Putative binding sites for arachidonic acid on the human cardiac Kv1.5 channel

Abstract

Background and Purpose In human heart, the Kv1.5 channel contributes to repolarization of atrial action potentials. This study examined the electrophysiological and molecular mechanisms underlying arachidonic acid (AA)-induced inhibition of the human Kv1.5 (hKv1.5) channel. Experimental Approach Site-directed mutagenesis was conducted to mutate amino acids that reside within the pore domain of the hKv1.5 channel. Whole-cell patch-clamp method was used to record membrane currents through wild type and mutant hKv1.5 channels heterologously expressed in CHO cells. Computer docking simulation was conducted to predict the putative binding site(s) of AA in an open-state model of the Kv1.5 channel. Key Results The hKv1.5 current was minimally affected at the onset of depolarization but was progressively reduced during depolarization by the presence of AA, suggesting that AA acts as an open-channel blocker. AA itself affected the channel at extracellular sites independently of its metabolites and signalling pathways. The blocking effect of AA was attenuated at pH 8.0 but not at pH 6.4. The blocking action of AA developed rather rapidly by co-expression of Kvβ1.3. The AA-induced block was significantly attenuated in H463C, T480A, R487V, I502A, I508A, V512A and V516A, but not in T462C, A501V and L510A mutants of the hKv1.5 channel. Docking simulation predicted that H463, T480, R487, I508, V512 and V516 are potentially accessible for interaction with AA. Conclusions and Implications AA itself interacts with multiple amino acids located in the pore domain of the hKv1.5 channel. These findings may provide useful information for future development of selective blockers of hKv1.5 channels.