Abstract 1098: Probing Mechanisms for Phosphorylation-Dependent Drug Sensitivity of IKs Block
KCNQ1 encodes the α-subunit underlying IKs, enhanced by Protein Kinase A (PKA) activation. We have previously shown that PKA-stimulated IKs and IKCNQ1 are ~3-fold less sensitive to block by drugs such as quinidine than basal currents, and that the onset of drug block is markedly slowed by PKA stimulation of the channel. These data suggest two competing hypotheses: channel phosphorylation either (1) modulates access of blocking drugs to a binding site, or (2) destabilizes the drug-channel interaction. To distinguish between these hypotheses, we studied recovery from block by quinidine in wild-type KCNQ1 and channels in which three cytoplasmic serine/threonine residues targeted by PKA were mutated to a bulky permanent negative charge (aspartic acid, D). The triple mutant displayed resistance to quinidine block similar to that seen with PKA-stimulated wild-type channels. To study recovery from block, we compared activating current at the end of a 5-sec pulse to +60 mV from -80 mV, either with or without a 5-sec prepulse to a hyperpolarizing potential of -120 mV. In the absence of PKA stimulation, quinidine blocked wild-type current to a similar extent with and without the prepulse (93±2% of pre-drug current vs 95±1%, ±SE, n=4 each). By contrast, with PKA-stimulated wild-type channels, there was greater relief of block with the hyperpolarization (75±2% vs 58±3%, P<0.05), and this effect was also seen with the triple-D mutant (76±3% vs 56±5%, p<0.05). These data indicate that insertion of bulky negative charges by phosphorylation relieves drug block. We conclude that PKA stimulation destabilizes the interaction between KCNQ1 channels and blocking drugs.