaxation.
Cell Culture, Clone, and Transfection
All animal experiments were conducted in accordance with the regulation of the National Institutes of Health, publication 8523. Primary cultured mesenteric artery endothelial cells (MAECs) were dissociated from mesenteric arteries by collagenase. Rat MAECs and human umbilical vein endothelial cells (HUVECs) were cultured in endothelial cell growth medium supplemented with 1% bovine brain extract. HEK 293 cells were cultured in DMEM supplemented with 10% FBS.
The human TRPC1 gene (NM_003304) and the mouse TRPV4 gene (NM_022017) were cloned into pcDNA6 and pCAGGS vectors, respectively. HEK cells were transfected using a reagent (Lipofectamine 2000). HUVECs were transfected by electroporation using Nucleofector II. All genes except PKG1α were transiently transfected into targeted cells. Functional studies were performed 3 days after transfection. TRPC1 point mutations were generated by a kit (QuickChange Site-Directed Mutagenesis kit; Stratagene, Santa Clara, Calif). TRPC1-small interfering RNA (siRNA) (GGAUGUGCGGGAGGUGAAGtt), TRPV4-siRNA (GUCUUCAACCGGCCUAUCCuu), and scramble controls were from Ambion, Austin, Tex.
Subcellular Colocalization and Fluorescence Resonance Energy Transfer
For subcellular colocalization in HEK cells, cyan fluorescent protein (CFP)-tagged TRPV4 and yellow fluorescent protein (YFP)-tagged TRPC1 were cotransfected into the cells. For colocalization in rat MAECs, a double immunofluorescence assay was performed by incubating the cells with a mixture of anti-TRPC1 and anti-TRPV4 antibodies, followed by fluorescence-labeled secondary antibodies. Fluorescence signals were detected by an FV1000 laser scanning confocal system.
For fluorescence resonance energy transfer (FRET), CFP (or YFP)-tagged TRPV4 and YFP (or CFP)-tagged TRPC1 were cotransfected into HEK cells. An inverted microscope equipped with 3-cube FRET filters and a charge-coupled device camera was used to measure the FRET ratio, as described elsewhere.12
Immunoprecipitation and Immunoblots
TRPV4 or TRPC1 proteins were immunoprecipitated by incubating the extracted proteins with anti-TRPV4 (model ACC-034; Alomone Laboratory, Jerusalem, Israel) or anti-TRPC1 (model ACC-010; Alomone Laboratory) antibody. The immunoprecipitates were purified by protein A agarose and resolved on gel. For immunoblots, the polyvinylidene difluoride membrane carrying the transferred proteins was incubated with the primary antibodies (Alomone Laboratory) at a 1:200 dilation.
[Ca2+]i Measurement and Pressure Myograph
Cultured cells were loaded with 10-μmol/L of fluorescent dye (Fura-2/AM). Flow was initiated by pumping normal physiological saline solution to a specially designed parallel plate flow chamber, in which cells were adhered to the bottom.13 Normal physiological saline solution contained the following (in mmol/L): NaCl, 140; KCl, 5; CaCl2, 1; MgCl2, 1; glucose, 10; and Hepes, 5 (pH, 7.4). In experiments using mesenteric arterial segments, the endothelial cell layer was intraluminally loaded with 20-μmol/L of fluorescent dye (Fluo-4/AM). Fluorescent dye (Fura-2 and Fluo-4) signals were measured using a fluorescence imaging system (Olympus) and a confocal system (model FV1000), respectively. Changes in [Ca2+]i were displayed as a change in the Fura-2 ratio (F340/F380) (for Fura-2 dye) or as a relative fluorescence intensity compared with the value before flow (F1/F0, for Fluo-4 dye).
For the flow dilation study, the arteries were preconstricted with phenylephrine, and changes in the external diameter of arteries were recorded by a pressure myograph.
If needed, cultured cells were pretreated at 37°C for 1 hour with T1E3 (1:100) or preimmune IgG (1:100), or at room temperature for 10 minutes with RuR, 8-bromoguanosine-3′,5′ (8-Br)–cGMP, or KT5823. Artery segments were pretreated with T1E3 (1:50) or preimmune IgG (1:50) at 4°C overnight. T1E3 antibody was increased in rabbits, as previously described.14 IgG was purified from both the T1E3 antiserum and the preimmune serum using a protein G column.
