Cis‐isomerism and other chemical requirements of steroidal agonists and partial agonists acting at TRPM3 channels

Abstract

BACKGROUND AND PURPOSE The transient receptor potential melastatin‐3 (TRPM3) channel forms calcium‐permeable, non‐selective, cationic channels that are stimulated by pregnenolone sulphate (PregS). Here, we aimed to define chemical requirements of this acute steroid action and potentially reveal novel stimulators with physiological relevance. EXPERIMENTAL APPROACH We used TRPM3 channels over‐expressed in HEK 293 cells, with intracellular calcium measurement and whole‐cell patch‐clamp recording techniques. KEY RESULTS The stimulation of TRPM3 channels was confined to PregS and closely related steroids and not mimicked by other major classes of steroids, including progesterone. Relatively potent stimulation of TRPM3‐dependent calcium entry was observed. A sulphate group positioned at ring A was important for strong stimulation but more striking was the requirement for a cis (β) configuration of the side group, revealing previously unrecognized stereo‐selectivity and supporting existence of a specific binding site. A cis‐oriented side group on ring A was not the only feature necessary for high activity because loss of the double bond in ring B reduced potency and loss of the acetyl group at ring D reduced efficacy and potency. Weak steroid stimulators of TRPM3 channels inhibited effects of PregS, suggesting partial agonism. In silico screening of chemical libraries for non‐steroid modulators of TRPM3 channels revealed the importance of the steroid backbone for stimulatory effects. CONCLUSIONS AND IMPLICATIONS Our data defined some of the chemical requirements for acute stimulation of TRPM3 channels by steroids, supporting the existence of a specific and unique steroid binding site. Epipregnanolone sulphate was identified as a novel TRPM3 channel stimulator.

Transient Receptor Potential Melastatin-3 (TRPM3)–Induced Activation of AP-1 Requires Ca2+Ions and the Transcription Factors c-Jun, ATF2, and Ternary Complex Factor

“…In contrast, in HEK293 cells engineered to express TRPM3 channels, gene transcription is activated following stimulation of TRPM3 stimulation with pregnenolone sulfate, independently of L-type voltage-gated Ca 21 channels (Lesch et al, 2014). Thus, TRPM3 functions in this heterologous system in the absence of L-type voltage-gated Ca 21 channels as a ligand-activated ionotropic receptor, leading to an influx of Ca 21 into the cells following activation (Wagner et al, 2008;Majeed et al, 2010). To elucidate the signaling pathway connecting TRPM3 stimulation with enhanced gene transcription of AP-1 responsive genes, we used this engineered HEK293 cell line, in which expression of TRPM3 is induced by adding tetracycline to the culture medium, as shown previously (Lesch et al, 2014).…”

“…TRPM3 activation has frequently been monitored using either intracellular Ca 21 measurement with Ca 21 indicators and/or a whole-cell patch clamp to measure the cationic membrane current (Wagner et al, 2008;Majeed et al, 2010). Thus, the influx of Ca 21 ions into the cells and the subsequent rise in intracellular Ca 21 concentration was used as an indication for an activated TRPM3 channel.…”

Rapid and Contrasting Effects of Rosiglitazone on Transient Receptor Potential TRPM3 and TRPC5 Channels

“…Human TRPM2, TRPM3, and TRPC5 were expressed as described previously (McHugh et al, 2003;Zeng et al, 2004;Majeed et al, 2010). TRP channel cDNA, stably incorporated into human embryonic kidney 293 cells, was under the control of a tetracycline-inducible promoter such that addition of 1 g/ml tetracycline (Tetϩ) induced expression of channels.…”

“…The strongest known activator of TRPM3 channels is pregnenolone sulfate (Wagner et al, 2008;Majeed et al, 2010). However, the concentrations of pregnenolone sulfate required for activation are supraphysiological (Wagner et al, 2008;Naylor et al, 2010).…”

The safety, efficacy and neuromotor effects of the neurosteroid anaesthetic alfaxalone in rats

“…The neurosteroid pregnenolone sulphate rapidly activated TRPM3 channels in pancreatic β cells, indicative of direct neurosteroid binding to TRPM3 (Nilius and Voets, 2008, Wagner et al, 2008, Majeed et al, 2010, Majeed et al, 2012. It was also reported that stimulation of TRPC6 channels in cath.a differentiated (CAD) neuronal cells increased 2-AG release, which suggests that TRPC6 channels could activate endocannabinoid synthesis (Bardell and Barker, 2010).…”