Differential Effects of Isoflurane on Mutant Cardiac IKs Channels

Abstract

The slow delayed-rectifier potassium (IKs) channel mediates repolarization of the cardiac action potential, and its dysfunction can lead to the long QT syndrome (LQTS). The IKs channel consists of two subunits, the pore-forming α-subunit, KCNQ1, and the auxiliary β-subunit, KCNE1. Volatile anesthetics have significant inhibitory effects on the IKs current, but their underlying molecular mechanism remains to be determined. We have previously shown that F340 was a critical residue involved in this interaction, similar to the observation reported for other putative IKs antagonists. In order to further elucidate this mechanism, we investigated the effects of a volatile anesthetic, isoflurane, on mutant constructs of the IKs channel. Two KCNQ1 mutants, A341C and A344C, were constructed by site-directed mutagenesis. These two residues were chosen due to their vicinity to F340, and to their roles in inherited LQTS. Whole-cell current was recorded from transiently transfected HEK-293 cells in the absence and presence of isoflurane (0.52 ± 0.01 mM). Isoflurane inhibited wild-type- (WT) KCNQ1 by 62.1 ± 1.9% (mean±SEM, n=9). This inhibition was significantly attenuated when WT-KCNQ1 was cotransfected with WT-KCNE1 (40.7 ±4.5%; n=9). In contrast, isoflurane inhibited the mutant A344C current by 25.5 ± 2.0% (n=13), but this inhibition was not affected by the cotransfection with WT-KCNE1 (26.3 ± 3.2%, n=13). The A341C mutant alone did not express any current. However, when A341C was cotransfected with WT-KCNE1, an IKs-like current was elicited that was inhibited by isoflurane by 63.1 ± 2.9% (n =11). These results show that in addition to F340, the A341 and A344 residues also contribute to the anesthetic binding environment. Furthermore, the differential impact of KCNE1 on the isoflurane effects on WT-KCNQ1 and the mutant constructs suggests a complex role for the β-subunit in modulating the pharmacology of the IKs channel.