TRPC3 properties of a native constitutively active Ca2+-permeable cation channel in rabbit ear artery myocytes
1 Ion Channels and Cell Signalling Research Centre, Division of Basic Medical Sciences, St George's, University of London, Cranmer Terrace, London SW17 ORE, UK
Abstract
Previously we have described a constitutively active, Ca2+-permeable, non-selective cation channel in freshly dispersed rabbit ear artery myocytes which has similar properties to some of the canonical transient receptor potential (TRPC) channel proteins. In the present work we have compared the properties of constitutive channel activity with known properties of TRPC proteins by investigating the effect of selective anti-TRPC antibodies and pharmacological agents on whole-cell and single cation channel activity. Bath application of anti-TRPC3 antibodies markedly reduced channel activity in inside-out patches and also produced a pronounced reduction of both current amplitude and variance of constitutively active whole-cell cation currents whereas anti-TRPC1/4/5/6/7 antibodies had no effect on channel activity. In the presence of antigenic peptide, anti-TRPC3 antibodies had no effect on whole-cell or single cation channel activity. Bath application of flufenamic acid, Gd3+, La3+ and Ca2+ inhibited spontaneous channel activity in outside-out patches with IC50 values of 6.8 μM, 25 nM, 1.5 μM and 0.124 mM, respectively, which are similar values to those against TRPC3 proteins. Immunocytochemical studies combined with confocal microscopy showed expression of TRPC3 proteins in ear artery myocytes, and these were predominately distributed at, or close to, the plasma membrane. These data provide strong evidence that native constitutively active cation channels in rabbit ear artery myocytes have similar properties to TRPC3 channel proteins and indicate that these proteins may have an important role in mediating this conductance.
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Introduction
In freshly dispersed rabbit ear artery smooth muscle cells we have described a constitutively active Ca2+-permeable non-selective cation current (Icat), which has been proposed to contribute to resting membrane conductance and basal Ca2+ influx (Albert et al. 2003). The spontaneous nature of this ion channel appears to reside in constitutive Gi/Go subunits of G-proteins which stimulate phospholipase D (PLD) to cleave phosphatidylcholine to produce phosphatidic acid. Subsequently phosphatidic acid is converted to diacylglycerol (DAG), which initiates channel opening via a protein kinase C (PKC)-independent mechanism (Albert & Large, 2004; Albert et al. 2005). In parallel there is an inhibitory signalling pathway in which Gq/G11 couples to U73122-sensitive phospholipase C (PLC) to produce DAG, which reduces open probability of ion channels by a PKC-dependent mechanism (Albert & Large, 2004; see Fig. 2 of Albert & Large, 2006). Moreover the neurotransmitter noradrenaline also increases Icat (Albert & Large, 2004).
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A, shows that inclusion of anti-TRPC3a antibodies at 1: 200 in the patch pipette solution markedly reduced both the amplitude (i) and variance (ii) of a constitutively active whole-cell cation current held at –50 mV. The inset shows individual current–voltage curves from Ai. B, shows that inclusion of anti-TRPC3a antibodies following preincubation with its antigenic peptide in the patch pipette solution had no effect on both the amplitude (i) or variance (ii) of the whole-cell current. The asterisk denotes when whole-cell configuration was obtained and the dotted lines represent zero holding current. C and D, mean data showing that anti-TRPC3a antibodies significantly reduced, respectively, holding current and current variance of constitutively active whole-cell cation currents (n= 7, P < 0.01).
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A key question concerns the molecular identity of Icat. The observation that DAG stimulates this conductance via a PKC-independent mechanism suggests strongly that member(s) of the canonical transient receptor potential (TRPC) family of channel proteins are involved. To our knowledge these are the only non-selective cation channels that are stimulated by DAG in this manner. Specifically it is often stated that this is a key characteristic of the TRPC3/6/7 subfamily (e.g. Minke & Cooke, 2002; Beech et al. 2004; Desai & Clapham, 2005) although there is a report that DAG also activates mouse TRPC5 by a PKC-independent mechanism (Lee et al. 2003).
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Previously we have highlighted similarities and some notable differences between Icat in rabbit ear artery and the noradrenaline-evoked cation conductance in portal vein myocytes (Albert et al. 2003), which is thought to involve TRPC6 proteins (Inoue et al. 2001).