Whole-Cell Patch Clamp
Whole-cell current was measured with an EPC-9 patch clamp amplifier. The pipette solution contained the following (in mmol/L): CsCl, 20; cesium-aspartate, 100; MgCl2, 1; ATP, 4; CaCl2, 0.08; 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetate, 10; and Hepes, 10 (pH, 7.2). Bath solution contained the following (in mmol/L): NaCl, 150; CsCl, 6; MgCl2, 1; CaCl2, 1.5; glucose, 10; and Hepes, 10 (pH, 7.4).
Cells were clamped at 0 mV. Whole-cell current density (pA/pF) was recorded in response to successive voltage pulses of +80 and −80 mV for a 100-millisecond duration, and then plotted against time.
Expanded experimental procedures are described in the Supplemental Materials (available online at http://atvb.ahajournals.org).
Physical Association of TRPC1 With TRPV4 in Heterologously Expressed HEK Cells
Subcellular localization of TRPC1 and TRPV4 was examined in living HEK cells that were cotransfected with YFP-tagged TRPC1 and CFP-tagged TRPV4. As shown in Figure 1, TRPV4 (Figure 1A, green) and TRPC1 (Figure 1B, red) were localized in both the cytosol and the plasma membrane. There was strong overlapping of TRPV4 and TRPC1 fluorescence, as is apparent in merged images (Figure 1C, yellow) and the intensity profile of TRPV4-CFP and TRPC1-YFP (Figure 1D). These data suggest that TRPV4 is colocalized with TRPC1. TRPV4 fluorescence also overlapped with a fluorescence plasma membrane marker, Di-8-ANEPPS (Supplemental Figure I), confirming its plasma membrane localization.
Two antibodies for coimmunoprecipitation, anti-TRPC1 and anti-TRPV4 (Alomone Laboratory), were previously reported to be highly specific.15,16 This was verified by immunoblots (Supplemental Figure IIA and B). In coimmunoprecipitation experiments, the anti-TRPC1 antibody could pull down TRPV4 in the protein lysates freshly prepared from TRPV4-C1–coexpressing HEK cells (Figure 1E). Furthermore, an anti-TRPV4 antibody could reciprocally pull down TRPC1 (Figure 1F). In control experiments, in which immunoprecipitation was performed with the IgG purified from preimmune serum, no band was observed (Figure 1E and F). These data indicate that TRPV4 physically associates with TRPC1.
FRET is a sensitive reporter of the proximity of 2 fluorophores.17 It provides a noninvasive means of monitoring the subunit assembly of ion channels and other proteins.12,16 As shown in Figure 1G, a strong FRET signal was detected in HEK cells coexpressed with CFP-tagged TRPV4 and YFP-tagged TRPC1 in those cells coexpressed with YFP-tagged TRPV4 and CFP-tagged TRPC1. These data strongly suggest that TRPV4 directly interacted with TRPC1 to form a complex. No FRET signal was detected in 2 negative controls, 1 cotransfected with CFP plus YFP and the other cotransfected with CFP-tagged TRPV4 plus YFP-tagged GIRK4 (Figure 1G). G-protein-activated inwardly rectifying K+ channels (GIRK4) belongs to inwardly rectifying potassium channels bearing no similarity to TRP, and it served as a membrane protein control. In a positive control in which the cells were transfected with CFP-YFP concatemer, significant FRET signals were observed (Figure 1G). The time-resolved FRET experiments showed that the interaction of TRPV4 and TRPC1 exists with or without flow (Supplemental Figure III). A deletion was made to determine the responsible domains for TRPV4-C1 interaction. Deletion of either the N- or the C-terminal cytoplasmic domain of both channels abolished the FRET signals, indicating that the C- and N-terminal domains of both channels are required for their interaction (Supplemental Figure IV).
