[Frontiers in Bioscience E2, 739-751, January 1, 2010]

Immunolocalization of mitoKATP subunits in human osteoblast-like cells

Jonathan Butler 1, 2, Alice Warley 2, Ken Brady 2,Fraser McDonald 1

1 Department of Orthodontics, King's College London, United Kingdom, 2 Centre for Ultrastructural Imaging, King's College London, United Kingdom

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Cells and culture conditions
3.2. Antibodies and probes
3.3. Immunoelectron microscopy
4. Results
4.1. Rat cardiomyocytes
4.2. SaOS-2 cells
5. Discussion
6. Conclusions
7. Acknowledgement
8. References

1. ABSTRACT

The mitochondrial ATP-dependent K channel (mitoKATP) has been shown to play a role in cellular protection against apoptosis, or programmed cell death. This channel has been identified and characterized in a number of cell and tissue types but to date the possible existence of mitoKATP in osteoblastic cells has not been investigated. The aim of this investigation was to establish whether the mitochondria of human osteosarcoma-derived osteoblasts (SaOS-2 cells) contain the putative mitoKATP subunits Kir6.1 and Kir6.2. Ultrathin sections of SaOS-2 cells were prepared for transmission electron microscopy using an adaptation of the Tokuyasu method, and immunolabelled using goat anti-Kir6.1 or anti-Kir6.2 antisera as the primary label, and a 10nm colloidal gold-conjugated donkey anti-goat secondary antibody. The suitability of the antisera and the immunostaining protocol were confirmed by using a sample of rat cardiac muscle as a positive control. Ultrastructural analysis revealed that SaOS-2 cells contain Kir6.2 but not Kir6.1, and that Kir6.2 is present in the mitochondria, but in extremely low abundance. These findings suggest that human osteoblast-like cells might contain mitoKATP channels in which Kir6.2 is the pore-forming subunit, although it appears that these channels are likely to be present in extremely low abundance.