Ulrich H, Akk G, Nery A A, Trujillo C A, Rodriguez A D, Eterović V A
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil.
J Neurosci Res. 2008 Jan;86(1):93-107. doi: 10.1002/jnr.21468.
The mechanism of eupalmerin acetate (EUAC) actions on the embryonic muscle nicotinic acetylcholine receptor (nAChR) in BC3H-1 cells was studied by using whole-cell and single-channel patch-clamp current measurements. With whole-cell currents, EUAC did not act as an agonist on this receptor. Coapplication of 30 microM EUAC with 50 microM, 100 microM, or 500 microM carbamoylcholine (CCh) reversibly inhibited the current amplitude, whereas, with 20 microM CCh, current was increased above control values in the presence of EUAC. EUAC concentration curves (0.01-40 microM) obtained with 100 microM and 500 microM CCh displayed slope coefficients, n(H), significantly smaller than one, suggesting that EUAC bound to several sites with widely differing affinities on the receptor molecule. The apparent rate of receptor desensitization in the presence of EUAC and CCh was either slower than or equal to that obtained with CCh alone. The major finding from single-channel studies was that EUAC did not affect single-channel conductance or the ability of CCh to interact with the receptor. Instead, EUAC acted by increasing the channel closing rate constant. The results are not consistent with the competitive model for EUAC inhibition, with the sequential open-channel block model, or with inhibition by increased desensitization. The data are best accounted for by a model in which EUAC acts by closed-channel block at low concentrations, by positive modulation at intermediate concentrations, and by negative allosteric modulation of the open channel at high concentrations.
通过全细胞和单通道膜片钳电流测量，研究了乙酸优帕美林（EUAC）对BC3H-1细胞胚胎肌肉烟碱型乙酰胆碱受体（nAChR）的作用机制。对于全细胞电流，EUAC并非该受体的激动剂。将30μM的EUAC与50μM、100μM或500μM的氨甲酰胆碱（CCh）共同应用时，可可逆地抑制电流幅度，而当CCh浓度为20μM时，在EUAC存在下电流高于对照值。用100μM和500μM CCh获得的EUAC浓度曲线（0.01 - 40μM）显示斜率系数n(H)显著小于1，表明EUAC以广泛不同的亲和力与受体分子上的多个位点结合。在EUAC和CCh存在下受体脱敏的表观速率要么比单独使用CCh时慢，要么与之相等。单通道研究的主要发现是，EUAC不影响单通道电导或CCh与受体相互作用的能力。相反，EUAC通过增加通道关闭速率常数起作用。这些结果与EUAC抑制的竞争模型、顺序开放通道阻断模型或脱敏增加抑制模型均不一致。数据最能由以下模型解释：EUAC在低浓度下通过关闭通道阻断起作用，在中等浓度下通过正性调节起作用，在高浓度下通过对开放通道的负性变构调节起作用。
