Tricarico Domenico, Barbieri Mariagrazia, Mele Antonietta, Carbonara Giuseppe, Camerino Diana Conte
Unit of Pharmacology, Department of Pharmacobiology, Faculty of Pharmacy, University of Bari, Bari, Italy.
FASEB J. 2004 Apr;18(6):760-1. doi: 10.1096/fj.03-0722fje. Epub 2004 Feb 6.
Carbonic-anhydrase (CA) inhibitors are used in the treatment of hypokalaemic periodic paralysis (hypoPP) and related channelopathies but their mechanism of action is unknown. Patch-clamp experiments and molecular modeling investigations were performed to evaluate the mechanism of actions of CA inhibitors on skeletal muscle Ca2+-activated-K+ (BK) channel of K+-deficient rats used as animal model of hypoPP. CA inhibitors showing different degree of CA inhibition such as acetazolamide (ACTZ), dichlorphenamide (DCP), hydrochlorthiazide (HCT), etoxzolamide (ETX), methazolamide (MTZ), and bendroflumethiazide (BFT), which lacks inhibitory effects on CA enzymes, were tested in vitro on BK channels. The application of ACTZ, BFT, ETX, and DCP to excised patches activated the BK channel with potency: ACTZ(DE50=7.3x10(-6)M)>BFT(DE50=5.93x10(-5)M)>ETX(DE50=1.17x10(-4)M)>>DCP. In contrast, MTZ and HCT failed to activate the BK channel. Molecular modeling studies showed that the capability of CA inhibitors to open the BK channel was related to the presence in their structures of an intra-molecular hydrogen bond with calculated inter-atomic distances ranging between 1.82 A degrees and 3.01 A degrees and of an aromatic ring poor of electrons. ACTZ, BFT, ETX, and DCP showed these pharmacofores, while MTZ and HCT did not. Our data indicate that the activation of BK channel is a property of CA inhibitors that interact with the channel subunit/s and that this effect is not related to their capability to inhibit the CA enzymes.
碳酸酐酶（CA）抑制剂可用于治疗低钾性周期性麻痹（低钾型PP）及相关离子通道病，但其作用机制尚不清楚。本研究进行了膜片钳实验和分子模拟研究，以评估CA抑制剂对作为低钾型PP动物模型的低钾大鼠骨骼肌钙激活钾（BK）通道的作用机制。选用了不同程度抑制CA的抑制剂，如乙酰唑胺（ACTZ）、二氯苯磺胺（DCP）、氢氯噻嗪（HCT）、依他唑胺（ETX）、甲醋唑胺（MTZ）以及对CA酶无抑制作用的苄氟噻嗪（BFT），在体外对BK通道进行测试。将ACTZ、BFT、ETX和DCP应用于切除的膜片上可激活BK通道，其效力为：ACTZ（DE50 = 7.3×10⁻⁶M）> BFT（DE50 = 5.93×10⁻⁵M）> ETX（DE50 = 1.17×10⁻⁴M）>> DCP。相反，MTZ和HCT未能激活BK通道。分子模拟研究表明，CA抑制剂打开BK通道的能力与其结构中存在分子内氢键（计算出的原子间距离在1.82 Å至3.01 Å之间）和缺电子芳香环有关。ACTZ、BFT、ETX和DCP具有这些药效基团，而MTZ和HCT则没有。我们的数据表明，BK通道的激活是CA抑制剂与通道亚基相互作用的特性，且这种效应与其抑制CA酶的能力无关。