In the present work we have investigated the effect of anti-TRPC antibodies on ion channel activity in rabbit ear artery myocytes. Immunopharmacological approaches have been used to study the roles of many types of ion channels including TRPC channel proteins in neurones (Kim et al. 2003; Dallas et al. 2005) and vascular myocytes (Xu & Beech, 2001). In addition we used immunocytochemical studies with confocal imaging to probe the cellular distribution of TRPC proteins and studied the inhibitory action of several multivalent cations and other pharmacological agents for comparison with expressed TRPC channels. The results from these studies suggest that the properties of Icat are similar to those of TRPC3 channel proteins, which indicates that TRPC3 may be a significant molecular constituent of this native conductance.
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Methods
Cell isolation
New Zealand White rabbits (2–3 kg) were killed by an I.V. injection of sodium pentobarbitone (120 mg kg–1, in accordance with the UK Animals (Scientific Procedures) Act 1986) and ear arteries from both ears were removed. The ear arteries were freshly dispersed using procedures and solutions previously described (Albert et al. 2003; Albert & Large, 2004).
Electrophysiology
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Whole-cell and single channel currents were recorded with an Axopatch 200B patch clamp amplifier (Axon Instruments, Inc., Union City, CA, USA) at room temperature using whole-cell recording, outside-out and inside-out configurations of the patch clamp technique and data acquisition and analysis protocols as previously described (see Supplemental material and Helliwell & Large, 1998; Albert et al. 2003; Albert & Large, 2004).
Immunocytochemistry
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Freshly dispersed myocytes were fixed by 4% paraformaldehyde in physiological saline solution (PSS, see Albert et al. 2003) containing penicillin (20 U ml–1) and streptomycin (20 μg ml–1) for 10 min at room temperature. The myocytes were then processed for TRPC protein staining and imaged using laser scanning confocal microscope as described in Supplemental material and Saleh et al. (2005).
Solutions and drugs
The bathing and patch pipette solutions for whole-cell recording, outside-out patches and inside-out patches were K+ free as previously described (Albert et al. 2003, 2005; Albert & Large, 2004; see Supplemental material).
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Flufenamic acid (FFA), GdCl3 and LaCl3 were dissolved in distilled H2O at a stock concentration of 10 mM. External 1.5 mM CaCl2 was replaced with either 10 μM, 100 μM or 10 mM CaCl2 and in the Ca2+-free external solution CaCl2 was omitted and 1 mM BAPTA was added (< 10 nM free Ca2+ concentration). Anti-TRPC antibodies were obtained from Alomone Laboratories (Jerusalem, Israel; defined as TRPCa), Santa Cruz Biotechnology (Santa Cruz, CA, USA; defined as TRPC7sc) and also from Professor W. P. Schilling (defined as hTRPC; see Goel et al. (2002) and Supplemental material). The values are the mean of n cells ±S.E.M. and statistical analysis was carried out using Student's t test (paired and unpaired) with the level of significance set at P < 0.05.
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Results
Effect of selective anti-TRPC antibodies on constitutively active cation channel activity in inside-out patches from rabbit ear artery myocytes
In the first series of experiments, we investigated the effect of selective anti-TRPC antibodies on constitutively active cation channels in inside-out patches from rabbit ear artery myocytes. The inside-out patch configuration allows anti-TRPC antibodies, raised against putative intracellular epitopes of human and mouse TRPC channel proteins (see Methods and Supplemental material), to be applied directly to the intracellular surface of the plasma membrane.
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A and B, bath application of, respectively, anti-TRPC3a antibodies at 1 : 200 dilution and anti-hTRPC3 antibodies at 0.3 μg ml–1 reversibly inhibited constitutively active channel activity in two different patches held at –50 mV. Insets show individual channel currents on a faster time scale. C, mean data showing the effect of anti-TRPC antibodies on constitutive channel activity represented as NPo. The data using anti-TRPC antibodies from different sources have been pooled; note that the anti-TRPC3a antibody had no effect on channel activity following preincubation with its antigenic peptide (P < 0.001).
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We also studied the effect of selective anti-TRPC antibodies raised against all TRPC channel proteins (except TRPC2, which is considered a pseudogene in man) on constitutive channel activity, and for each TRPC protein, antibodies from two different sources were used (see Methods). Figure 1C shows that only anti-TRPC3 antibodies had a significant effect on channel activity and that antibodies raised against TRPC1/4/5/6/7 had no effect on channel activity in inside-out patches.
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These data indicate that TRPC3 proteins have an important role in mediating constitutive channel activity in rabbit ear artery myocytes.
