Premium
Novel Approach for The Electrophysiological Characterization of Mitochondrial Calcium Uniporter
Author(s) -
Polina Iuliia,
Xie An,
Mishra Jyotsna,
Adhikari Neeta,
Clements Richard,
Jhun Bong Sook,
Dudley Samuel,
OUchi Jin
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.04443
Subject(s) - uniporter , mitochondrion , microbiology and biotechnology , biology , hek 293 cells , chemistry , biochemistry , cytosol , gene , enzyme
The recent discovery of the molecular identity of the mitochondrial Ca 2+ uniporter pore (MCU) opens new possibilities for applying genetic approaches to study mitochondrial Ca 2+ regulation in various cell types. Recently, we reported that the CCDC109A gene encodes a transcript variant shorter than the originally reported MCU (renamed “MCU‐L”). Importantly, this short MCU variant (renamed “MCU‐S”) lacks the mitochondrial target sequence at N‐terminal but maintains its protein sequence identical to MCU‐L. Hypothesis/Aim MCU‐S variant can form functional Ca 2+ ‐permeable channels similar to MCU‐L that are targeted to membranes other than the mitochondria, such as the plasma membrane (PM). Methods HEK293T cells were transfected with MCU variants and used for biochemical (cell surface protein biotinylation and protein fractionation), cell biological (confocal imaging of MCUs), and physiological assays (Ca 2+ imaging and whole‐cell patch‐clamp). Results Overexpressed MCU‐S was preferentially localized in the PM rather than in the mitochondria, while the MCU‐L is exclusively expressed in the mitochondria. Using live‐cell Ca 2+ imaging, we demonstrated that overexpression of MCU‐S induces larger Ca 2+ influx across the PM compared to control cells, which is blocked by the conventional MCU blocker Ru360. Moreover, by whole‐cell patch clamping, we found that cells overexpressing MCU‐S exhibit an Ru360‐sensitive inward current with a similar current‐voltage relationship reported in MCU‐L recording from mitoplasts. Lastly, co‐transfection of MCU‐S with proto‐oncogene tyrosine‐protein kinase (c‐Src) and proline‐rich tyrosine kinase 2 (Pyk2) significantly increased whole‐cell MCU‐S current compared to cells co‐transfected with control vectors. Conclusion MCU‐S forms a Ca 2+ channel in the plasma membrane with similar characteristics of MCU‐L. The physiological significance of this finding has yet to be determined, but our experiments suggest that it is possible to study MCU with a heterologous expression system, which uses considerably easier conditions compared to studying MCU and its regulation in the native inner mitochondrial membrane.