Received for publication January 31, 2008.
Revised March 24, 2008.
Accepted for publication March 25, 2008.

PROBING THE BINDING SITES AND MECHANISMS OF ACTION OF TWO hERG CHANNEL ACTIVATORS, NS1643 AND PD307243

Abstract

We studied the mechanisms and sites of activator actions of PD(307243) and NS(1643) on the 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 K_d values of 3 and 20 uM, 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 appeared 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, a peptide toxin BeKm-1 that 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. PD's activator effect 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.

Key words: Ion channel regulation, Potassium, Antiarrhythmic drugs, Func. analysis receptor/ion channel mutants

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