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Obligatory role for phosphatidylinositol 4,5-bisphosphate in activation of native TRPC1 store-operated channels in vascular myocytes.

Saleh SN, Albert AP, Large WA - J. Physiol. (Lond.) (2008)

Bottom Line: However, these reagents did not alter the ability of PIP(2) to activate SOCs in inside-out patches.Co-immunoprecipitation experiments provide evidence that PKC-dependent phosphorylation of TRPC1 occurs constitutively and was increased by CPA and PDBu but decreased by chelerythrine.These novel results show that PIP(2) can activate TRPC1 SOCs in native vascular myocytes and plays an important role in SOC activation by CPA, BAPTA-AM and PDBu.

View Article: PubMed Central - PubMed

Affiliation: Ion Channels and Cell Signalling Research Centre, Division of Basic Medical Sciences, St George's, University of London, Cranmer Terrace, London, UK.

ABSTRACT
In the present study the effect of phosphatidylinositol 4,5-bisphosphate (PIP(2)) was studied on a native TRPC1 store-operated channel (SOC) in freshly dispersed rabbit portal vein myocytes. Application of diC8-PIP(2), a water soluble form of PIP(2), to quiescent inside-out patches evoked single channel currents with a unitary conductance of 1.9 pS. DiC8-PIP(2)-evoked channel currents were inhibited by anti-TRPC1 antibodies and these characteristics are identical to SOCs evoked by cyclopiazonic acid (CPA) and BAPTA-AM. SOCs stimulated by CPA, BAPTA-AM and the phorbol ester phorbol 12,13-dibutyrate (PDBu) were reduced by anti-PIP(2) antibodies and by depletion of tissue PIP(2) levels by pre-treatment of preparations with wortmannin and LY294002. However, these reagents did not alter the ability of PIP(2) to activate SOCs in inside-out patches. Co-immunoprecipitation techniques demonstrated association between TRPC1 and PIP(2) at rest, which was greatly decreased by wortmannin and LY294002. Pre-treatment of cells with PDBu, which activates protein kinase C (PKC), augmented SOC activation by PIP(2) whereas the PKC inhibitor chelerythrine decreased SOC stimulation by PIP(2). Co-immunoprecipitation experiments provide evidence that PKC-dependent phosphorylation of TRPC1 occurs constitutively and was increased by CPA and PDBu but decreased by chelerythrine. These novel results show that PIP(2) can activate TRPC1 SOCs in native vascular myocytes and plays an important role in SOC activation by CPA, BAPTA-AM and PDBu. Moreover, the permissive role of PIP(2) in SOC activation requires PKC-dependent phosphorylation of TRPC1.

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Anti-PIP2 antibodies inhibit TRPC1 SOC activityA, bath application of anti-PIP2 antibodies (1: 200 dilution) to the cytosolic surface of inside-out patches held at –80 mV markedly inhibited SOC activity initially induced by 10 μm CPA (a) or 50 μm BAPTA-AM (b) in cell-attached patches and 1 μm PDBu (c) applied to a quiescent inside-out patch. B, mean data showing that anti-PIP2 antibodies significantly reduced SOC activity evoked by CPA, BAPTA-AM and PDBu (*P < 0.05). C, co-immunoprecipitation experiments where tissue lysates from portal vein were immunoprecipitated (IP) with an anti-TRPC1 antibody (Santa Cruz) and then Western blotted (WB) with an anti-PIP2 antibody. In control conditions a band of ∼70 kDa was observed (see Methods), which was absent after pre-treatment of the anti-TRPC1 antibody with its antigenic peptide (AgP). Note that bands detected with an anti-β-actin antibody were unaffected by pretreatment with the antigenic peptide.
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fig02: Anti-PIP2 antibodies inhibit TRPC1 SOC activityA, bath application of anti-PIP2 antibodies (1: 200 dilution) to the cytosolic surface of inside-out patches held at –80 mV markedly inhibited SOC activity initially induced by 10 μm CPA (a) or 50 μm BAPTA-AM (b) in cell-attached patches and 1 μm PDBu (c) applied to a quiescent inside-out patch. B, mean data showing that anti-PIP2 antibodies significantly reduced SOC activity evoked by CPA, BAPTA-AM and PDBu (*P < 0.05). C, co-immunoprecipitation experiments where tissue lysates from portal vein were immunoprecipitated (IP) with an anti-TRPC1 antibody (Santa Cruz) and then Western blotted (WB) with an anti-PIP2 antibody. In control conditions a band of ∼70 kDa was observed (see Methods), which was absent after pre-treatment of the anti-TRPC1 antibody with its antigenic peptide (AgP). Note that bands detected with an anti-β-actin antibody were unaffected by pretreatment with the antigenic peptide.

