![]() |
ИСТИНА |
Войти в систему Регистрация |
ФНКЦ РР |
||
Nonsteroidal anti-inflammatory drugs besides of suppression of prostaglandins synthesis may modulates a variety of ion channels including receptors of γ-aminobutyric acid type A (GABAAR). We combined electrophysiological and modeling approaches to study the mechanisms of mefenanic acid (MFA) interaction with GABAAR. Patch-clamp recordings showed that MFA strongly potentiates GABA-induced currents in acutely isolated rat Purkinje cells. At concentration of 3-100 µM MFA increased the amplitude of currents with EC50 +- 15 µM and maximal potentiation up to 700%. The further growth of MFA concentration caused an inhibitory effect on GABAAR which was dependent on membrane voltage. We found that potentiating effects of MFA and general anesthetic etomidate (ETM) were non-additive, suggesting that MFA acts through the binding site targeted by etomidate. We built the model of open α1β2γ2 GABAAR based on the GluCl X-ray structure and used Monte-Carlo energy minimization to predict the MFA binding site(s). There are experimental data indicating that etomidate efficacy strongly depends on mutations of α1 M236 and β2 M286 residues. We imposed distant constraints to find the ETM and MFA binding modes in the transmembrane β(+)/α(-) interface. We showed that both ETM and MFA form H-bonds with β2 M2 R269 (19′) and N265 (15′) residues, van-der-Waals contacts with hydrophobic residues β2 M3 Met286, Phe289 and α1 M1 Leu232, Pro233, Met236. In order to find an inhibitory site we pulled (and simultaneously rotated) MFA through the pore. Our calculations predict the existence of two binding sites located at 6 and 31 Å of the pore depth. In the upper site MFA mainly interacts with charged and polar residues 20′-21′. In the lower site hydrophobic residues from -2′, 2′ and 5′ rings and polar residues 2′, 6′ are strongly contributed in MFA stabilization. The complete pore occlusion in the upper site is achieved by binding of two MFA molecules simultaneously.