RT Journal Article
SR Electronic
T1 Probing the Binding Sites and Mechanisms of Action of Two Human Ether-a-go-go-Related Gene Channel Activators, 1,3-bis-(2-Hydroxy-5-trifluoromethyl-phenyl)-urea (NS1643) and 2-[2-(3,4-Dichloro-phenyl)-2,3-dihydro-1H-isoindol-5-ylamino]-nicotinic acid (PD307243)
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 1709
OP 1721
DO 10.1124/mol.108.045591
VO 73
IS 6
A1 Xulin Xu
A1 Maurizio Recanatini
A1 Marinella Roberti
A1 Gea-Ny Tseng
YR 2008
UL http://molpharm.aspetjournals.org/content/73/6/1709.abstract
AB We studied the mechanisms and sites of activator actions of 2-[2-(3,4-dichloro-phenyl)-2,3-dihydro-1H-isoindol-5-ylamino]-nicotinic acid [PD307243 (PD)] and 1,3-bis-(2-hydroxy-5-trifluoromethyl-phenyl)-urea [NS1643 (NS)] on the human ether-a-go-go-related gene (hERG) channel expressed in oocytes and COS-7 cells. PD and NS affected hERG in a concentration-dependent manner, reaching a maximal increase in current amplitude by 100% and ≥300% (1-s test pulse to 0 mV), with apparent Kd values of 3 and 20 μM, respectively. Both drugs slowed hERG inactivation. NS additionally shifted the activation curve in the negative direction, accelerated activation, and slowed deactivation. Kinetic model simulations suggested that the activator effects of PD and NS could be largely accounted for by their effects on the hERG gating kinetics. Both drugs worked from outside the cell membrane but their binding sites seemed to be distinctly different. Perturbing the conformation of outer vestibule/external pore entrance (by cysteine substitution at high-impact positions or cysteine side chain modification at intermediate-impact positions) prevented the activator effect of NS but not that of PD. Furthermore, the peptide toxin BeKm-1, which bound to the outer mouth of the hERG channel, suppressed NS effect but potentiated PD effect. We propose that NS is a “gating-modifier”: it binds to the outer vestibule/pore entrance of hERG and increases current amplitudes by promoting channel activation while retarding inactivation. The activator effect of PD was prevented by external quaternary ammonium cations or dofetilide, which approached the hERG selectivity filter from opposite sides of the membrane and depleted K+ ions in the selectivity filter. We suggest that PD may work as a “pore-modifier” of the hERG channel. The American Society for Pharmacology and Experimental Therapeutics