F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

Summary

A method to isolate submitochondrial vesicles enriched in F1FO ATP synthase complexes from rat brain is described. These vesicles allow the study of the activity of F1FO ATPase complex and its modulation using the technique of patch clamp recording.

Abstract

Mitochondria are involved in many important cellular functions including metabolism, survival¹, development and, calcium signaling². Two of the most important mitochondrial functions are related to the efficient production of ATP, the energy currency of the cell, by oxidative phosphorylation, and the mediation of signals for programmed cell death³.

The enzyme primarily responsible for the production of ATP is the F1FO-ATP synthase, also called ATP synthase^4-5. In recent years, the role of mitochondria in apoptotic and necrotic cell death has received considerable attention. In apoptotic cell death, BCL-2 family proteins such as Bax enter the mitochondrial outer membrane, oligomerize and permeabilize the outer membrane, releasing pro-apoptotic factors into the cytosol⁶. In classic necrotic cell death, such as that produced by ischemia or excitotoxicity in neurons, a large, poorly regulated increase in matrix calcium contributes to the opening of an inner membrane pore, the mitochondrial permeability transition pore or mPTP. This depolarizes the inner membrane and causes osmotic shifts, contributing to outer membrane rupture, release of pro-apoptotic factors, and metabolic dysfunction. Many proteins including Bcl-xL⁷ interact with F1FO ATP synthase, modulating its function. Bcl-xL interacts directly with the beta subunit of F1FO ATP synthase, and this interaction decreases a leak conductance within the F1FOATPasecomplex, increasing the net transport of H+ by F1FO during F1FO ATPase activity⁸ and thereby increasing mitochondrial efficiency. To study the activity and modulation of the ATP synthase, we isolated from rodent brain submitochondrial vesicles (SMVs) containing F1FO ATPase. The SMVs retain the structural and functional integrity of the F1FO ATPase as shown in Alavian et al. Here, we describe a method that we have used successfully for the isolation of SMVs from rat brain and we delineate the patch clamp technique to analyze channel activity (ion leak conductance) of the SMVs.

Protocol

1. Brain Mitochondrial Isolation (Adapted from Brown M.R. et al.⁹)

1. Sacrifice the rat using methods approved by the Institutional Animal Care and Use Committee (IACUC).
2. Cut the head of the animal by decapitation, cut the skin and expose the skull.
3. Open the skull gently by cutting with a scissor or rongeur. Remove the brain.
4. Mince finely the brain without cerebellum in Isolation Buffer (see Table 1) and transfer it to a 5 ml glass/teflon homogenizer (see equipment list).
5. Homogenize tissue gently 10 times (no bubbles), approximately 5 min.
6. Centrifuge samp....

Results

The first step of our protocol allows for isolation of purified mitochondria as shown by Western blot in Figure 1. In Figure 2 is shown an example of a brain-derived submitochondrial vesicle patch recording. Using the inside-out patch configuration we demonstrate channel activity modulated by ATP. The control (CTL) recording (left) shows multi-conductance channel activity with a peak conductance of 600 pS on average. that was immediately decreased upon addition of 1 mM ATP to the ba.......

Discussion

The methods described herein enable the isolation of pure mitochondria at the end of step 1 and submitochondrial vesicles (SMVs) after step 2 from whole brain without distinction of cell phenotypes.SMVspurified by this method are essentially free of contamination by other subcellular organelles as shown in Figure 1 and our previous work (Alavian KN et al.⁸) and retain their structural and functional integrity prior to freezing. After freezing and thawing, isolated mitochondria o.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Potter-Elvehjem Tissue Grinder withPTFEPestle	Krackeler Scientific, Inc.	1-7725T-5
Eppendorf Centrifuge 5424	Eppendorf	5424 000.410
4639 Cell Disruption Vessel	Parr Instrument Company	4639
Ficoll	Sigma-Aldrich	F5415
Polycarbonate centrifuge tubes	Beckman Coulter	P20314
SW-50.1 rotor	Beckman Coulter
L8-70M Ultracentrifuge	Beckman Coulter
Digitonin	Sigma-Aldrich	D5628
Lubrol PX (C12E9)	Calbiochem	205534
Axopatch 200B	Axon Instruments
Digidata 1440A	Molecular Device
pClamp10.0	Molecular Device
Manipulator	Sutter Instrument
Borosilicate glass capillary	World Precision Instruments	1308325
Flaming/Brown Micropipette Puller Model P-87	Sutter Instrument