BLOCKADE OF TRPC4 CHANNELS BY THE PICO145 INHIBITOR: AN EXPERIMENTAL AND MOLECULAR STUDY
DOI:
https://doi.org/10.17721/1728.2748.2026.104.25-31Keywords:
TRPC4, Pico145, mICAT, molecular docking, smooth muscleAbstract
Background. Canonical transient receptor potential channel 4 (TRPC4) in intestinal smooth muscle is activated via interaction with M2/M3 muscarinic receptors and activated by these receptors G-proteins and represents an important component of calcium signaling. Blockade of these channels by highly potent inhibitors such as Pico145 is of considerable interest for the development of new pharmacological approaches. The aim of this study was to investigate the effect of Pico145 on muscarinic receptor–induced cation current (mICAT) in mouse ileal smooth muscle cells and to elucidate the molecular mechanism of Pico145 binding to the TRPC4 channel.
Methods. Electrophysiological experiments were performed using the patch-clamp technique on isolated smooth muscle cells from mouse ileum. Molecular modeling was conducted by docking Pico145 into the cryo-EM structure of TRPC4 (PDB: 5Z96) following validation on the reference TRPC1/TRPC4:Pico145 complex (PDB: 8WPM). Binding energies and spatial correspondence of the ligand pose relative to the experimentally determined Pico145 position in the TRPC1/TRPC4 channel were evaluated.
Results. Pico145 was shown to markedly suppress the amplitude of mICAT in intestinal smooth muscle cells. et a concentration of 100 pM, the inhibitor reduced the current by more than 80 % (p < 0.05) compared with control, consistent with the established contribution of TRPC4 to the generation of mICAT. Molecular docking confirmed that Pico145 binds within a conserved intersubunit hydrophobic pocket of the TRPC4 transmembrane domain, which corresponds to the binding site previously identified in the structurally related TRPC5 protein. The modeled conformation of Pico145 almost completely reproduces the experimentally determined ligand pose in the TRPC4 channel. Minor differences in the amino acid composition of the pocket (Thr599 in mouse TRPC4 versus Val624 in human TRPC4) do not affect the binding mode, confirming the conserved nature of the inhibition mechanism.
Conclusions. Pico145 effectively blocks receptor-operated TRPC4 channels in smooth muscle, leading to a pronounced inhibition of mICAT. Molecular modeling revealed that Pico145 binds to a conserved intramembrane site in the TRPC4 channel that coincides with the binding site in structurally related TRPC1/4/5 channels. These results support the common inhibitory mechanism of the TRPC4 by Pico145 and may facilitate the rational design of new selective TRPC channel blockers.
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