1. Academic Validation
  2. Pore Polarity and Charge Determine Differential Block of Kir1.1 and Kir7.1 Potassium Channels by Small-Molecule Inhibitor VU590

Pore Polarity and Charge Determine Differential Block of Kir1.1 and Kir7.1 Potassium Channels by Small-Molecule Inhibitor VU590

  • Mol Pharmacol. 2017 Sep;92(3):338-346. doi: 10.1124/mol.117.108472.
Sujay V Kharade 1 Jonathan H Sheehan 1 Eric E Figueroa 1 Jens Meiler 1 Jerod S Denton 2
Affiliations

Affiliations

  • 1 Department of Anesthesiology (S.V.K., E.E.F., J.S.D.), Department of Pharmacology (E.E.F., J.S.D.), Department of Biochemistry (J.H.S., J.M.), Center for Structural Biology (J.H.S., J.M.), Department of Chemistry (J.M.), Institute of Chemical Biology (J.S.D.), Vanderbilt University, Nashville, Tennessee.
  • 2 Department of Anesthesiology (S.V.K., E.E.F., J.S.D.), Department of Pharmacology (E.E.F., J.S.D.), Department of Biochemistry (J.H.S., J.M.), Center for Structural Biology (J.H.S., J.M.), Department of Chemistry (J.M.), Institute of Chemical Biology (J.S.D.), Vanderbilt University, Nashville, Tennessee jerod.s.denton@vanderbilt.edu.
Abstract

VU590 was the first publicly disclosed, submicromolar-affinity (IC50 = 0.2 μM), small-molecule inhibitor of the inward rectifier potassium (Kir) channel and diuretic target, Kir1.1. VU590 also inhibits Kir7.1 (IC50 ∼ 8 μM), and has been used to reveal new roles for Kir7.1 in regulation of myometrial contractility and melanocortin signaling. Here, we employed molecular modeling, mutagenesis, and patch clamp electrophysiology to elucidate the molecular mechanisms underlying VU590 inhibition of Kir1.1 and Kir7.1. Block of both channels is voltage- and K+-dependent, suggesting the VU590 binding site is located within the pore. 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 block. In contrast, substituting a negatively charged residue at the equivalent position in Kir7.1 enhances block by VU590, suggesting the VU590 binding mode is different. 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. 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.

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