Mentions: In the first experiments SOC activity was induced in cell-attached patches by CPA or BAPTA-AM and then patches were excised and anti-PIP2 antibodies were applied to the internal membrane surface of the patches. Figure 2Aa and b and B shows that mean NPo of SOC activity, initially induced by 10 μm CPA or 50 μm BAPTA-AM, was significantly inhibited by anti-PIP2 antibodies (1: 200 dilution) by 91 ± 3% (n= 7) and 82 ± 9% (n= 7), respectively. This effect was not seen when the anti-PIP2 antibody was denatured prior to application by boiling for a period of 30 min (n= 4, data not shown). In addition Fig. 2Ac and B show that anti-PIP2 antibodies also produced marked inhibition of SOC activity induced by the PKC-activating phorbol ester phorbol 12,13-dibutyrate (PDBu, 1 μm), which has been previously shown to activate SOCs in this preparation (Albert & Large, 2002b; Saleh et al. 2008). In these experiments inside-out patches were prepared from quiescent cells before application of PDBu and anti-PIP2 antibodies, which significantly reduced activity by 92 ± 2% (n= 5). Figure 2C shows that tissue lysates from portal vein immunoprecipitated with anti-TRPC1 antibodies then Western blotted with anti-PIP2 antibodies detected a band of ∼70 kDa, which is the predicted band for the PIP2 complex with this antibody (see Methods). Moreover Fig. 2C shows that preincubation of anti-TRPC1 antibodies with its antigenic peptide (AgP) reduced detection of the band with anti-PIP2 antibodies without affecting expression levels of β-actin proteins.


Obligatory role for phosphatidylinositol 4,5-bisphosphate in activation of native TRPC1 store-operated channels in vascular myocytes.

Saleh SN, Albert AP, Large WA - J. Physiol. (Lond.) (2008)

Anti-PIP2 antibodies inhibit TRPC1 SOC activityA, bath application of anti-PIP2 antibodies (1: 200 dilution) to the cytosolic surface of inside-out patches held at –80 mV markedly inhibited SOC activity initially induced by 10 μm CPA (a) or 50 μm BAPTA-AM (b) in cell-attached patches and 1 μm PDBu (c) applied to a quiescent inside-out patch. B, mean data showing that anti-PIP2 antibodies significantly reduced SOC activity evoked by CPA, BAPTA-AM and PDBu (*P < 0.05). C, co-immunoprecipitation experiments where tissue lysates from portal vein were immunoprecipitated (IP) with an anti-TRPC1 antibody (Santa Cruz) and then Western blotted (WB) with an anti-PIP2 antibody. In control conditions a band of ∼70 kDa was observed (see Methods), which was absent after pre-treatment of the anti-TRPC1 antibody with its antigenic peptide (AgP). Note that bands detected with an anti-β-actin antibody were unaffected by pretreatment with the antigenic peptide.
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Related In: Results  -  Collection

