A TRPA1 channel agonist (Figure 4–figure supplement 3D). Although greater concentrations of AITC (100 mM), have been reported to also activate TRPV1 (Everaerts et al., 2011), only 7 out of 62 AITC-responsive cells responded to the TRPV1 agonist capsaicin, and in the similar experiments 35 cells responded to 0.5 mM capsaicin but not to AITC, which is consistent with AITC especially activating TRPA1 at this concentration. Functional GABAB receptors are obligate heterodimers of GABAB1 and GABAB2 receptors (Padgett and Slesinger, 2010). To test in the event the effect of baclofen is determined by the presence of heterodimeric GABAB receptors, we co-expressed GABAB1 and GABAB2 receptors, with TRPM3 channelsBadheka et al. eLife 2017;six:e26147. DOI: 10.7554/eLife.9 ofResearch articleNeurosciencein HEK293 cells (Figure five). When both the GABAB1 and GABAB2 receptors have been co-expressed with TRPM3, PregS-induced Ca2+ signals were nearly completely eliminated (Figure 5A). Upon washout of baclofen and PregS, a clear raise in Ca2+ (off-response) was observed in most cells. The impact of baclofen was strongly alleviated by co-expression from the Gbg sink bARK-CT (Figure 5A), indicating the involvement of Gbg. Baclofen also essentially eliminated heat-induced Ca2+ signals (Figure 5B); in these cells a marked off-response was also observed upon washout of baclofen. In cells expressing TRPM3 and only the GABAB1 (Figure 5C) or only the GABAB2 (Figure 5D) receptors,A3.BPregS Baclofen Ratio (340/380 nm)three.2.2.5Ratio (340/380 nm)two.two.n=1.51.n=197 n=22 n=1.1.n=0.0.five 0.Control Bac 0 100 200 Bac +ARK-CT 300GABAB1 + B2 + TRPMBaclofenControl Bac0 one hundred 2000.GABAB1 + B2 + TRPM400 500Time (s)Time (s)C3.PregS BaclofenD3.PregS Baclofen2.two.Ratio (340/380 nm)Ratio (340/380 nm)n=2.two.n=1.n=1.1.n=68 n=1.0.5 0.Handle Bac resp 0 100 200 Bac non resp 300GABAB1 + TRPM0.n=Control Bac resp Bac non resp 200 3000.GABAB2 + TRPMTime (s)Time (s)Figure five. Baclofen inhibits PregS-induced Ca2+ signals in HEK cells expressing the GABAB1 and GABAB2 receptors in a Gbg-dependent manner. Ca2+ imaging experiments in HEK cells had been performed as described in Components and solutions. Typical traces SEM showing the effect of three consecutive applications of 12.5 mM PregS and also the effect of 25 mM baclofen. The cells have been transfected with mTRPM3 plus (A, B) GABAB1 + GABAB2 receptor, and in a subset of cells the Gbg sink bARK-CT (blue trace in panel A), (C) GABAB1 receptor, (D) GABAB2 receptor. In panel A, note the practically complete inhibition of PregS-induced Ca2+ signal by baclofen, plus the increase of Ca2+ following washout of baclofen (`off’ effect). In panel B, Ca2+ responses to three consecutive heat pulses are shown (temperature: blue curve), note the marked off-response soon after washout of baclofen. In panels C and D the baclofen treated cells were subdivided into cells displaying no response to baclofen (Bac non-resp), and cells in which baclofen induced a Disopyramide Autophagy partial reduction in the PregS-induced Ca2+ signals (Bac resp). DOI: 10.7554/eLife.26147.Badheka et al. eLife 2017;6:e26147. DOI: ten.7554/eLife.10 ofTemperature (C)Research articleNeurosciencebaclofen therapy only resulted inside a 208260-29-1 Protocol little partial inhibition of PregS-induced Ca2+ signals in a subset of cells. Our data indicate that activation of 3 distinct endogenous Gi-coupled receptors inhibits native TRPM3 channels in DRG neurons. Ca2+ signals, on the other hand, are not a linear readout of channel activity, therefore we also performed whole-cell patch clamp experiments to confirm that acti.
