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Review
. 2021 Jun 1;320(6):C1125-C1140.
doi: 10.1152/ajpcell.00548.2020. Epub 2021 Apr 7.

Next-generation inward rectifier potassium channel modulators: discovery and molecular pharmacology

C David Weaver  1   2   3 , Jerod S Denton  1   3   4
Affiliations
Review

Next-generation inward rectifier potassium channel modulators: discovery and molecular pharmacology

C David Weaver et al. Am J Physiol Cell Physiol. .

Abstract

Inward rectifying potassium (Kir) channels play important roles in both excitable and nonexcitable cells of various organ systems and could represent valuable new drug targets for cardiovascular, metabolic, immune, and neurological diseases. In nonexcitable epithelial cells of the kidney tubule, for example, Kir1.1 (KCNJ1) and Kir4.1 (KCNJ10) are linked to sodium reabsorption in the thick ascending limb of Henle's loop and distal convoluted tubule, respectively, and have been explored as novel-mechanism diuretic targets for managing hypertension and edema. G protein-coupled Kir channels (Kir3) channels expressed in the central nervous system are critical effectors of numerous signal transduction pathways underlying analgesia, addiction, and respiratory-depressive effects of opioids. The historical dearth of pharmacological tool compounds for exploring the therapeutic potential of Kir channels has led to a molecular target-based approach using high-throughput screen (HTS) of small-molecule libraries and medicinal chemistry to develop "next-generation" Kir channel modulators that are both potent and specific for their targets. In this article, we review recent efforts focused specifically on discovery and improvement of target-selective molecular probes. The reader is introduced to fluorescence-based thallium flux assays that have enabled much of this work and then provided with an overview of progress made toward developing modulators of Kir1.1 (VU590, VU591), Kir2.x (ML133), Kir3.X (ML297, GAT1508, GiGA1, VU059331), Kir4.1 (VU0134992), and Kir7.1 (ML418). We discuss what is known about the small molecules' molecular mechanisms of action, in vitro and in vivo pharmacology, and then close with our view of what critical work remains to be done.

Keywords: drug discovery; high-throughput screening; medicinal chemistry; small molecules.

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Conflict of interest statement

No conflicts of interest, financial or otherwise, are declared by the authors.

Figures

Figure 1.
Figure 1.
Overview of inward rectifier potassium channel structure and function. A: side- (top) and top-down (bottom) views of a Kir2.2 crystal structure model (7). Each of the four subunits is shown with a different color. The top-down view highlights the central location of ion-conduction pore. B: current-to-voltage relationship of Kir2.1 (strong rectifier) and Kir1.1 (weak rectifier) current recorded between -120 mV and +120 mV in the whole cell configuration of the patch-clamp technique. The cartoon figures show the direction of potassium ion flow at voltages more negative (bottom left) or more positive (top) than the Nernst potential for potassium, before (top left) and after (top right) intracellular block by magnesium (blue sphere) present in the pipette solution. The voltage-dependent block of Kir2.1 and positive voltages is responsible for the observed inward rectification. The weaker pore block by magnesium is responsible for weaker rectification observed in Kir1.1.
Figure 2.
Figure 2.
Molecular physiology of ion transport in the nephron. A: sodium chloride reabsorption in the thick ascending limb of Henle is mediated by the luminal sodium-potassium-chloride co-transporter, NKCC2. Kir1.1 (ROMK) recycles potassium into the tubule lumen to energize NKCC2. Sodium and chloride exit the basolateral membrane via the sodium pump and ClC-Kb chloride channels, respectively. B: the sodium-chloride co-transporter (NCC) mediates sodium chloride reabsorption in the distal convoluted tubule. Basolateral Kir4.1/5.1. channels hyperpolarize the membrane potential and promote chloride exit through ClC-Kb, which in turn stimulate WNK-SPAK kinase-dependent phosphorylation and activity of NCC. C: in the cortical collecting duct, luminal Kir1.1 channels hyperpolarize the apical membrane potential and create a favorable electrochemical driving force for sodium reabsorption through epithelial sodium channels (ENaC). Basolateral Kir4.1/5.1 and Kir7.1 channels also hyperpolarize the membrane potential and recycle potassium across the membrane to maintain the activity of the sodium pump. NKCC2, sodium-potassium-2-chloride.
Figure 3.
Figure 3.
Summary of next generation Kir channel modulators. Kir, inward rectifier potassium. (Reference sources listed in rightmost column in numerical order: , , , , , , , , , , .)
Figure 4.
Figure 4.
VU590 functional binding sites in Kir1.1 and Kir7.1. Site-directed mutagenesis, voltage-clamp electrophysiology, and molecular modeling were used to identify Kir1.1-N171 (A) and Kir7.1-E149 and A150 (B) as essential for optimal block of the channels. These studies also revealed that Kir7.1-T153 creates an energetic barrier to small-molecule inhibitors accessing their deeper binding site near E149 and A150. Kir, inward rectifier potassium. [Reproduced from Kharade et al. (19) with permission.]
Figure 5.
Figure 5.
Identification of the VU591 functional binding site in Kir1.1. A: homology modeling and in silico docking were used to identify energetically favorable VU591 docking sites in putative “upper” and “lower” pore locations that were subsequently tested with mutagenesis and patch-clamp electrophysiology. No mutations in the lower site alter VU591 block; however, mutation of V168 and N171 led to a loss of VU591 potency. B: close-up view of VU591 docked into the upper site interacting with V168 and N171. Kir, inward rectifier potassium. [Reproduced from Swale et al. (21) with permission from Elsevier.]
Figure 6.
Figure 6.
Identifications of amino acids critical for activation of GIRK channels by ML297. Shown in A are the aligned amino acid sequences of GIRK1 and GIRK2 highlighting the two positions in the sequence that were determined as critical for ML297 efficacy. Shown in B are the results of thallium flux assays from HEK-293 cells co-expressing wild-type GIRK2 along with either wild-type GIRK1, wild-type GIRK2, mutant GIRK1, or mutant GIRK2 channels. Mutation of either phenylalanine F137 or aspirate D173 in GIRK1 resulted in loss of ML297 efficacy. ML297 efficacy was only observed with co-expression of either wild-type GIRK2 along with wild-type GIRK1 or wild-type GIRK2 along with a GIRK2 mutant containing both residues, F148 and D184 (correspond to residues F137 and F173 found in GIRK1). Shown in C are the positions of GIRK1 F137 and D173 in a computer model of heteromeric GIRK1/GIRK2 based on the crystal structure of homomeric GIRK2. GIRK, G protein-coupled inward rectifier potassium. [Elements of this figure were derived from information previously published by Wydeven et al. (90).]
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