Computational and functional analyses of a small-molecule binding site in ROMK

Area of Science:

• Biophysics
• Molecular Pharmacology
• Renal Physiology

Background:

• The renal outer medullary potassium channel (ROMK) is crucial for regulating electrolyte and water transport in the kidneys, influencing blood pressure.
• Loss-of-function mutations in KCNJ1 (ROMK) cause salt wasting and hypotension, driving interest in ROMK-targeting diuretics.
• Small-molecule ROMK inhibitors offer potential for novel antihypertensive therapies.

Purpose of the Study:

• To computationally identify and experimentally validate binding sites for the ROMK inhibitor VU591 within the ROMK channel pore.
• To elucidate the structural basis of selective ROMK inhibition by small molecules.

Main Methods:

• Comparative molecular modeling and in silico ligand docking of VU591 to ROMK homology models.
• Site-directed mutagenesis of putative binding sites within the ROMK channel.
• Patch-clamp electrophysiology to assess the functional impact of mutations on VU591 sensitivity.

Main Results:

• Computational analysis identified two potential VU591 binding sites within the ROMK transmembrane pore.
• Mutagenesis of the upper binding site, specifically at Val(168) or Asn(171), dramatically reduced ROMK channel sensitivity to VU591.
• Mutations in the lower putative binding site did not affect VU591 sensitivity.

Conclusions:

• The upper pore region, containing unique residues Val(168) and Asn(171), is critical for VU591 binding and ROMK inhibition.
• Computational modeling is a valuable tool for predicting ligand-channel interactions and guiding drug design for ROMK.
• This study proposes a mechanism for ROMK inhibition and provides a structural basis for developing selective ROMK-targeting antihypertensive drugs.