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 evoked by 50 μM PregS, currents are plotted at −100 and 100 mV (lower and upper traces), dashed lines show zero current. (A–B) Representative traces for inhibition by 100 μM carbachol (CCh), without (A) or with 100 μM diC8 PI(4,5)P2 (B) in the whole-cell patch pipette in cells expressing M1 muscarinic receptors. (C) Summary of the data (n = 5 for control and n = 7 for PI(4,5)P2, ns: p=0.103, two sample t-test). (D) Representative trace showing inhibition by 5 μM ACh, in a cell expressing M1 muscarinic receptors (E) similar experiment in a cell co-expressing the C-terminus of βARK which binds to Gβγ. (F) Summary data (n = 6 for control and n = 7 for βARK-CT, ***p=0.00032, two sample t-test). (G) Representative trace showing inhibition by 5 μM ACh in a cell expressing the Gi-coupled M2 muscarinic receptors and mTRPM3α2, (H) similar experiment in a cell co-expressing the C-terminus of βARK. (I) Summary data, (n = 4 for control, n = 4 for βARK-CT, n = 3 for G203A). ***p=0.000003 and p=0.000022, one-way analysis of variance with Bonferroni post hoc comparison.