Inhibition of Transient Receptor Potential Melastatin 3 ion channels by G-protein βγ subunits
- New Jersey Medical School, Rutgers, the State University of New Jersey, United States
- Weis Center for Research, Geisinger Clinic, United States
Figures
Figure 1 with 3 supplements
Inhibition of TRPM3 by Gq-coupled M1 and Gi-coupled M2 muscarinic receptors via Gβγ.
Whole-cell patch clamp experiments on HEK cells expressing mTRPM3α2 and Gq-coupled M1 or Gi-coupled M2 muscarinic receptors were performed as described in Materials and methods. TRPM3 currents were …
Figure 1—figure supplement 1
Activation of M1, but not M2 muscarinic receptors induces PI(4,5)P2 hydrolysis.
FRET-based PI(4,5)P2 measurements were performed as described in the Materials and methods section, downward deflection of the FRET ratio curve indicates a decrease in plasma membrane PI(4,5)P2 …
Figure 1—figure supplement 2
Activation of GPCRs inhibit TRPM3 currents in various conditions.
Whole-cell patch clamp experiments in HEK cells transfected with mTRPM3α2 were performed as described in Materials and methods; TRPM3 currents were evoked by 50 μM PregS, currents are plotted at …
Figure 1—figure supplement 3
PLCγ activation by the PDGFRβ inhibits TRPM3 activity.
TEVC measurements were performed as described in Materials and methods, in Xenopus oocytes injected with cRNA encoding hTRMP3 and either the wild-type PDGFRβ (A) or the Y1009F-Y1021F mutant (B), …
Figure 2 with 1 supplement
Co-expressed Gβ1γ2, but not Gαi or Gαo inhibits TRPM3 currents.
TEVC measurements in Xenopus oocytes expressing hTRPM3 were performed as described in Materials and methods; currents are plotted at 100 mV (upper traces) and −100 mV (lower trace). Currents were …
Figure 2—figure supplement 1
Co-expressed Gβ1γ2, but not Gαi or Gαo inhibits hTRPM3 currents; box and scatter plots.
Box and scatter plot of data from Figure 2D. Boxes show 25th and 75th percentiles, whiskers 90 and 10 percentiles.
Figure 3 with 1 supplement
Purified recombinant Gβ1γ2 inhibits TRPM3 currents in excised patches.
(A–C) Excised inside-out patch clamp experiments were performed in Xenopus oocytes expressing hTRPM3, with 100 μM PregS in the patch pipette, as described in Materials and methods, currents at −100 …
Figure 3—figure supplement 1
Inhibition TRPM3 in excised patches by Gβγ purified from bovine brain.
Excised inside-out patch clamp experiments were performed in Xenopus oocytes expressing hTRPM3, with 100 μM PregS in the patch pipette, as described in Materials and methods and as shown in Figure 3.…
Figure 4 with 3 supplements
PregS-induced Ca2+ signals are inhibited by agonists of Gi-coupled receptors in DRG neurons.
Ca2+ imaging experiments in DRG neurons were performed as described in Materials and methods. (A) Average trace ± SEM showing the effect of three consecutive applications of 12.5 μM PregS from …
Figure 4—figure supplement 1
Distribution of PregS responsive and non-responsive DRG neurons of TRPM8-GFP reporter mice.
(A) Pie chart of PregS-responsive and non-responsive neurons based on analysis of Ca2+ responses to 12.5 μM PregS of 1063 KCl responsive cells. The GFP-positive population was divided into smaller …
Figure 4—figure supplement 2
Individual traces and representative images for Ca2+ imaging experiments.
(A, B) Individual 340/380 nm ratios traces for Fura2-loaded DRG neurons; application of 12.5 μM PregS and 1 μM somatostatin (SST) are indicated by the horizontal lines. (A) SST non-responsive, (B) …
Figure 4—figure supplement 3
Baclofen does not inhibit PregS-induced Ca2+ signals in non-neuronal cells, and Ca2+ signals in DRG neurons evoked by KCl, the TRPM8 agonist WS12, or the TRPA1 agonist AITC.
(A) Ca2+ signals (mean ± SEM of 340/380 nm ratios) in Fura2-loaded KCl-responsive and KCl non-responsive cells induced by three consecutive applications of 12.5 μM PregS. The Ca2+ traces for …
Figure 5
Baclofen inhibits PregS-induced Ca2+ signals in HEK cells expressing the GABAB1 and GABAB2 receptors in a Gβγ-dependent manner.
Ca2+ imaging experiments in HEK cells were performed as described in Materials and methods. Average traces ± SEM showing the effect of three consecutive applications of 12.5 μM PregS and the effect …
Figure 6
The GABAB receptor agonist baclofen inhibits inward currents induced by the TRPM3 channel agonist CIM0216.
(A–B) Whole-cell patch clamp measurements in small GFP-positive DRG neurons were performed as described in Materials and methods at −60 mV holding potential in nominally Ca2+ free solution. The …
Figure 7
Baclofen inhibits nocifensive behavioral responses induced by the TRPM3 channel agonist CIM0216, but not responses to the TRPA1 agonist AITC.
(A–B) Nocifensive responses to the injection of CIM0216 (50 nmol/paw) were recorded as described in Materials and methods in control animals, and in animals where 12.5 nmol/paw baclofen was also …