TRPC1 Alters the Kinetics of Flow-Induced [Ca2+]i Transient and 4α-PDD–Stimulated Whole-Cell Current and Enables the PKG Modulation of [Ca2+]i Transient in HEK Cells
Consistent with another report,8 flow induced a [Ca2+]i transient in TRPV4-expressing HEK cells (Figure 2A). After [Ca2+]i reached its peak, it decayed, with a half-life of 78±7 seconds (n=6) (the half-life is defined as the duration for [Ca2+]i to reduce to 50% of its peak value). Intriguingly, coexpression of TRPC1 with TRPV4 markedly prolonged the flow-induced [Ca2+]i transient (Figure 2B). In fact, no apparent decay in [Ca2+]i was observed within the duration of experiments of approximately 10 minutes (Figure 2B). In a whole-cell patch clamp, a TRPV4-specific agonist, 4α-PDD, 5 μmol/L, activated a transient current (Supplemental Figure VA), with a decay half-life of 41±4 seconds (n=14). TRPC1 coexpression also markedly slowed down the decay of this current transient (Supplemental Figure VB). In addition, TRPC1 coexpression caused a small, but statistically significant, increase in the peak amplitude of [Ca2+]i transient (Figure 2C) and whole-cell current (Supplemental Figure VC). In controls, both flow-induced [Ca2+]i transient and 4α-PDD–stimulated current transient were absent in wild-type HEK and TRPC1-expressing HEK cells (X Yao, PhD, unpublished data, 2008). Furthermore, a TRPV antagonist RuR, 5 μmol/L, inhibited the [Ca2+]i increase (Figure 2C) and the whole-cell current increase (Supplemental Figure VC), confirming a requirement for TRPV.
To study PKG regulation, HEK cells were stably transfected with PKG1α, then transiently transfected with either TRPV4 or TRPV4 plus TRPC1. In TRPV4-expressing cells, pretreatment with a PKG activator, 8-Br-cGMP, 2 mmol/L, for 10 minutes had no effect on the flow-induced [Ca2+]i increase (Figure 2D and F). In contrast, in TRPV4-C1–coexpressing cells, 8-Br-cGMP, 2 mmol/L, markedly reduced the magnitude of the [Ca2+]i increase (Figure 2E and F). KT5823, 1 μmol/L, a potent and highly specific PKG inhibitor, abolished the inhibitory action of 8-Br-cGMP (Figure 2E and F). In cells cotransfected with TRPV4 and mutant TRPC1 that were mutated at putative PKG phosphorylation sites (TRPC1S172A or TRPC1T313A), the inhibitory action of 8-Br-cGMP, 2 mmol/L, was reduced (Figure 2F). These data suggest that PKG does not act on TRPV4 itself. Instead, it inhibits the function of TRPV4-C1 complex by phosphorylating on serine 172 and threonine 313 of TRPC1.
T1E3 and TRPC1Δ567-793 Diminish the TRPV4-Mediated [Ca2+]i Increase in TRPV4-C1–Overexpressing HEK Cells
A polyclonal anti–TRPC1 antibody T1E3, which can plug the ion permeation pore of TRPC1,14 was used. In TRPV4-C1–coexpressing HEK cells, a preincubation with T1E3 (1:100) for 1 hour at 37°C diminished the flow-induced [Ca2+]i increase (Figure 3A and B). The specificity of T1E3 was verified by immunoblot (Supplemental Figure IIC) and patch clamp (Supplemental Figure VI). In the patch clamp, T1E3 had no direct action on 4α-PDD–stimulated current in TRPV4-expressing cells, confirming a lack of direct T1E3 action on TRPV4 (Supplemental Figure VI). TRPC1Δ567-793, a mutant with deletion in the pore regions of TRPC1,18 was also used. Cotransfection of TRPC1Δ567-793 with TRPV4 diminished the flow-induced [Ca2+]i increase (Figure 3A and B). These data indicate that interfering TRPC1 with T1E3 or TRPC1Δ567-793 resulted in an alteration in flow-induced [Ca2+]i response mediated by TRPV4, supporting a functional interaction of TRPC1 with TRPV4.
Physical Association of TRPC1 With TRPV4 in the Primary Cultured Rat MAECs
In double-labeling immunofluorescence experiments, rat MAECs were stained for TRPC1 with Alexa fluor 488 (green) and TRPV4 with Alexa fluor 546 (red). As shown in Figure 4A and B, both TRPC1 and TRPV4 had significant distribution on the plasma membrane, with some intracellular distribution. On merged images, there was strong overlapping of TRPC1 and TRPV4 fluorescence (yellow) (Figure 4C). No staining was observed in the control in which the primary antibodies were preabsorbed with excessive amounts of respective antigens (Figure 4E and F).
