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. 2008 May;43(5):506-14.
doi: 10.1016/j.ceca.2007.09.001. Epub 2007 Oct 17.

Complex regulation of the TRPC3, 6 and 7 channel subfamily by diacylglycerol and phosphatidylinositol-4,5-bisphosphate

Loïc Lemonnier  1 , Mohamed Trebak, James W Putney Jr
Affiliations

Complex regulation of the TRPC3, 6 and 7 channel subfamily by diacylglycerol and phosphatidylinositol-4,5-bisphosphate

Loïc Lemonnier et al. Cell Calcium. 2008 May.

Abstract

TRPC3, 6 and 7 channels constitute a subgroup of non-selective, calcium-permeable cation channels within the TRP superfamily that are activated by products of phospholipase C-mediated breakdown of phosphatidylinositol-4,5-bisphosphate (PIP(2)). A number of ion channels, including other members of the TRP superfamily, are regulated directly by PIP(2). However, there is little information on the regulation of the TRPC channel subfamily by PIP(2). Pretreatment of TRPC7-expressing cells with a drug that blocks the synthesis of polyphosphoinositides inhibited the ability of the synthetic diacylglycerol, oleyl-acetyl glycerol, to activate TRPC7. In excised patches, TRPC7 channels were robustly activated by application of PIP(2) or ATP, but not by inositol 1,4,5-trisphosphate. Similar results were obtained with TRPC6 and TRPC3, although the effects of PIP(2) were somewhat less and with TRPC3 there was no significant effect of ATP. In the cell-attached configuration, TRPC7 channels could be activated by the synthetic diacylglycerol analog, oleyl-acetyl glycerol. However, this lipid mediator did not activate TRPC7 channels in excised patches. In addition, channel activation by PIP(2) in excised patches was significantly greater than that observed with oleyl-acetyl glycerol in the cell-attached configuration. These findings reveal complex regulation of TRPC channels by lipid mediators. The results also reveal for the first time direct activation by PIP(2) of members of the TRPC ion channel subfamily.

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Figures

Figure 1
Figure 1. The inositol lipid kinase inhibitor, LY294002, inhibits Ca2+ entry responses of TRPC7-expressing HEK293 cells to OAG
A: Time course of average fluorescence ratio in TRPC7-HEK cells loaded with Fura-2 and pretreated (LY, n=55, 2 independent experiments) or not (Con, n=74, 2 independent experiments) with 100 µM LY294002. LY294002 was added 5 minutes prior to the beginning of data collection and was present throughout. Cells were exposed to 30 µM OAG as indicated by the horizontal bar. Results are presented as mean ± SEM, and are representative of a total of 6 independent experiments. B: The protocol was similar to that for A, except LY294002 was added after activation of Ca2+ entry by OAG, as indicated (n=20, from one experiment, representative of four).
Figure 2
Figure 2. Effects of LY294002 and PIP2 on whole-cell TRPC7 currents
A: Average time courses (mean ± SEM) of whole-cell current development at −100 and +100 mV in TRPC7-expressing HEK293 cells pretreated (LY, solid circle, n=5) or not (Con, open circle, n=5) with 100 µM LY294002 for 5 minutes before break-in, and then exposed to 30 µM OAG as indicated by the horizontal bar. As in A, LY294002 was maintained in the bath during the whole course of the recording. B: Current-voltage relationships for OAG-activated TRPC7 currents. The IV relationships are representative of TRPC7-HEK cells pretreated (LY) or not with 100 µM LY294002 and then sequentially exposed to a control (gray traces) or a 30 µM OAG-containing solution (black traces). C: Average time courses (mean ± SEM) of whole-cell current development at −100 and +100 mV in TRPC7-expressing HEK293 cells activated by 30 µM OAG. Shown are average current from Control experiments (Con, open circles, n=5), and from experiments in which the pipette solution contained 100µM PIP2 (PIP2, solid circles, n=5).
Figure 3
Figure 3. PIP2 activates TRPC7 channels in inside-out patches
A: Histogram summarizing TRPC7 channel open probability over time at +60 mV during repeated applications of a 10 µM PIP2-containing solution. The figure is representative of results obtained in 12 independent inside-out patches. B: Examples of currents recorded in an inside-out patch clamped at +60 mV and exposed to a standard (Con, top panel) or a 10 µM PIP2-containing solution (bottom panel). “c” and “o” denote respectively the closed and open states. C and D: All-points histograms summarizing single channel amplitudes at +60 mV in the inside-out configuration before (Control) and after addition of 10 µM PIP2.
Figure 4
Figure 4. MgATP activates TRPC7 channels in inside-out patches
A: Histogram summarizing TRPC7 channel open probability over time at +60 mV when challenged with 1 mM MgATP. The figure is representative of results obtained in 9 independent inside-out patches. B: Representative traces of currents recorded in an inside-out patch clamped at +60 mV and exposed to a control (top panel) or 1 mM MgATP-containing solution (bottom panel). “c” and “o” denote respectively the closed and open states. C: All-points histograms summarizing single channel amplitudes at +60 mV in the inside-out configuration before (Con) and after addition of 1 mM MgATP (ATP).
Figure 5
Figure 5. Summarized properties of TRPC7 channels
Histograms summarizing TRPC7 single channel activity at +60 mV in the inside-out configuration when challenged with 1 mM MgATP (n=9 patches), 10 µM PIP2 (n=12 patches) or 10 µM IP3 (n=4 patches) (C = Control). Results are presented as mean ± SEM.
Figure 6
Figure 6. TRPC6 channels in inside-out patches are activated by PIP2
A and B: All-points histograms summarizing single channel amplitudes at +60 mV in the inside-out configuration before (A, Control) and after addition of 10 µM PIP2 (B, PIP2). C: Examples of TRPC6 currents recorded in an inside-out patch clamped at +60 mV and exposed to a standard (top panel) or 10 µM PIP2-containing solution (bottom panel). “c” and “o” denote respectively the closed and open states. D: Histograms summarizing TRPC6 single channel activity at +60 mV in the inside-out configuration when challenged with 1 mM MgATP (n=5 patches), 10 µM PIP2 (n=8 patches) or 10 µM IP3 (n=5 patches). Results are presented as mean ± SEM.
Figure 7
Figure 7. TRPC3 channels in inside-out patches are not significantly affected by PIP2
A and B: All-points histograms summarizing single channel amplitudes at +60 mV in the inside-out configuration before (A, Control) and after addition of 10 µM PIP2 (B, PIP2). C: Examples of TRPC3 currents recorded in an inside-out patch clamped at +60 mV and exposed to a regular (top panel) or a 10 µM PIP2-containing solution (bottom panel). “c” and “o” denote respectively the closed and open states. D: Histograms summarizing the variations of TRPC3 single channel activity at +60 mV in the inside-out configuration when challenged with 1 mM MgATP (n=5 patches), 10 µM PIP2 (n=10 patches) or 10 µM IP3 (n=5 patches). Results are presented as mean ± SE.
Figure 8
Figure 8. Effects of OAG on TRPC7 channel activity in cell attached and excised patch configurations
Histograms summarizing effects of 30 µM OAG (Con = controls) on TRPC7 single channel activity (Po = open probability) at +60 mV in the cell attached or excised, inside out patch configuration. Shown are averages ± SEM for 9 experiments for cell attached patch, and 10 experiments for excised patch.
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