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. 2022 Oct 24:14:1033434.
doi: 10.3389/fnagi.2022.1033434. eCollection 2022.

The identification of the key residues E829 and R845 involved in transient receptor potential melastatin 2 channel gating

Yuhuan Luo  1 , Shijia Chen  2 , Fei Wu  3 , Chunming Jiang  1 , Marong Fang  4
Affiliations

The identification of the key residues E829 and R845 involved in transient receptor potential melastatin 2 channel gating

Yuhuan Luo et al. Front Aging Neurosci. .

Abstract

Transient receptor potential melastatin 2 (TRPM2), a non-selective cation channel, is involved in many physiological and pathological processes, including temperature sensing, synaptic plasticity regulation, and neurodegenerative diseases. However, the gating mechanism of TRPM2 channel is complex, which hinders its functional research. With the discovery of the Ca2+ binding site in the S2-S3 domain of TRPM2 channel, more and more attention has been drawn to the role of the transmembrane segments in channel gating. In this study, we focused on the D820-F867 segment around the S2 domain, and identified the key residues on it. Functional assays of the deletion mutants displayed that the deletions of D820-W835 and L836-P851 destroyed channel function totally, indicating the importance of these two segments. Sequence alignments on them found three polar and charged residues with high conservation (D820, E829, and R845). D820A, E829A, and R845A which removed the charge and the side chain of the residues were tested by 500 μM adenosine diphosphate-ribose (ADPR) or 50 mM Ca2+. E829A and R845A affected the characteristic of channel currents, while D820A behaved similarly to WT, indicating the participations of E829 and R845 in channel gating. The charge reversing mutants, E829K and R845D were then constructed and the electrophysiological tests showed that E829A and E829K made the channel lose function. Interestingly, R845A and R845D exhibited an inactivation process when using 500 μM ADPR, but activated normally by 50 mM Ca2+. Our data suggested that the negative charge at E829 took a vital part in channel activation, and R845 increased the stability of the Ca2+ combination in S2-S3 domain, thus guaranteeing the opening of TRPM2 channel. In summary, our identification of the key residues E829 and R845 in the transmembrane segments of TRPM2. By exploring the gating process of TRPM2 channel, our work helps us better understand the mechanism of TRPM2 as a potential biomarker in neurodegenerative diseases, and provides a new approach for the prediction, diagnosis, and prognosis of neurodegenerative diseases.

Keywords: TRPM2; activation; gating mechanism; inactivation; key residues; transmembrane segments.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Functional assay of WT transient receptor potential melastatin 2 (TRPM2) and three deletion mutants. (A–D) Representative whole-cell recordings of TRPM2 channel currents induced by intracellular solution (ICS) of 500 μM adenosine diphosphate-ribose (ADPR) from Human Embryonic Kidney 293 T (HEK293T) cells expressing (A) WT, (B) △D820-W835, (C) △L836-P851, and (D) △D852-F867. The arrow in each panel indicates the time point at which whole cell configuration was established. (E) Summary of the current density induced in cells expressing WT and deletion mutants. In each case, six cells were tested. ***p < 0.001 compared with WT.
Figure 2
Figure 2
Sequence alignments between hTRPM2, mTRPM2, and mTRPM8. The sequence alignments were done for the residues in the red dashed box. Apart from the residues constitute the Ca2+ binding site (E843 and Q846), residues with high conservation were marked in red, and the polar and charged ones among them were highlighted in yellow (D820, E829, and R845).
Figure 3
Figure 3
Functional assay of WT TRPM2 and its alanine mutants. (A–D) Representative whole-cell recordings of TRPM2 channel currents induced by ICS of 500 μM ADPR from HEK293T cells expressing (A) WT, (B) D820A, (C) E829A, and (D) R845A. The arrow in each panel indicates the time point at which whole cell configuration was established. (E) Summary of the current density induced in cells expressing WT and the mutants. In each case, six cells were tested. ***p < 0.001 compared with WT.
Figure 4
Figure 4
Functional assay of WT TRPM2 and its alanine mutants. (A–D) Representative whole-cell recordings of TRPM2 channel currents induced by ICS of 50 mM Ca2+ from HEK293T cells expressing (A) WT, (B) D820A, (C) E829A, and (D) R845A. The arrow in each panel indicates the time point at which whole cell configuration was established. (E) Summary of the current density induced in cells expressing WT and the mutants. In each case, six cells were tested. ***p < 0.001 compared with WT.
Figure 5
Figure 5
Functional assay of WT TRPM2 and mutants at E829. (A–C) Representative whole-cell recordings of TRPM2 channel currents induced by ICS of 500 μM ADPR from HEK293T cells expressing (A) WT, (B) E829A, (C) E829K, and (D) Summary of the current density induced in cells expressing WT and mutants. In each case, six cells were tested. (E–H) Representative whole-cell recordings of TRPM2 channel currents induced by ICS of 50 mM Ca2+ from HEK293T cells expressing (E) WT, (F) E829A, (G) E829K, and (H) Summary of the current density induced in cells expressing WT and mutants. The arrow in each panel indicates the time point at which whole cell configuration was established. In each case, six cells were tested. ***p < 0.001 compared with WT.
Figure 6
Figure 6
Functional assay of WT TRPM2 and mutants at R845. (A–C) Representative whole-cell recordings of TRPM2 channel currents induced by ICS of 500 μM ADPR from HEK293T cells expressing (A) WT, (B) R845A, (C) R845D, and (D) Summary of the current density induced in cells expressing WT and mutants. In each case, six cells were tested. (E–H) Representative whole-cell recordings of TRPM2 channel currents induced by ICS of 50 mM Ca2+ from HEK293T cells expressing (E) WT, (F) R845A, (G) R845D, and (H) Summary of the current density induced in cells expressing WT and mutants. The arrow in each panel indicates the time point at which whole cell configuration was established. In each case, six cells were tested.
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