Abstract

VU590 was the first publicly disclosed, sub-micromolar-affinity (IC50=0.2 µM), small-molecule inhibitor of the inward rectifier potassium (Kir) channel, Kir1.1, an emerging diuretic target for the treatment of hypertension. VU590 also inhibits Kir7.1, albeit with 40-fold lower potency (IC50~8 µM), and has been used as a Kir7.1 tool compound to uncover new roles of the channel in regulation of myometrial contractility and melanocortin signaling in the brain. Here, we employed molecular modeling, site-directed mutagenesis, and patch clamp electrophysiology to elucidate the molecular mechanisms underlying inhibition of Kir1.1 and Kir7.1 by VU590. Block of Kir1.1 and Kir7.1 is voltage dependent and reduced by increasing the electrochemical driving force for K+ influx, suggesting the VU590 binding site is located within the pore of both channels. Scanning mutagenesis analysis in Kir1.1 revealed that asparagine 171 (N171) is the only pore-lining residue required for high-affinity block, and that substituting negatively charged residues (N171D, N171E) at this position dramatically weakens VU590 block. In striking contrast, substituting a negatively charged residue at the equivalent position in Kir7.1 enhances block by VU590, suggesting the VU590 binding mode is different in Kir1.1 and Kir7.1. Interestingly, mutations of threonine 153 (T153) in Kir7.1 that reduce constrained polarity at this site (T153C, T153V, T153S) make wild type and binding site mutants (E149Q, A150S) more sensitive to block by VU590. The Kir7.1-T153C mutation enhances block by the structurally unrelated inhibitor VU714, but not by a higher-affinity analog ML418, suggesting that the polar side chain of T153 creates a barrier to low-affinity ligands that interact with E149 and A150 in Kir7.1. Reverse mutations in Kir1.1 suggest that this mechanism is conserved in other Kir channels. This study reveals a previously unappreciated role of membrane pore polarity in determination of Kir channel inhibitor pharmacology.