Drews A, Mohr F, Rizun O, Wagner T F J, Dembla S, Rudolph S, Lambert S, Konrad M, Philipp S E, Behrendt M, Marchais-Oberwinkler S, Covey D F, Oberwinkler J
Experimentelle und Klinische Pharmakologie und Toxikologie, Universität des Saarlandes, Homburg, Germany.
Br J Pharmacol. 2014 Feb;171(4):1019-32. doi: 10.1111/bph.12521.
Transient receptor potential melastatin 3 (TRPM3) proteins form non-selective but calcium-permeable membrane channels, rapidly activated by extracellular application of the steroid pregnenolone sulphate and the dihydropyridine nifedipine. Our aim was to characterize the steroid binding site by analysing the structural chemical requirements for TRPM3 activation.
Whole-cell patch-clamp recordings and measurements of intracellular calcium concentrations were performed on HEK293 cells transfected with TRPM3 (or untransfected controls) during superfusion with pharmacological substances.
Pregnenolone sulphate and nifedipine activated TRPM3 channels supra-additively over a wide concentration range. Other dihydropyridines inhibited TRPM3 channels. The natural enantiomer of pregnenolone sulphate was more efficient in activating TRPM3 channels than its synthetic mirror image. However, both enantiomers exerted very similar inhibitory effects on proton-activated outwardly rectifying anion channels. Epiallopregnanolone sulphate activated TRPM3 almost equally as well as pregnenolone sulphate. Exchanging the sulphate for other chemical moieties showed that a negative charge at this position is required for activating TRPM3 channels.
Our data demonstrate that nifedipine and pregnenolone sulphate act at different binding sites when activating TRPM3. The latter activates TRPM3 by binding to a chiral and thus proteinaceous binding site, as inferred from the differential effects of the enantiomers. The double bond between position C5 and C6 of pregnenolone sulphate is not strictly necessary for the activation of TRPM3 channels, but a negative charge at position C3 of the steroid is highly important. These results provide a solid basis for understanding mechanistically the rapid chemical activation of TRPM3 channels.
瞬时受体电位香草酸亚家族成员3（TRPM3）蛋白形成非选择性但对钙通透的膜通道，可被细胞外施加的甾体硫酸孕烯醇酮和二氢吡啶硝苯地平快速激活。我们的目的是通过分析TRPM3激活的结构化学要求来表征甾体结合位点。
在用药理物质进行灌流期间，对转染了TRPM3的HEK293细胞（或未转染的对照细胞）进行全细胞膜片钳记录和细胞内钙浓度测量。
硫酸孕烯醇酮和硝苯地平在很宽的浓度范围内超相加地激活TRPM3通道。其他二氢吡啶抑制TRPM3通道。硫酸孕烯醇酮的天然对映体在激活TRPM3通道方面比其合成镜像更有效。然而，两种对映体对质子激活的外向整流阴离子通道具有非常相似的抑制作用。硫酸表异孕烯醇酮激活TRPM3的效果几乎与硫酸孕烯醇酮相同。将硫酸根换成其他化学基团表明，该位置的负电荷是激活TRPM3通道所必需的。
我们的数据表明，硝苯地平和硫酸孕烯醇酮在激活TRPM3时作用于不同的结合位点。从对映体的不同作用推断，后者通过与手性且因此是蛋白质性质的结合位点结合来激活TRPM3。硫酸孕烯醇酮C5和C6位之间的双键对于TRPM3通道的激活并非严格必需，但甾体C3位的负电荷非常重要。这些结果为从机制上理解TRPM3通道的快速化学激活提供了坚实的基础。