In coimmunoprecipitation experiments, the anti–TRPC1 antibody could pull down TRPV4 (Figure 4G) and the anti–TRPV4 antibody could reciprocally pull down TRPC1 (Figure 4H). Rat MAECs also express TRPC3, TRPC4, TRPC5, and TRPC6, but the anti–TRPV4 antibody was unable to pull down these TRPCs (Supplemental Figure VII).
T1E3 Reduces Flow-Induced Endothelial [Ca2+]i Increase and Vascular Dilation in Isolated Rat Small Mesenteric Artery Segments
A flow-induced [Ca2+]i increase was studied in endothelial cells in isolated small mesenteric artery segments. The time course of flow-induced [Ca2+]i transient in the endothelial cells (Figure 5B) appeared to lie in between those of TRPV4-expressing HEK cells (Figure 2A) and TRPV4-C1–coexpressing HEK cells (Figure 2B). This [Ca2+]i increase consisted of a transient peak followed by a relatively sustained phase, which was prolonged and lasted within the experimental duration of approximately 8 to 10 minutes (Figure 5B). T1E3 (1:50, overnight) and RuR (5 μmol/L, 10 minutes) inhibited this [Ca2+]i increase (Figure 5B and C).
Consistent with our previous report,4 flow elicited vascular dilation in isolated small mesenteric artery segments. The dilation consisted of an initial transient peak followed by a sustained phase (Figure 5D). Both phases were inhibited by T1E3 and RuR (Figure 5E and F). These data suggest an involvement of TRPV4-C1 complex in flow-induced endothelial [Ca2+]i increase and subsequent vascular dilation.
Effect of T1E3, TRPC1Δ567-793, TRPC1-siRNA, and TRPV4-siRNA on Flow-Induced [Ca2+]i Increase and 4α-PDD–Stimulated Whole-Cell Current in HUVECs
HUVECs express both TRPV4 and TRPC1 (Supplemental Figure VIII). Flow-induced [Ca2+]i increase (Figure 6A and B) and 4α-PDD (5 μmol/L)–stimulated current (Supplemental Figure IXA and B) in HUVECs were inhibited by T1E3 and TRPC1Δ567-793, respectively. TRPC1-siRNA effectively reduced TRPC1 expression, but had no effect on TRPV4 expression (Figure 6C). Functionally, this siRNA reduced the magnitude of flow-induced [Ca2+]i increase (Figure 6D) and 4α-PDD–stimulated whole-cell current (Supplemental Figure IXD to E). More important, TRPC1-siRNA also accelerated the decay of [Ca2+]i transient (Figure 6D) and whole-cell current (Supplemental Figure IXD). In TRPC1-siRNA–treated cells, residual [Ca2+]i transient (Figure 6D) and whole-cell current (Supplemental Figure IXD and E) were inhibited by RuR. TRPV4-specific siRNA also diminished the flow-induced [Ca2+]i increase (Figure 6B) and 4α-PDD–stimulated current (Supplemental Figure IXB). These data support the notion that the interaction of TRPC1 with TRPV4 prolongs the flow-induced [Ca2+]i transient.
Previous studies showed that flow activates TRPV4 in TRPV4-overexpressing HEK cells.8 However, the detailed activation mechanism remains obscure. In the present study, we found that coexpression of TRPC1 with TRPV4 altered the decay kinetics of flow-induced [Ca2+]i increase and 4α-PDD–stimulated whole-cell current; it also enabled the [Ca2+]i transient to be negatively regulated by PKG. Interfering TRPC1 with T1E3 or TRPC1Δ567-793 diminished the [Ca2+]i increase in TRPV4-C1–coexpressing HEK cells. Coimmunoprecipitation, FRET, and subcellular colocalization experiments demonstrated a close physical association of TRPV4 with TRPC1. These data strongly suggest a physical interaction of TRPC1 with TRPV4 and an important functional role of this interaction in flow-induced Ca2+ influx.
