Kidney Week

Abstract: TH-PO325

Discovery and Characterization of VU0493206, the First Small-Molecule Activator of Kir4.1/Kir5.1 Channels

Session Information

• Fluid, Electrolyte, and Acid-Base Disorders: Basic
  November 03, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
  Abstract Time: 10:00 AM - 12:00 PM

Category: Fluid‚ Electrolyte‚ and Acid-Base Disorders

• 1001 Fluid‚ Electrolyte‚ and Acid-Base Disorders: Basic

Authors

• Mcclenahan, Samantha J., Vanderbilt University Medical Center, Nashville, Tennessee, United States

Samantha J. Mcclenahan,

• Isaeva, Elena, Medical College of Wisconsin, Milwaukee, Wisconsin, United States

Elena Isaeva,

• Klemens, Christine Anne, University of South Florida, Tampa, Florida, United States

Christine Anne Klemens,

• Staruschenko, Alexander, University of South Florida, Tampa, Florida, United States

Alexander Staruschenko,

• Denton, Jerod S., Vanderbilt University Medical Center, Nashville, Tennessee, United States

Jerod S. Denton,

Background

Heteromeric K_ir4.1/K_ir5.1 potassium channels play key roles in regulating distal tubule potassium sensing and sodium chloride transport. Mutations in KCNJ10 and KCNJ16, encoding K_ir4.1 and K_ir5.1, respectively, lead to several renal tubulopathies. Given the importance of K_ir4.1/K_ir5.1 in maintenance of renal salt and water homeostasis, we sought to develop pharmacological tools for exploring its physiology, druggability, and therapeutic potential.

Methods

Fluorescence-based thallium flux assays were used for high-throughput screening (HTS) of small-molecule libraries for novel K_ir4/1/K_ir5.1 activators. Whole-cell and cell-attached patch clamp techniques were used to characterize effects of VU0493206 on K_ir4.1/K_ir5.1 channel activity. A voltage-sensitive dye was used to characterize effects of VU0493206 on mCCD_Cl1 cell membrane potential.

Results

We performed a HTS of 87,475 compounds for novel channel modulators, leading to the discovery of 427 inhibitors and 107 activators of heteromeric K_ir4.1/K_ir5.1 channels. Here, we report the discovery and characterization of VU0493206, the first small-molecule activator of K_ir4.1/K_ir5.1 channels. In whole-cell patch clamp experiments, VU0493206 activates K_ir4.1/K_ir5.1-mediated currents by approximately 600% at -120 mV with an EC₅₀ of 11 µM. Cell-attached patch clamp recordings indicates activation is mediated by an increase in single channel current amplitude and number of open channels. VU0493206 hyperpolarizes the membrane potential of mCCD_Cl1 renal epithelial cells, consistent with activation of endogenously expressed K_ir4.1/K_ir5.1 channels. At doses up to 30 µM, VU0493206 is selective for K_ir4.1/K_ir5.1 over 11 other members of the K_ir channel family. Depletion of plasmalemmal PIP₂ and mutation of PIP₂-binding sites on the channel prevents K_ir4.1/K_ir5.1 activation by VU0493206. Heteromeric channels carrying a novel loss-of-function mutation in K_ir5.1 (T64I) are activated by VU0493206, suggesting that some disease-causing mutations in K_ir4.1/K_ir5.1 may be rescued with activators.

Conclusion

We anticipate that VU0493206 and other small-molecule modulators will be useful for exploring the integrative physiology and therapeutic potential of K_ir4.1/K_ir5.1 heteromeric channels.

Funding

• NIDDK Support