Evolution of Temperature Dependence of TRPM2 Channel Gating and Inactivation in Vertebrates

• Human TRPM2 (hsTRPM2) channels are highly sensitive temperature sensors in the brain. • Zebrafish are ectothermic, their brain temperature varies between ∼15°C and ∼37°C. • We show here that zebrafish TRPM2 (drTRPM2) gating is insensitive to temperature. • Inactivation rate is temperature sensitive for both drTRPM2 and hsTRPM2. • hsTRPM2 inactivates ∼20x faster than drTRPM2 due to specific changes in pore sequence. TRPM2 is a Ca 2+ permeable cation channel activated by cytosolic Ca 2+ and ADP ribose (ADPR). In contrast to invertebrate orthologs, vertebrate TRPM2 channels inactivate due to evolutionary alterations in amino acid sequence around the selectivity filter. Human TRPM2 (hsTRPM2) serves as a deep-brain temperature sensor important for body temperature regulation, and its gating is exquisitely temperature dependent. To address whether TRPM2 is temperature sensitive also in ectotherms that lack body temperature regulation, here we investigated the functional properties of zebrafish ( Danio rerio ) TRPM2 (drTRPM2) across the temperature range of 15°C to 37°C. The rate of ion permeation through the open pore was weakly temperature sensitive (Q 10 ∼1.3) as expected for a diffusion-limited process. In the presence of saturating concentrations of ligands (ADPR and Ca 2+ ) drTRPM2 open probability showed no temperature dependence. Moreover, for both ADPR and Ca 2+ , the apparent affinities for channel activation were unaffected by temperature, reporting a standard enthalpy of opening near zero for drTRPM2. Inactivation of drTRPM2 is an order of magnitude slower than that of hsTRPM2. To address whether in both orthologs the same mechanism underlies inactivation, we studied its temperature sensitivity. For both drTRPM2 and hsTRPM2 inactivation rate was modestly temperature dependent (Q 10 ∼2.7 and ∼4.4). A triple substitution which converts the post-filter sequence of drTRPM2 into the corresponding human sequence accelerated drTRPM2 inactivation by 10-20-fold. The data suggest that temperature dependence of hsTRPM2 gating evolved in the course of vertebrate evolution, whereas inactivation was temperature dependent already when it first appeared in early vertebrates.