Effect of anti-TRPC3 antibodies on constitutively active whole-cell cation currents
To further investigate the potential role of TRPC3 proteins in constitutive channel activity, we studied the effect of anti-TRPC3a antibodies using whole-cell recording. Figure 2A shows the effect of including of anti-TRPC3a antibody at 1 : 200 dilution in the patch pipette solution on the properties of basal whole-cell currents. Figure 2Ai illustrates that immediately after whole-cell configuration was obtained a characteristic ‘noisy’ holding current was observed which declined in amplitude and ‘noisy’ appearance over about 5 min. The inset in Fig. 2A shows current–voltage (I–V) relationships evoked after 1 min (a) and 5 min (b), which had similar reversal potentials of about 0 mV but after 5 min the I–V relationship was reduced at all membrane potentials and exhibited less current fluctuations at negative potentials. The characteristic ‘noisy’ appearance of the constitutive whole-cell currents probably reflects the spontaneous opening and closing of cation channels and therefore we examined the effect of anti-TRPC3a antibodies on the variance of these whole-cell currents using noise analysis. Figure 2Aii illustrates that anti-TRPC3a antibodies produced a marked reduction in variance of the whole-cell current shown in Fig. 2Ai, and Fig. 2C and D shows that inclusion of anti-TRPC3a antibodies in the patch pipette solution induced a significant reduction in both the mean amplitude and variance of constitutively active whole-cell cation currents at –50 mV after 5 min.
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Effect of pharmacological agents on constitutive cation channel activity in outside-out patches
To further investigate whether TRPC3 proteins are components of these native cation channels we carried out experiments using the pharmacological agents flufenamic acid (FFA), Gd3+ and La3+, which have been previously shown to have differential effects on expressed TRPC channel proteins in cell lines (see Table 1) and therefore may be useful tools for distinguishing between various TRPC proteins involved in native cation conductances.
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A, shows that bath application of 10 μM flufenamic acid (FFA) reduced and 100 μM FFA completely blocked constitutive cation channel activity at –50 mV. B, C, D and E, respectively, show mean concentration–effect curves for FFA, Gd3+, La3+ and [Ca2+]o on constitutive channel activity at –50 mV.
Changing external Ca2+ concentration ([Ca2+]o) has been shown to have differential effects on the activity of expressed TRPC channel proteins (see Table 1), and therefore we further investigated this effect of [Ca2+]o on constitutive channel activity in outside-out patches. Figure 3E shows that [Ca2+]o had a pronounced inhibitory effect on channel activity in ear artery myocytes with an IC50 value of 0.124 mM.
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Immunocytochemical evidence for the presence of TRPC3 channel proteins in rabbit ear artery myocytes
In the next series of experiments we investigated whether TRPC3 proteins are present in ear artery myocytes using immunocytochemical techniques and confocal microscopy, which enable the expression and location of TRPC3 proteins to be examined using anti-TRPC3 antibodies.
A shows a single confocal plane fluorescence image of a myocyte labelled with anti-TRPC3a antibodies (1 : 200) and B shows another myocyte labelled with anti-TRPC3a antibodies (1 : 200) preincubated with their antigenic peptide (1 : 100). Insets in A and B show transmitted light image of these myocytes. White circles in A indicate Regions 1 and 2, which were used to analyse the localization of fluorescence (see Methods and Supplemental material). A dotted line was used in B to outline the contour of a cell, due to its low fluorescence. C, mean data showing the localization of fluorescence related to binding of anti-TRPC3a antibodies. D, mean data showing intensity of fluorescence, expressed as average pixel fluorescence (intensity units per pixel). The values of all pixels in the confocal plane of the myocytes were added up and then divided by the number of pixels. The specificity of labelling was confirmed by greatly reduced fluorescence after preincubation with antigenic peptide or by virtual lack of fluorescence in the absence of primary antibodies (P < 0.001).
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We also investigated the expression of other TRPC proteins in rabbit ear artery myocytes and found that anti-TRPC1a and anti-TRPC7sc antibodies stained ear artery myocytes predominantly at the plasma membrane whereas anti-TRPC6a antibodies stained myocytes throughout the cell (data not shown). Anti-TRPC4a and anti-TRPC5a antibodies did not produce significant specific staining of ear artery myocytes (data not shown).
