Abstract
Mitochondria act as ‘sinks’ for Ca2+ signaling, with mitochondrial Ca2+ uptake linking physiological stimuli to increased ATP production. However, mitochondrial Ca2+ overload can induce a cellular catastrophe by opening of the mitochondrial permeability transition pore (mPTP). This pore is a large conductance pathway in the inner mitochondrial membrane that causes bioenergetic collapse and appears to represent a final common path to cell death in many diseases. The role of the mPTP as a determinant of disease outcome is best established in ischemia/reperfusion injury in the heart, brain, and kidney, and it is also implicated in neurodegenerative disorders and muscular dystrophies. As the probability of pore opening can be modulated by drugs, it represents a useful pharmacological target for translational research in drug discovery. Described in this unit is a protocol utilizing isolated mitochondria to quantify this phenomenon and to develop a high‐throughput platform for phenotypic screens for Ca2+ dyshomeostasis. © 2019 The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Highlights
As a universal second messenger, Ca2+ plays a critical role in a wide range of cellular processes
Cell-permeant fluorescent dyes can assist in capturing mitochondrial permeability transition pore (mPTP) opening in an intact cell, the results obtained can be variable, as protocols both to induce and to inhibit mPTP opening have proven to be unreliable. mPTP opening has long been studied in isolated mitochondria through very robust protocols that rely on measurements of light scattering or fluorescence measurements following bolus additions of calcium
Mitochondrial quality and functional integrity can be assessed using support protocols for the following: measurement of mitochondrial membrane potential ( m), which is essential for Ca2+ uptake, using the fluorescence indicator rhodamine123 (Support Protocol 1); measurement of the oxygen consumption rate (OCR) using a Clark-type electrode (Support Protocol 2); and immunoblotting for various mitochondrial proteins as an additional surrogate for mitochondrial content and function (Support Protocol 3)
Summary
As a universal second messenger, Ca2+ plays a critical role in a wide range of cellular processes. Intracellular [Ca2+] gradients are tightly regulated; whereas extracellular [Ca2+] is at ß1 mM, cytosolic [Ca2+] ([Ca2+]c) and mitochondrial [Ca2+] ([Ca2+]m) are maintained at close to 100 nM. This unique electrochemical gradient allows the low [Ca2+]c to undergo a substantial proportional increase, with a small and energetically inexpensive absolute change, in response to a stimulus. Such changes in [Ca2+]c are a fundamental aspect of cell physiology in all tissues, underlying excitation contraction coupling, secretion, and motility (Denton & McCormack, 1985; Duchen, 1992; Duchen, Leyssens, & Crompton, 1998; Prudent et al, 2016)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
