Meclofenamate binding to TRPM4 is largely abolished by p.L907A and p.S924A mutations, which increased the IC50 by 4.8-fold and 3.6-fold, respectively, without altering Ca2+ sensitivity.
Identification of L907 and S924 residues in the TRPM4 channel as the binding pocket for meclofenamate provides a structural basis for developing novel anti-arrhythmic therapies.
Effect estimate: 4.8x increase (p.L907A) and 3.6x increase (p.S924A)
Abstract Introduction Cardiac arrhythmias pose a major health threat, yet often prove difficult to prevent and treat. Current anti-arrhythmic treatments lack efficacy and have considerable adverse effects, including drug-induced pro-arrhythmia. We have recently identified meclofenamate as a potent TRPM4 inhibitor. We showed that inhibition of TRPM4 with meclofenamate suppresses cardiac arrhythmias in living mice in a TRPM4-dependent manner via suppression of a Ca2+-dependent background current, which contributes to cardiac cellular excitability. In order to further contribute to drug development targeting TRPM4, we investigated the binding pocket of meclofenamate in TRPM4. Methods We first performed in silico docking of meclofenamate and other compounds in TRPM4. Afterwards, results were verified using the whole-cell and inside-out patch clamp technique on HEK-293T cells transiently transfected with either wildtype or mutant TRPM4 channels. Results Using whole-cell patch clamp, we observed that binding of meclofenamate was largely abolished in cells expressing either the p.L907A mutation or the p.S924A mutation. Inside-out patch clamp recordings showed a 4.8x increase in the IC50 value of meclofenamate by the p.L907A mutant and a 3.6x increase by the p.S924A mutant. Furthermore, despite location of the L907 residue in the vicinity of a Ca2+ binding site, we showed that meclofenamate does not interfere with Ca2+ sensitivity of the human TRPM4 channel. Conclusion The presented data establishes the involvement of L907 and S924 residues in the binding pocket of meclofenamate in TRPM4, informing further drug development targeting TRPM4 in suppressing cardiac arrhythmias.
Pironet et al. (Fri,) conducted a other in Cardiac arrhythmias. Meclofenamate vs. Wildtype TRPM4 channels was evaluated on IC50 value of meclofenamate (4.8x increase (p.L907A) and 3.6x increase (p.S924A)). Meclofenamate binding to TRPM4 is largely abolished by p.L907A and p.S924A mutations, which increased the IC50 by 4.8-fold and 3.6-fold, respectively, without altering Ca2+ sensitivity.
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