Discussion
, 百拇医药 In the present study we have used electrophysiological and immunocytochemical techniques to investigate the possibility that TRPC channel proteins constitute native spontaneously active Ca2+-permeable non-selective cation channels in freshly dispersed rabbit ear artery smooth muscle cells. The data provide strong evidence TRPC3 is involved in forming the functional ion channel.
Experiments with anti-TRPC antibodies
These data clearly shows that two anti-TRPC3 antibodies raised against different epitopes within the TRPC3 protein markedly reduced channel activity in inside-out patches by about 90%. It is not evident how the anti-TRPC3 antibodies inhibit channel activity but the inhibitory response was completely blocked by preincubation with an antigenic peptide. It is possible that attachment of the antibody to the channel protein inhibits channel opening by preventing essential conformational changes (see Dallas et al. 2005). In contrast antibodies to other TRPC proteins (two for each subtype) had no significant effect on channel activity. This is good evidence that TRPC3 molecules are components of the functioning ion channel. Also inclusion of an anti-TRPC3 antibody reduced both constitutively active whole-cell conductance and current variance. These data are in keeping with the hypothesis that the stochastic opening and closing of TRPC3 channels contribute to the resting membrane conductance of these myocytes.
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Immunocytochemical experiments demonstrated the expression of TRPC3 proteins in ear artery myocytes and that these proteins were preferentially localized to the cell membrane, which would be expected for a functional plasmalemmal ion channel. TRPC1 and TRPC7 antibody staining showed a similar pattern of distribution, and whereas TRPC6 was present it appeared to be evenly distributed throughout the cell. However anti-TRPC1/C6 and C7 antibodies had no effect on channel activity. An important caveat is that we have not tested the antibodies on expressed TRPC channel proteins to confirm that these antibodies have an inhibitory effect on the respective channels. Therefore the present work does not rule out that other TRPC subunits may contribute to the functioning channel.
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Pharmacology of Icat
In addition we studied the inhibitory actions of flufenamic acid (FFA), Gd3+, La3+ and [Ca2+]o on channel activity for comparison with TRPC channels expressed in cell lines. A summary of these and other properties are shown in Table 1. There is a close similarity between the characteristics of native channels in ear artery and expressed TRPC3 channels although FFA, Gd3+ and La3+ were slightly more potent in ear artery. Since TRPC3/6/7 are members of the same subgroup it is pertinent to note that flufenamic acid inhibits NPo in ear artery but potentiates TRPC6 currents. Also Gd3+ and La3+ are several orders more potent against channels in ear artery compared to TRPC7 currents. Although TRPC1 appeared to be localized to the membrane in ear artery myocytes it should be noted that PKC activates TRPC1 but inhibits Icat in ear artery and TRPC3 currents. Moreover Gd3+, La3+ and [Ca2+]o have been shown to potentiate TRPC4/5 currents whereas these agents inhibit Icat in ear artery and TRPC3 currents (Table 1).
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Comparison with other native TRPC3 channels in vascular smooth muscle
Experiments with antisense oligodeoxy-nucleotides have suggested that TRPC3 proteins mediates pyrimidine-induced depolarization in rat cerebral arteries (Reading et al. 2005). It is interesting that in this study the resting membrane potential was not altered in TRPC3 antisense-treated arteries. These data suggest that TRPC3 is not involved in the resting membrane conductance of this preparation or that another conductance was increased to compensate for the knock-down of TRPC3. In contrast in a TRPC6-deficient mouse model the resting membrane potential of cerebral arteries was more depolarized than wild-type preparations and this was accounted for by increased expression of TRPC3 in TRPC6–/– mice (Dietrich et al. 2005).
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It is well known that TRPC molecules form heteromeric channels and we cannot rule out that other TRPC subunits are involved in the channel described in the present work. Nevertheless the properties of Icat are closest to TRPC3 and if other TRPC molecules are involved their presence does not confer significant known functional characteristics on the constitutive channel in ear artery myocytes.
Conclusions
The present results indicate that TRPC3 channel proteins are important constituents of spontaneously active cation channels in rabbit ear artery myocytes. The physiological importance of these channels is due to their contribution to the resting membrane conductance. Increased channel activity will lead to a more depolarized resting membrane potential and consequently enhance activity of voltage-gated Ca2+ channels resulting in increased vascular tone and excitability.
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Supplemental material
The online version of this paper can be accessed at:
DOI: 10.1113/jphysiol.2005.102780
http://jp.physoc.org/cgi/content/full/jphysiol.2005.102780/DC1
and contains supplemental material.
