Probing the mechanisms underlying modulation of quinidine sensitivity to cardiac IKs block by protein kinase A-mediated IKs phosphorylation

Abstract

Background and purpose: Cardiac IKs is enhanced by protein kinase A (PKA) stimulation. And PKA-stimulated IKs is about threefold less sensitive to quinidine block than basal current. In this study, we further tested two competing hypotheses: IKs phosphorylation either (i) modulates access of blocking drugs to a binding site; or (ii) destabilizes the drug-channel interaction. Experimental approach: To distinguish between these hypotheses, we studied quinidine block of IKs channels in which three PKA site residues of the a-subunit KCNQ1 were mutated with a bulky negative charged aspartic acid (D). To study alleviation of IKs block by quinidine, we compared activating current at +60 mV, either with or without 5 s hyperpolarizing prepulses to-120 mV.Key results: Without PKA stimulation, quinidine (100 mM) blocked wild-type current to a similar extent with and without the prepulse (93 2% of pre-drug current at +60 mV vs. 95 1%). With PKA-stimulated wild-type channels, however, there was less block with the hyperpolarization to-120 mV: at +60 mV, block was 71 2% (-prepulse) versus 58 3% (+prepulse). Individual D-mutations and the triple-D mutant were resistant to quinidine block similar to that seen with PKA-stimulated wild-type IKs.Conclusions and implications: We conclude that phosphorylation-induced insertion of bulky negative charges alleviates quinidine block and that PKA-induced stimulation, by conferring negative charges to the channels, blunts IKs block as the interaction between the channels and blockers becomes destabilized. These effects would be of clinical significance in providing protective mechanisms against pro-arrhythmias caused by drug-induced inhibition of IKs and IKr. © 2009 The British Pharmacological Society All rights reserved.