Balázs Tóth, László Csanády
1 Semmelweis University, Department of Medical Biochemistry , Budapest
2 Semmelweis University, Department of Medical Biochemistry , Budapest
Human TRPM2 (hTRPM2) is a non-selective Ca2+ permeable cation channel, expressed abundantly in brain, immune cells, and pancreatic β-cells. Under physiological conditions it plays a role in glucose-induced insulin secretion, and in the immune response. Besides, it enhances the sensitivity of cells toward oxidative stress induced damage. Thus, under pathological conditions, hTRPM2 is responsible for neuronal cell death during ischemia/reperfusion in the brain.
The TRPM2 channel has appeared a long time ago. Its ancestors can be found even in very simple multicellular organisms, such as Nematostella vectensis (nvTRPM2). Comparison of the biophysical behaviors of nvTRPM2 and hTRPM2 provides insight into TRPM2 evolution.
We have used electrophysiology, molecular biology and protein biochemistry approaches to compare biophysical properties of the two channels.
Both channels are co-activated by intracellular Ca2+ and ADP-ribose (ADPR). ADPR binds to the C-terminal NUDT9-H domain, whereas the Ca2+ binding sites have not yet been identified. The apparent affinities of the two orthologs for ADPR are similar, but channel closure in response to sudden removal of ADPR is ~20x slower in hTRPM2. This suggests that the open conformation is more stable in hTRPM2. Interestingly, evolution has compensated the longer open times and the consequently higher channel activity of hTRPM2 with a reduced Ca2+ permeability, lower Ca2+ and Na+ conductances, and the appearance of a fast irreversible inactivation mechanism.
In contrast, nvTRPM2 displays a 10x higher apparent affinity for Ca2+ than hTRPM2. Based on our cryo-EM structure of the nvTRPM2 channel, we could identify the amino acid sidechains which compose the Ca2+ binding site. Due to better expression levels of nvTRPM2 we could remove these sidechains by site directed mutagenesis and confirm their importance in inside-out patch-clamp experiments.
Aside from minor differences, nvTRPM2 is a good model for investigation of biophysical properties of hTRPM2, and is much easier to study.
Postdoctoral fellow with UNKP fellowship
Supervisor: László Csanády