Mefenamic Acid Binding and Effect on IKs Channel Gating

Abstract

The pairing of KCNQ1 and KCNE1 in the heart, together mediates the slow delayed rectifier current (IKs), which is partly responsible for cardiomyocyte repolarization. The nonsteroidal-anti-inflammatory drug, mefenamic acid, has been identified as an IKs activator. Here, we provide a biophysical and pharmacological characterization of mefenamic acid's effect on IKs. Using whole-cell patch-clamp, we show that mefenamic acid enhances IKsactivity in a dose-dependent manner by changing the slowly activating IKs current into an almost linear current with instantaneous onset which, can in turn, be inhibited by the IKs blocker, HMR1556. Both single channel studies which reveal no changes in the maximum conductance, and whole-cell studies which reveal a dramatically altered G-V relationship despite increasingly longer interpulse intervals, suggest that mefenamic acid decreases the voltage sensitivity of the IKschannel and causes a hyperpolarizing shift in the voltage-dependence of channel activation. Since the stoichiometry of KCNQ1 to KCNE1 subunits has the potential to vary up to a ratio of 4:4, it was important to examine the effect of other IKs stoichiometries on the action of mefenamic acid. No effect on KCNQ1 alone was found and the degree of current activation was dependent on the number of KCNE1 subunits present, which suggested a binding site on KCNE1 may exist. Consistent with this, mutational analysis revealed an extracellular site of significance on KCNE1. The charge on residue Lys41 and, potentially other surrounding residues, are critical for the activating effect of mefenamic acid on IKs. Overall, unlike most other IKs activators which have little effect on IKs channels with increasingly saturated stoichiometries,wehave shown that mefenamic acid is able to enhance all Iks channel complexes containing different KCNQ1:KCNE1 stoichiometries which suggest it may represent a promising therapeutic approach to treating long QT syndromes.