This material can also be found as part of the full-text HTML version available from http://www.blackwell-synergy.com
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Footnotes
Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial explotation.
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Abstract
Previously we have described a constitutively active, Ca2+-permeable, non-selective cation channel in freshly dispersed rabbit ear artery myocytes which has similar properties to some of the canonical transient receptor potential (TRPC) channel proteins. In the present work we have compared the properties of constitutive channel activity with known properties of TRPC proteins by investigating the effect of selective anti-TRPC antibodies and pharmacological agents on whole-cell and single cation channel activity. Bath application of anti-TRPC3 antibodies markedly reduced channel activity in inside-out patches and also produced a pronounced reduction of both current amplitude and variance of constitutively active whole-cell cation currents whereas anti-TRPC1/4/5/6/7 antibodies had no effect on channel activity. In the presence of antigenic peptide, anti-TRPC3 antibodies had no effect on whole-cell or single cation channel activity. Bath application of flufenamic acid, Gd3+, La3+ and Ca2+ inhibited spontaneous channel activity in outside-out patches with IC50 values of 6.8 μM, 25 nM, 1.5 μM and 0.124 mM, respectively, which are similar values to those against TRPC3 proteins. Immunocytochemical studies combined with confocal microscopy showed expression of TRPC3 proteins in ear artery myocytes, and these were predominately distributed at, or close to, the plasma membrane. These data provide strong evidence that native constitutively active cation channels in rabbit ear artery myocytes have similar properties to TRPC3 channel proteins and indicate that these proteins may have an important role in mediating this conductance.
, 百拇医药
Introduction
In freshly dispersed rabbit ear artery smooth muscle cells we have described a constitutively active Ca2+-permeable non-selective cation current (Icat), which has been proposed to contribute to resting membrane conductance and basal Ca2+ influx (Albert et al. 2003). The spontaneous nature of this ion channel appears to reside in constitutive Gi/Go subunits of G-proteins which stimulate phospholipase D (PLD) to cleave phosphatidylcholine to produce phosphatidic acid. Subsequently phosphatidic acid is converted to diacylglycerol (DAG), which initiates channel opening via a protein kinase C (PKC)-independent mechanism (Albert & Large, 2004; Albert et al. 2005). In parallel there is an inhibitory signalling pathway in which Gq/G11 couples to U73122-sensitive phospholipase C (PLC) to produce DAG, which reduces open probability of ion channels by a PKC-dependent mechanism (Albert & Large, 2004; see Fig. 2 of Albert & Large, 2006). Moreover the neurotransmitter noradrenaline also increases Icat (Albert & Large, 2004).
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A, shows that inclusion of anti-TRPC3a antibodies at 1: 200 in the patch pipette solution markedly reduced both the amplitude (i) and variance (ii) of a constitutively active whole-cell cation current held at –50 mV. The inset shows individual current–voltage curves from Ai. B, shows that inclusion of anti-TRPC3a antibodies following preincubation with its antigenic peptide in the patch pipette solution had no effect on both the amplitude (i) or variance (ii) of the whole-cell current. The asterisk denotes when whole-cell configuration was obtained and the dotted lines represent zero holding current. C and D, mean data showing that anti-TRPC3a antibodies significantly reduced, respectively, holding current and current variance of constitutively active whole-cell cation currents (n= 7, P < 0.01).
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A key question concerns the molecular identity of Icat. The observation that DAG stimulates this conductance via a PKC-independent mechanism suggests strongly that member(s) of the canonical transient receptor potential (TRPC) family of channel proteins are involved. To our knowledge these are the only non-selective cation channels that are stimulated by DAG in this manner. Specifically it is often stated that this is a key characteristic of the TRPC3/6/7 subfamily (e.g. Minke & Cooke, 2002; Beech et al. 2004; Desai & Clapham, 2005) although there is a report that DAG also activates mouse TRPC5 by a PKC-independent mechanism (Lee et al. 2003).
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Previously we have highlighted similarities and some notable differences between Icat in rabbit ear artery and the noradrenaline-evoked cation conductance in portal vein myocytes (Albert et al. 2003), which is thought to involve TRPC6 proteins (Inoue et al. 2001).
