VU0134992 exhibits voltage-dependent block of Kir4.1 (A) Representative whole-cell current responses to voltage steps (1 second) from a holding potential (−80 mV for 5 mM K⁺ or −25 mV for 50 mM K⁺) in the presence or absence of 30 µM VU0134992. (B) Percentage of VU0134992 inhibition was calculated at each voltage with 5 and 50 mM extracellular K⁺. The VU0134992-induced block of Kir4.1 was statistically significantly lower at −120 and −100 mV in 50 mM K⁺ compared with 5 mM K⁺ containing bath solution. *P < 0.05, statistically significantly different from the 5 mM K⁺ extracellular solution control (n ≥ 5).

E158 and I159 are required for effective block of Kir4.1 by VU0134992. (A) (Left) Kir4.1 homology model showing the location of E158 (green), where it is predicted to interact with VU0134992. (Right) Magnified section of the Kir4.1 homology model (red square on left) showing the interaction of VU013492 with the pore-lining E158 residue in Kir4.1. (B) Mutagenesis studies were carried out to test the prediction of the homology model. Bar graph showing the percentage of inhibition of Kir4.1-WT (solid gray bar), and various mutant channels (colored or open bars) at 3 µM VU0134992. Kir4.1-carrying mutations at the E158 and I159 residue showed statistically significantly reduced sensitivity to VU0134992. (C) Representative traces showing the inhibition of WT and E158N/Q/D and I159S mutants in the absence (blue) or presence (red) of 3 µM VU0134992 and 2 mM BaCl₂ (black). (D) VU0134992 CRCs comparing Kir4.1-WT, Kir1.1, and Kir4.1-E158N, E158D, E158Q and I159S mutants at −120 mV. (E) CRCs for the indicated channels measured at +120 mV. *P < 0.05, statistically significantly different from the WT control (n ≥ 5). NF, not functional.

Overview of VU0134992 SAR. Inhibition of Tl⁺ flux toward Kir4.1 was evaluated for each compound synthesized according to Supplemental Figs. 1–3. Specific figures for IC₅₀ values are described in Supplemental Tables 1–3. Veh, vehicle.

Dose-dependent effects of VU0134992 on renal excretion. Rats were given oral gavage of either vehicle alone or the indicated doses of VU0134992 followed by saline volume load and were placed in metabolic cages for collection of urine over a 4-hour period. VU0134992 statistically significantly (P < 0.05) increased urine volume (A), urine Na⁺ (B), and urine K⁺ (C) at doses of 50 and 100 mg/kg as compared with the vehicle control. *P < 0.05, statistically significantly different from the vehicle control (n ≥ 4).