Probing Mitochondrial Permeability Transition Pore Activity in Nucleated Cells and Platelets by High-Throughput Screening Assays Suggests Involvement of Protein Phosphatase 2B in Mitochondrial Dynamics

Assay Drug Dev Technol. 2018 Dec;16(8):445-455. doi: 10.1089/adt.2018.872. Epub 2018 Nov 27.

Abstract

Mitochondrial permeability transition pore (mPTP) formation is well documented in isolated mitochondria. However, convincing detection of mPTP in whole cells remains elusive. In this study, we describe a high-throughput assay for Ca2+-activated mPTP opening in platelets using HyperCyt flow cytometry. In addition, we demonstrate that in several nucleated cells, using multiple approaches, the detection of cyclophilin D-dependent mPTP opening is highly challenging. Results with the mitochondrial-targeted Ca2+-sensing green fluorescent protein (mito-Case12) suggest the involvement of protein phosphatase 2B (PP2B; calcineurin) in regulating mitochondrial dynamics. Our results highlight the danger of relying on cyclosporine A alone as a pharmacological tool, and the need for comprehensive studies of mPTP in the cell.

Keywords: Case12; PP2B; calcineurin; calcium; cyclosporine A; mitochondria permeability transition pore; platelet.

MeSH terms

  • Animals
  • Blood Platelets / cytology*
  • Blood Platelets / enzymology*
  • Calcineurin / metabolism*
  • HEK293 Cells
  • High-Throughput Screening Assays*
  • Humans
  • Mitochondria, Liver / metabolism*
  • Mitochondrial Dynamics*
  • Mitochondrial Membrane Transport Proteins / metabolism*
  • Mitochondrial Permeability Transition Pore
  • Rats
  • Tacrolimus / pharmacology

Substances

  • Mitochondrial Membrane Transport Proteins
  • Mitochondrial Permeability Transition Pore
  • Calcineurin
  • Tacrolimus