In the present work we have investigated the effect of anti-TRPC antibodies on ion channel activity in rabbit ear artery myocytes. Immunopharmacological approaches have been used to study the roles of many types of ion channels including TRPC channel proteins in neurones (Kim et al. 2003; Dallas et al. 2005) and vascular myocytes (Xu & Beech, 2001). In addition we used immunocytochemical studies with confocal imaging to probe the cellular distribution of TRPC proteins and studied the inhibitory action of several multivalent cations and other pharmacological agents for comparison with expressed TRPC channels. The results from these studies suggest that the properties of Icat are similar to those of TRPC3 channel proteins, which indicates that TRPC3 may be a significant molecular constituent of this native conductance.
, 百拇医药
Methods
Cell isolation
New Zealand White rabbits (2–3 kg) were killed by an I.V. injection of sodium pentobarbitone (120 mg kg–1, in accordance with the UK Animals (Scientific Procedures) Act 1986) and ear arteries from both ears were removed. The ear arteries were freshly dispersed using procedures and solutions previously described (Albert et al. 2003; Albert & Large, 2004).
Electrophysiology
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Whole-cell and single channel currents were recorded with an Axopatch 200B patch clamp amplifier (Axon Instruments, Inc., Union City, CA, USA) at room temperature using whole-cell recording, outside-out and inside-out configurations of the patch clamp technique and data acquisition and analysis protocols as previously described (see Supplemental material and Helliwell & Large, 1998; Albert et al. 2003; Albert & Large, 2004).
Immunocytochemistry
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Freshly dispersed myocytes were fixed by 4% paraformaldehyde in physiological saline solution (PSS, see Albert et al. 2003) containing penicillin (20 U ml–1) and streptomycin (20 μg ml–1) for 10 min at room temperature. The myocytes were then processed for TRPC protein staining and imaged using laser scanning confocal microscope as described in Supplemental material and Saleh et al. (2005).
Solutions and drugs
The bathing and patch pipette solutions for whole-cell recording, outside-out patches and inside-out patches were K+ free as previously described (Albert et al. 2003, 2005; Albert & Large, 2004; see Supplemental material).
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Flufenamic acid (FFA), GdCl3 and LaCl3 were dissolved in distilled H2O at a stock concentration of 10 mM. External 1.5 mM CaCl2 was replaced with either 10 μM, 100 μM or 10 mM CaCl2 and in the Ca2+-free external solution CaCl2 was omitted and 1 mM BAPTA was added (< 10 nM free Ca2+ concentration). Anti-TRPC antibodies were obtained from Alomone Laboratories (Jerusalem, Israel; defined as TRPCa), Santa Cruz Biotechnology (Santa Cruz, CA, USA; defined as TRPC7sc) and also from Professor W. P. Schilling (defined as hTRPC; see Goel et al. (2002) and Supplemental material). The values are the mean of n cells ±S.E.M. and statistical analysis was carried out using Student's t test (paired and unpaired) with the level of significance set at P < 0.05.
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Results
Effect of selective anti-TRPC antibodies on constitutively active cation channel activity in inside-out patches from rabbit ear artery myocytes
In the first series of experiments, we investigated the effect of selective anti-TRPC antibodies on constitutively active cation channels in inside-out patches from rabbit ear artery myocytes. The inside-out patch configuration allows anti-TRPC antibodies, raised against putative intracellular epitopes of human and mouse TRPC channel proteins (see Methods and Supplemental material), to be applied directly to the intracellular surface of the plasma membrane.
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A and B, bath application of, respectively, anti-TRPC3a antibodies at 1 : 200 dilution and anti-hTRPC3 antibodies at 0.3 μg ml–1 reversibly inhibited constitutively active channel activity in two different patches held at –50 mV. Insets show individual channel currents on a faster time scale. C, mean data showing the effect of anti-TRPC antibodies on constitutive channel activity represented as NPo. The data using anti-TRPC antibodies from different sources have been pooled; note that the anti-TRPC3a antibody had no effect on channel activity following preincubation with its antigenic peptide (P < 0.001).
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We also studied the effect of selective anti-TRPC antibodies raised against all TRPC channel proteins (except TRPC2, which is considered a pseudogene in man) on constitutive channel activity, and for each TRPC protein, antibodies from two different sources were used (see Methods). Figure 1C shows that only anti-TRPC3 antibodies had a significant effect on channel activity and that antibodies raised against TRPC1/4/5/6/7 had no effect on channel activity in inside-out patches.
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These data indicate that TRPC3 proteins have an important role in mediating constitutive channel activity in rabbit ear artery myocytes.
