Structure‐activity Relationship of the Transient Receptor Potential Melastatin 2 (TRPM2) and a Novel Peptide Antagonist Tat‐M2NX: Potential Therapeutic Target in Cerebral Ischemia

TRPM2 are calcium (Ca 2+ )‐permeable ion channels highly expressed in the brain, playing a critical role in neuronal injury and death following cerebral ischemia caused by stroke or cardiac arrest (CA). CA leads to impairment of long‐term potentiation (LTP), which is a cellular correlate for enhanced synaptic communication in neurons, for at least 30 days. This impairment is associated to cognitive and memory deficits in patients. Our preliminary data suggest that TRPM2 inhibition reverses LTP impairment at acute and delayed time points (24 hours to 7 days). We propose a model in which cerebral ischemia causes sustained TRPM2 activity leading to LTP impairment. However, the lack of specific antagonists hinder the study of TRPM2 in pathophysiology. Our lab design a peptide predicted to preclude ADPR binding and activation of TRPM2. We hypothesize that the peptide tat‐M2NX is a potent antagonist of TRPM2 with high binding affinity. We performed a structure‐activity relationship of tat‐M2NX to TRPM2 to understand the mechanism of action. Materials and Methods Expression of human TRPM2 channels in HEK‐293 cells was induced with doxycycline for 18–24 hours. Whole‐cell patch clamp recordings were performed in the presence of 100μM ADPR (agonist) and 0, 0.05, 0.15, 0.3, 0.5, 2, 5, and 10μM tat‐M2NX in the pipette. TRPM2 currents were quantified by maximal peak amplitude in the absence and presence of tat‐M2NX antagonism. The antagonism of tat‐M2NX was validated with the pore blocker clotrimazole (CTZ). The structure‐activity relationship was performed to test the antagonistic effects on TRPM2 using mutant versions of tat‐M2NX (2μM): scramble, single point mutations, and truncated tat‐M2NX. The exclusion criteria were established as access resistance of R a <15 MΩ in addition to a final leak current <350pA. Data was analyzed normalizing current amplitude to cell density and plotted as percent inhibition. The potency or IC 50 was calculated from the tat‐M2NX drug‐response curve. Statistical significance was established as p<0.05 for all groups using One‐Way Analysis of Variance. Results and conclusions The peptide tat‐M2NX antagonizes >90% of TRPM2 channel currents at 2, 5, and 10μM concentrations. No significance difference was observed at 0.05, 0.15, 0.3, and 0.5μM concentrations of tat‐M2NX suggesting that tat‐M2NX is a potent TRPM2 antagonist. The tat‐M2NX IC 50 is approximately 258nM. Furthermore, tat‐M2NX displays sequence specificity that is critical for pharmacological antagonism due to the lack of effect observed by a scramble tat‐M2NX peptide. The mutant versions of tat‐M2NX are unable to dampen TRPM2 currents indicative of the requirement of full tat‐M2NX for antagonism of TRPM2. Further studies are required to determine the mechanism of action of tat‐M2NX. The peptide tat‐M2NX is cell permeable representing a new tool for the study TRPM2 function in the pathophysiology of neurological disease, cancer, and other diseases. Support or Funding Information Funding: R01NS092645‐01A1 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .