<p>The Ca²⁺ permeable cation channel TRPM2 is expressed in neurons of the hypothalamic thermoregulatory center, and contributes to body temperature regulation. Its activation gate opens upon binding of cytosolic Ca²⁺ and ADP ribose, but its distinct inactivation gate closes upon prolonged ligand exposure. In contrast to inactivation of most other neuronal channels, inactivation of human TRPM2 (hsTRPM2) is irreversible, but neither the mechanistic reason nor the evolution of that unique feature is known. Here we find that for the zebrafish ortholog (drTRPM2) inactivation is readily reversible upon ligand removal. Using a combination of electrophysiology, kinetic modeling, and thermodynamic analysis we reveal strong allosteric coupling between the extracellular inactivation gate and the cytosolic Ca²⁺ binding site: inactivation gate closure traps Ca²⁺ in its binding site. Moreover, we demonstrate that for hsTRPM2 inactivation is also technically reversible, but hsTRPM2 channels have evolved to bind Ca²⁺ so tightly in the inactivated state, that recovery requires lowering free [Ca²⁺] to subnanomolar levels. These findings delineate the appearance and evolution of TRPM2 inactivation in vertebrates, and explain why the human channel remains trapped in the inactivated state under physiological conditions.</p><p></p>