Effect of anti-TRPC3 antibodies on constitutively active whole-cell cation currents
To further investigate the potential role of TRPC3 proteins in constitutive channel activity, we studied the effect of anti-TRPC3a antibodies using whole-cell recording. Figure 2A shows the effect of including of anti-TRPC3a antibody at 1 : 200 dilution in the patch pipette solution on the properties of basal whole-cell currents. Figure 2Ai illustrates that immediately after whole-cell configuration was obtained a characteristic ‘noisy’ holding current was observed which declined in amplitude and ‘noisy’ appearance over about 5 min. The inset in Fig. 2A shows current–voltage (I–V) relationships evoked after 1 min (a) and 5 min (b), which had similar reversal potentials of about 0 mV but after 5 min the I–V relationship was reduced at all membrane potentials and exhibited less current fluctuations at negative potentials. The characteristic ‘noisy’ appearance of the constitutive whole-cell currents probably reflects the spontaneous opening and closing of cation channels and therefore we examined the effect of anti-TRPC3a antibodies on the variance of these whole-cell currents using noise analysis. Figure 2Aii illustrates that anti-TRPC3a antibodies produced a marked reduction in variance of the whole-cell current shown in Fig. 2Ai, and Fig. 2C and D shows that inclusion of anti-TRPC3a antibodies in the patch pipette solution induced a significant reduction in both the mean amplitude and variance of constitutively active whole-cell cation currents at –50 mV after 5 min.
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Effect of pharmacological agents on constitutive cation channel activity in outside-out patches
To further investigate whether TRPC3 proteins are components of these native cation channels we carried out experiments using the pharmacological agents flufenamic acid (FFA), Gd3+ and La3+, which have been previously shown to have differential effects on expressed TRPC channel proteins in cell lines (see Table 1) and therefore may be useful tools for distinguishing between various TRPC proteins involved in native cation conductances.
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A, shows that bath application of 10 μM flufenamic acid (FFA) reduced and 100 μM FFA completely blocked constitutive cation channel activity at –50 mV. B, C, D and E, respectively, show mean concentration–effect curves for FFA, Gd3+, La3+ and [Ca2+]o on constitutive channel activity at –50 mV.
Changing external Ca2+ concentration ([Ca2+]o) has been shown to have differential effects on the activity of expressed TRPC channel proteins (see Table 1), and therefore we further investigated this effect of [Ca2+]o on constitutive channel activity in outside-out patches. Figure 3E shows that [Ca2+]o had a pronounced inhibitory effect on channel activity in ear artery myocytes with an IC50 value of 0.124 mM.
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Immunocytochemical evidence for the presence of TRPC3 channel proteins in rabbit ear artery myocytes
In the next series of experiments we investigated whether TRPC3 proteins are present in ear artery myocytes using immunocytochemical techniques and confocal microscopy, which enable the expression and location of TRPC3 proteins to be examined using anti-TRPC3 antibodies.
A shows a single confocal plane fluorescence image of a myocyte labelled with anti-TRPC3a antibodies (1 : 200) and B shows another myocyte labelled with anti-TRPC3a antibodies (1 : 200) preincubated with their antigenic peptide (1 : 100). Insets in A and B show transmitted light image of these myocytes. White circles in A indicate Regions 1 and 2, which were used to analyse the localization of fluorescence (see Methods and Supplemental material). A dotted line was used in B to outline the contour of a cell, due to its low fluorescence. C, mean data showing the localization of fluorescence related to binding of anti-TRPC3a antibodies. D, mean data showing intensity of fluorescence, expressed as average pixel fluorescence (intensity units per pixel). The values of all pixels in the confocal plane of the myocytes were added up and then divided by the number of pixels. The specificity of labelling was confirmed by greatly reduced fluorescence after preincubation with antigenic peptide or by virtual lack of fluorescence in the absence of primary antibodies (P < 0.001).
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We also investigated the expression of other TRPC proteins in rabbit ear artery myocytes and found that anti-TRPC1a and anti-TRPC7sc antibodies stained ear artery myocytes predominantly at the plasma membrane whereas anti-TRPC6a antibodies stained myocytes throughout the cell (data not shown). Anti-TRPC4a and anti-TRPC5a antibodies did not produce significant specific staining of ear artery myocytes (data not shown).
Discussion
, 百拇医药 In the present study we have used electrophysiological and immunocytochemical techniques to investigate the possibility that TRPC channel proteins constitute native spontaneously active Ca2+-permeable non-selective cation channels in freshly dispersed rabbit ear artery smooth muscle cells. The data provide strong evidence TRPC3 is involved in forming the functional ion channel.
Experiments with anti-TRPC antibodies
These data clearly shows that two anti-TRPC3 antibodies raised against different epitopes within the TRPC3 protein markedly reduced channel activity in inside-out patches by about 90%. It is not evident how the anti-TRPC3 antibodies inhibit channel activity but the inhibitory response was completely blocked by preincubation with an antigenic peptide. It is possible that attachment of the antibody to the channel protein inhibits channel opening by preventing essential conformational changes (see Dallas et al. 2005). In contrast antibodies to other TRPC proteins (two for each subtype) had no significant effect on channel activity. This is good evidence that TRPC3 molecules are components of the functioning ion channel. Also inclusion of an anti-TRPC3 antibody reduced both constitutively active whole-cell conductance and current variance. These data are in keeping with the hypothesis that the stochastic opening and closing of TRPC3 channels contribute to the resting membrane conductance of these myocytes.
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Immunocytochemical experiments demonstrated the expression of TRPC3 proteins in ear artery myocytes and that these proteins were preferentially localized to the cell membrane, which would be expected for a functional plasmalemmal ion channel. TRPC1 and TRPC7 antibody staining showed a similar pattern of distribution, and whereas TRPC6 was present it appeared to be evenly distributed throughout the cell. However anti-TRPC1/C6 and C7 antibodies had no effect on channel activity. An important caveat is that we have not tested the antibodies on expressed TRPC channel proteins to confirm that these antibodies have an inhibitory effect on the respective channels. Therefore the present work does not rule out that other TRPC subunits may contribute to the functioning channel.
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Pharmacology of Icat
In addition we studied the inhibitory actions of flufenamic acid (FFA), Gd3+, La3+ and [Ca2+]o on channel activity for comparison with TRPC channels expressed in cell lines. A summary of these and other properties are shown in Table 1. There is a close similarity between the characteristics of native channels in ear artery and expressed TRPC3 channels although FFA, Gd3+ and La3+ were slightly more potent in ear artery. Since TRPC3/6/7 are members of the same subgroup it is pertinent to note that flufenamic acid inhibits NPo in ear artery but potentiates TRPC6 currents. Also Gd3+ and La3+ are several orders more potent against channels in ear artery compared to TRPC7 currents. Although TRPC1 appeared to be localized to the membrane in ear artery myocytes it should be noted that PKC activates TRPC1 but inhibits Icat in ear artery and TRPC3 currents. Moreover Gd3+, La3+ and [Ca2+]o have been shown to potentiate TRPC4/5 currents whereas these agents inhibit Icat in ear artery and TRPC3 currents (Table 1).
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Comparison with other native TRPC3 channels in vascular smooth muscle
Experiments with antisense oligodeoxy-nucleotides have suggested that TRPC3 proteins mediates pyrimidine-induced depolarization in rat cerebral arteries (Reading et al. 2005). It is interesting that in this study the resting membrane potential was not altered in TRPC3 antisense-treated arteries. These data suggest that TRPC3 is not involved in the resting membrane conductance of this preparation or that another conductance was increased to compensate for the knock-down of TRPC3. In contrast in a TRPC6-deficient mouse model the resting membrane potential of cerebral arteries was more depolarized than wild-type preparations and this was accounted for by increased expression of TRPC3 in TRPC6–/– mice (Dietrich et al. 2005).
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It is well known that TRPC molecules form heteromeric channels and we cannot rule out that other TRPC subunits are involved in the channel described in the present work. Nevertheless the properties of Icat are closest to TRPC3 and if other TRPC molecules are involved their presence does not confer significant known functional characteristics on the constitutive channel in ear artery myocytes.
Conclusions
The present results indicate that TRPC3 channel proteins are important constituents of spontaneously active cation channels in rabbit ear artery myocytes. The physiological importance of these channels is due to their contribution to the resting membrane conductance. Increased channel activity will lead to a more depolarized resting membrane potential and consequently enhance activity of voltage-gated Ca2+ channels resulting in increased vascular tone and excitability.
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Supplemental material
The online version of this paper can be accessed at:
DOI: 10.1113/jphysiol.2005.102780
http://jp.physoc.org/cgi/content/full/jphysiol.2005.102780/DC1
and contains supplemental material.
This material can also be found as part of the full-text HTML version available from http://www.blackwell-synergy.com
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Footnotes
Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial explotation.
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