TRPV4 is abundantly expressed in vascular endothelial cells,19 and plays a key role in flow-induced endothelial Ca2+ influx and subsequent vascular dilation.2,3 Interestingly, flow-induced [Ca2+]i transient in native endothelial cells bears certain similarity to that in TRPV4-C1–coexpressing HEK cells. First, endothelial cells also displayed a relatively prolonged flow-induced [Ca2+]i transient and 4α-PDD–stimulated whole-cell current, and knocking down of TRPC1 with TRPC1-siRNA accelerated the decay of [Ca2+]i transient and whole-cell current in endothelial cells. Second, the [Ca2+]i transient and the cation current in endothelial cells were also inhibited by T1E3 and TRPC1Δ567-793. Moreover, T1E3 suppressed flow-induced vascular dilation. Coimmunoprecipitation and double-immunolabeling studies confirmed the physical interaction of TRPV4 with TRPC1 in endothelial cells. These data strongly suggest that TRPV4 also forms a complex with TRPC1 in native endothelial cells, where it plays a role in flow-induced endothelial Ca2+ influx and subsequent vascular dilation.
As previously mentioned, the physical association of TRPC1 with TRPV4 prolonged the flow-induced [Ca2+]i transient and enabled the [Ca2+]i transient to be negatively regulated by PKG. This has important significance for endothelial cell function. A prolonged Ca2+ influx would allow endothelial cells to produce more NO, enhancing vascular dilation.4,20 However, excessive [Ca2+]i and NO could lead to apoptosis and cell death.21 To protect from this, vascular endothelial cells possess a negative feedback mechanism, in which flow-induced Ca2+ influx is inhibited by [Ca2+]i via the Ca2+-NO-cGMP-PKG pathway.13 Unfortunately, the molecular identity of such flow-sensitive PKG-inhibitable Ca2+ influx channels is unknown. The present study strongly suggests that the TRPV4-C1 complex be the candidate. This TRPV4-C1 complex in endothelial cells prolongs the flow-induced Ca2+ influx, thus producing more NO; at the same time, it allows the [Ca2+]i influx to be negatively regulated by the [Ca2+]i level via the Ca2+-NO-cGMP-PKG pathway. Consistent with this notion, in native endothelial cells, flow elicits prolonged NO production (Supplemental Figure X); furthermore, an NO donor suppresses the flow-induced [Ca2+]i increase (presumably mediated by the TRPV4-C1 complex) (Supplemental Figure X). However, this negative feedback scheme cannot be applied to HEK cells, because these cells do not express NO synthases and guanylyl cyclases, which are required for the operation of the Ca2+-NO-cGMP-PKG pathway.
The results from the present study also provided some clue as to how TRPV4 and TRPC1 interact with each other. FRET data suggest that TRPV4 interacts directly with TRPC1 without the presence of a third protein between them. Two possibilities exist: (1) TRPV4 may coassemble with TRPC1 to form heterotetrameric channels. Heteromeric assembly across different TRP subfamilies has been reported between TRPC1 and TRPP222 and between TRPV4 and TRPP2.23 (2) TRPV4 and TRPC1 may each form homotetrameric channels, and 2 homotetrameric channels then interact with each other through their N- or C-terminus. Evidence from the present study favors the first possibility. First, T1E3, an antibody that plugs the pore of TRPC1,13 diminished the flow-induced Ca2+ influx that is known to be mediated by TRPV4. Second, coassembly of TRPV4 with TRPC1Δ567-793, a mutant with deletion in the TRPC1 pore-forming region,18 also disrupted the flow-induced Ca2+ influx. This evidence suggests that the pore regions of TRPC1 and TRPV4 are closely appositional to each other, likely forming heterotetrameric channels sharing a common pore, similar to the reported heterotetrameric channels of TRPP2-TRPC1.22 However, future patch clamp studies are needed to characterize the electrophysiological and/or pharmacological properties of TRPV4-C1 heteromeric channels.
In conclusion, we demonstrated that TRPC1 forms a functional complex with TRPV4. This association prolongs the flow-induced Ca2+ influx and enables this influx to be negatively regulated by PKG. In native endothelial cells, TRPV4-C1 complex is the main molecular entity that is responsible for flow-induced Ca2+ influx and subsequent vascular dilation.
We thank Bernd Nilius, PhD for the mouse TRPV4 clone, Indu Ambudkar, PhD (National Institutes of Health) for the TRPC1Δ567-793, and Q. Xia, PhD (Zhejiang University, Hangzhou, China) for help.
Sources of Funding
This study was supported by grants CUHK477307, CUHK477408, and CUHK479109 from the Hong Kong Research Grant Council (RGC); the Focused Investment Scheme of Chinese University of Hong Kong (CHUK); and Li Ka Shing Institute of Health Sciences.
Received on: April 30, 2009; final version accepted on: January 7, 2010.
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