Morris Kendall D W, Amin Jahanshah
University of South Florida, College of Medicine, Department of Pharmacology, MDC Box 9, 12901 Bruce B. Downs Blvd., Tampa, FL 33612, USA.
Mol Pharmacol. 2004 Jul;66(1):56-69. doi: 10.1124/mol.66.1.56.
Rho(1) receptor-channels (rho(1)Rs) are GABA-gated chloride channels that exhibit slow kinetics, little desensitization, and inert pharmacology to most anesthetics, except for neuroactive steroids (NSs). NSs differentially modulate rho(1)Rs dependent on the steric arrangement of the hydrogen atom at the fifth carbon position. In particular, the NS allotetrahydrodeoxycorticosterone (5alpha-THDOC) potentiates, whereas 5beta-pregnane-3alpha-ol-20-one (pregnanolone) and 5beta-dihydroprogesterone (5beta-DHP) inhibit rho(1) GABA currents. Here, we used Xenopus laevis oocytes expressing rho(1)Rs as a model system to study the mechanism of NS modulation. The second transmembrane residue, Ile307, was mutated to 16 amino acids. Subsequent testing of these mutants with 5alpha- and 5beta-NSs, at equivalent GABA activity, showed the following paradigm. For 5beta-DHP, Ile307 mutation either altered the degree of inhibition or entirely reversed the direction of modulation, rendering 5beta-DHP a potentiator. Dependent on the mutation, pregnanolone remained an inhibitor, transformed into a potentiator, or converted to inhibitor and potentiator based on concentration. The extent of mode reversal for both 5beta compounds showed a correlation with the side-chain hydrophilicity of the 307 residue. In contrast, Ile307 substitutions did not alter the direction of modulation for 5alpha-THDOC but caused a significant increase in the level of potentiation. Paradoxical to their impact on the mode and/or the degree of modulation, none of the mutations altered the concentration range producing the response significantly for any of the above NSs. Moreover, preincubation of Ile307 mutants with 5alpha or 5beta alone produced an equivalent effect on the activation time course. Based on the above data, a universal model is presented wherein anesthetic compounds like NSs can potentiate or inhibit the activity of ligand-gated ion channels distinct from interaction with alternative binding sites.
Rho(1)受体通道（rho(1)Rs）是γ-氨基丁酸（GABA）门控氯离子通道，其动力学缓慢，几乎不脱敏，并且对大多数麻醉剂（除了神经活性甾体（NSs））具有惰性药理学特性。NSs根据第五个碳位置上氢原子的空间排列差异调节rho(1)Rs。特别地，NS别四氢脱氧皮质酮（5α-THDOC）增强rho(1)Rs，而5β-孕烷-3α-醇-20-酮（孕烷醇酮）和5β-二氢孕酮（5β-DHP）抑制rho(1)GABA电流。在此，我们使用表达rho(1)Rs的非洲爪蟾卵母细胞作为模型系统来研究NS调节机制。第二个跨膜残基Ile307被突变为16种氨基酸。随后在等效GABA活性下用5α和5β NSs对这些突变体进行测试，结果如下。对于5β-DHP，Ile307突变要么改变抑制程度，要么完全逆转调节方向，使5β-DHP成为增强剂。根据突变情况，孕烷醇酮仍然是抑制剂、转变为增强剂或根据浓度转变为抑制剂和增强剂。两种5β化合物的调节模式逆转程度与307位残基的侧链亲水性相关。相比之下，Ile307替代并未改变5α-THDOC的调节方向，但导致增强水平显著增加。与它们对调节模式和/或调节程度的影响相悖的是，对于上述任何一种NSs，没有一种突变显著改变产生反应的浓度范围。此外，单独用5α或5β对Ile307突变体进行预孵育对激活时间进程产生等效影响。基于上述数据，提出了一个通用模型，其中像NSs这样的麻醉化合物可以增强或抑制配体门控离子通道的活性，这不同于与替代结合位点的相互作用。
