PhD Scientific Days 2023

Budapest, 22-23 June 2023

Molecular Sciences - Posters L

Molecular Characterization of Pathological and Tissue-specific TRPM2 Cation Channel Variants

Tóth Ádám Viktor1, Adam Bartok1, Laszlo Csanady1
1 Department of Biochemistry, Semmelweis University, Budapest, Hungary

Text of the abstract

TRPM2 is a temperature-sensitive, non-selective, Ca2+-permeable cation channel, activated by the simultaneous intracellular binding of ADPR, Ca2+ and PIP2. It is highly expressed in various cell types, including those in the central nervous system, bone marrow, immune system and pancreatic β-cells. Regarding its promiscuous expresstion profile, the channel can play a role in numerous physiological and pathological functions, such as body temperature regulation, cytokine production, migration, apoptosis, oxidative stress response inflammation, neurodegeneration. Genetic association studies have found linkage between point mutations in TRPM2 and bipolar disorder (D543E, R755C) or amyotrophic lateral sclerosis and Parkinson's dementia (P1018L). Additionally, alternative splicing results in tissue-specific variants in healthy neutrophils (ΔC-TRPM2) and in the striatum (SSF-TRPM2), which may modify the ligand specificity and function of the channel in a cell-specific manner.
Current investigation of the mentioned TRPM2 variants has primarily utilized fluorescent imaging techniques or whole-cell electrophysiological methods, which are limited in their ability to study the direct effect of intracellular ligands. The goal of this study is to investigate the molecular characteristics of the listed ion channel variants.
The TRPM2 variants are expressed in HEK-293 cells through transient transfection. Functional measurements are performed in a cell-free environment, using inside-out patch clamp configuration. This method enables the recording of micro- and macroscopic currents, as well as the efficient exchange of intracellular ligands. With this method, the ADPR and calcium sensitivity, gating parameters, inactivation kinetics, and temperature dependence of individual channel variants can be accurately defined.
Hitherto, mammalian expression vectors containing the variants were produced. Pivotal functional analysis of TRPM2-R755C showed no shift in ligand binding affinities. Further detailed analysis of the kinetic parameters of gating may reveal pathological phenotype. Detailed knowledge of the molecular characteristics of these variants may help to understand their role in pathomechanisms and tissue-specific variant functions.
This particular work is funded by Hungarian Centre of Excellence for Molecular Medicine (H-CEMM), János Bolyai Research Scholarship of the Hungarian Academy of Sciences (BO/00103/20) and MTA Lendület grant LP2017-14/2017.