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fig02: Anti-PIP2 antibodies inhibit TRPC1 SOC activityA, bath application of anti-PIP2 antibodies (1: 200 dilution) to the cytosolic surface of inside-out patches held at –80 mV markedly inhibited SOC activity initially induced by 10 μm CPA (a) or 50 μm BAPTA-AM (b) in cell-attached patches and 1 μm PDBu (c) applied to a quiescent inside-out patch. B, mean data showing that anti-PIP2 antibodies significantly reduced SOC activity evoked by CPA, BAPTA-AM and PDBu (*P < 0.05). C, co-immunoprecipitation experiments where tissue lysates from portal vein were immunoprecipitated (IP) with an anti-TRPC1 antibody (Santa Cruz) and then Western blotted (WB) with an anti-PIP2 antibody. In control conditions a band of ∼70 kDa was observed (see Methods), which was absent after pre-treatment of the anti-TRPC1 antibody with its antigenic peptide (AgP). Note that bands detected with an anti-β-actin antibody were unaffected by pretreatment with the antigenic peptide.
Mentions: In the first experiments SOC activity was induced in cell-attached patches by CPA or BAPTA-AM and then patches were excised and anti-PIP2 antibodies were applied to the internal membrane surface of the patches. Figure 2Aa and b and B shows that mean NPo of SOC activity, initially induced by 10 μm CPA or 50 μm BAPTA-AM, was significantly inhibited by anti-PIP2 antibodies (1: 200 dilution) by 91 ± 3% (n= 7) and 82 ± 9% (n= 7), respectively. This effect was not seen when the anti-PIP2 antibody was denatured prior to application by boiling for a period of 30 min (n= 4, data not shown). In addition Fig. 2Ac and B show that anti-PIP2 antibodies also produced marked inhibition of SOC activity induced by the PKC-activating phorbol ester phorbol 12,13-dibutyrate (PDBu, 1 μm), which has been previously shown to activate SOCs in this preparation (Albert & Large, 2002b; Saleh et al. 2008). In these experiments inside-out patches were prepared from quiescent cells before application of PDBu and anti-PIP2 antibodies, which significantly reduced activity by 92 ± 2% (n= 5). Figure 2C shows that tissue lysates from portal vein immunoprecipitated with anti-TRPC1 antibodies then Western blotted with anti-PIP2 antibodies detected a band of ∼70 kDa, which is the predicted band for the PIP2 complex with this antibody (see Methods). Moreover Fig. 2C shows that preincubation of anti-TRPC1 antibodies with its antigenic peptide (AgP) reduced detection of the band with anti-PIP2 antibodies without affecting expression levels of β-actin proteins.

Bottom Line: However, these reagents did not alter the ability of PIP(2) to activate SOCs in inside-out patches.Co-immunoprecipitation experiments provide evidence that PKC-dependent phosphorylation of TRPC1 occurs constitutively and was increased by CPA and PDBu but decreased by chelerythrine.These novel results show that PIP(2) can activate TRPC1 SOCs in native vascular myocytes and plays an important role in SOC activation by CPA, BAPTA-AM and PDBu.

View Article: PubMed Central - PubMed

Affiliation: Ion Channels and Cell Signalling Research Centre, Division of Basic Medical Sciences, St George's, University of London, Cranmer Terrace, London, UK.

ABSTRACT
In the present study the effect of phosphatidylinositol 4,5-bisphosphate (PIP(2)) was studied on a native TRPC1 store-operated channel (SOC) in freshly dispersed rabbit portal vein myocytes. Application of diC8-PIP(2), a water soluble form of PIP(2), to quiescent inside-out patches evoked single channel currents with a unitary conductance of 1.9 pS. DiC8-PIP(2)-evoked channel currents were inhibited by anti-TRPC1 antibodies and these characteristics are identical to SOCs evoked by cyclopiazonic acid (CPA) and BAPTA-AM. SOCs stimulated by CPA, BAPTA-AM and the phorbol ester phorbol 12,13-dibutyrate (PDBu) were reduced by anti-PIP(2) antibodies and by depletion of tissue PIP(2) levels by pre-treatment of preparations with wortmannin and LY294002. However, these reagents did not alter the ability of PIP(2) to activate SOCs in inside-out patches. Co-immunoprecipitation techniques demonstrated association between TRPC1 and PIP(2) at rest, which was greatly decreased by wortmannin and LY294002. Pre-treatment of cells with PDBu, which activates protein kinase C (PKC), augmented SOC activation by PIP(2) whereas the PKC inhibitor chelerythrine decreased SOC stimulation by PIP(2). Co-immunoprecipitation experiments provide evidence that PKC-dependent phosphorylation of TRPC1 occurs constitutively and was increased by CPA and PDBu but decreased by chelerythrine. These novel results show that PIP(2) can activate TRPC1 SOCs in native vascular myocytes and plays an important role in SOC activation by CPA, BAPTA-AM and PDBu. Moreover, the permissive role of PIP(2) in SOC activation requires PKC-dependent phosphorylation of TRPC1.

Show MeSH