Table S1 List of all 1,4-dihydropyridine derivatives used in this study together with their chemical structures.

Table S2 List of all steroidal compounds used in this study together with their chemical structures.

Table S3 Listing of the numerical values used to draw the bar plots of the various figures in this paper.

Figure S1 Stably transfected myc-tagged mouse TRPM3 is expressed in HEK cells even at high passage numbers. Untransfected HEK293 cells (control) and HEK293 cells stably transfected with myc-TRPM3 (passage number 23) were solubilized as described (Behrendt et al., 2009) and equal amounts of protein were loaded on 6% and 12% slab gels. Subsequently, SDS-PAGE and Western blotting were performed according to standard procedures. Nitrocellulose membranes were incubated with either (A) anti-Akt (Cell Signalling #9272, 1:500) or (B) anti-c-myc (Roche, clone 9E10, 1:500) antibodies. Blots were visualized by utilizing IRDye® 800CW labelled secondary antibodies and the ODYSSEY® Sa infrared imaging system (LI-COR Biosciences).

Figure S2 Current-voltage relationships of the recordings presented in Figure 1B (A), Figure 2C (B), Figure 4A (C), Figure 5A (D), Figure 5D (E), Figure 6D (F) and Figure 7B (G).

Figure S3 The effects of methyl-β-cyclodextrin and cholesterol on the activity of mouse TRPM3 channels. Ca2+ imaging experiments during the application of 100 μM PS on TRPM3 expressing cells (stably transfected) that were untreated controls (black trace) or pretreated with a cholesterol: methyl-β-cyclodextrin complex (at a concentration ratio of 1:5 mM) at 37°C for 1 h during Fura2 loading (blue trace) or methyl-β-cyclodextrin (2.78 mM) alone (red trace). Shown is the averaged response of 3 different experiments per trace (n = 203–365 in total). The differences between the experimental traces and the untreated controls were highly significant (P < 0.001, two-tailed unpaired Student's t-test). The results confirm the findings of Naylor et al. (2010), and show that also the activity of mouse TRPM3 channels is reduced by cholesterol and enhanced by the removal of cholesterol. The extracellular solution used for this experiment contained 135 mM NaCl, 5.4 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 20 mM glucose, 10 mM HEPES, (pH 7.25 adjusted with NaOH, 315 mosmol kg-1).

Figure S4 Pairwise alignment of steroidal compounds. The panels on the right side show exactly the same alignment but rotated by approximately 90°. (A) Comparison of PS with epiallopregnanolone sulphate (3β,5α). (B) Comparison of PS with epipregnanolone sulphate (3β,5β). (C) Comparison of PS with pregnanolone sulphate (3α,5β). (D) Comparison of PS (nat-PS, 3β) with ent-PS (3α). (E) The same comparison of PS (nat-PS, 3β) with ent-PS (3α), but ent-PS was flipped by 180° along its long axis (extending approximately from C3 to C17). Note that the sulphate moiety at the C3 position can rotate rather freely in all substances. Its position is therefore not the focus of these comparisons. The alignments were generated by manually selecting pairs of atoms to align. For panels B and C, the carbon atoms of the C and D ring, together with C20 were used. For panels A and D, carbon atoms throughout the steroid backbone were used (and C20 for A). For aligning the flipped ent-PS (E), the sulphur and the oxygen atom attached to C3, C3, C9 and C10 were selected.

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