Cholinergic Ligand–dependent Modulation of Oxidative Phosphorylation Coupling in Digitonin-permeabilized BE(2)-C Neuroblastoma Cells
April 28th, 2026
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We present a protocol to selectively permeabilize plasma membrane, enabling targeted delivery of substrates and inhibitors to assess mitochondrial oxidative phosphorylation (OXPHOS) in cultured cells, overcoming plasma membrane transport barriers that typically hinder such analysis. Using this technique, we demonstrated how OXPHOS function is modulated by muscarinic agonists and antagonists.
Overview
This study evaluates oxidative phosphorylation (OXPHOS) function in cultured cells by using defined substrate–inhibitor combinations, while preserving cellular structure and cytosolic context. The approach overcomes limitations of intact cell assays by employing digitonin-mediated selective plasma membrane permeabilization, enabling direct assessment of mitochondrial respiration through specific electron transport chain (ETC) entry points.
Key Study Components
Area of Science
- Cellular bioenergetics
- Mitochondrial physiology
- Pharmacology
Background
- OXPHOS function is commonly assessed in isolated mitochondria, which lose cellular context.
- Intact cells are poorly permeable to several Krebs cycle intermediates, limiting substrate-supported respiration studies.
- Selective permeabilization can expand substrate accessibility while retaining cellular architecture.
- Pharmacologic modulation of mitochondrial respiration is relevant for understanding drug effects.
Purpose of Study
- To assess OXPHOS function in cultured cells using defined substrate–inhibitor combinations.
- To retain cellular structure and cytosolic context lost in isolated mitochondrial preparations.
- To evaluate the impact of selective permeabilization on substrate accessibility and pharmacologic modulation.
Methods Used
- Digitonin-mediated selective plasma membrane permeabilization of cultured cells.
- Extracellular flux analyzer–based coupling and electron flow assays.
- Empirical titration of digitonin in BE(2)-C neuroblastoma, HEK293, and primary rat dorsal root ganglion (DRG) neurons.
- Use of substrate–inhibitor combinations (e.g., succinate + rotenone) to isolate specific ETC-driven respiration.
Main Results
- Permeabilized cells showed increased Complex II–IV-driven oxygen consumption with succinate and Complex I inhibition, indicating improved substrate access.
- Substrates entering via endogenous transport pathways (e.g., pyruvate/malate) showed smaller differences between permeabilized and non-permeabilized cells.
- Muscarinic ligands produced agonist- versus antagonist-associated differences in oxygen consumption in coupling assays.
- Electron flow assays revealed minimal ligand-associated effects under tested conditions.
Conclusions
- Selective permeabilization enhances substrate accessibility in cultured-cell bioenergetic assays.
- This approach enables detailed analysis of pharmacologic modulation of mitochondrial respiration.
- Ligand effects are more detectable at the level of coupling-defined respiratory states than maximal electron transfer capacity.
Frequently Asked Questions
What is the main advantage of selective plasma membrane permeabilization in OXPHOS assays?
Selective permeabilization allows direct access of otherwise membrane-impermeant substrates to mitochondria, enabling more precise assessment of specific electron transport chain activities in cultured cells.
Why is digitonin used in this protocol?
Digitonin selectively permeabilizes the plasma membrane without disrupting mitochondrial membranes, preserving mitochondrial integrity while allowing substrate entry.
How does substrate choice affect respiration measurements in permeabilized versus intact cells?
Substrates that cannot cross intact plasma membranes (e.g., succinate) show increased respiration in permeabilized cells, while substrates that use endogenous transporters (e.g., pyruvate/malate) show less difference.
What cell types were tested in this study?
BE(2)-C neuroblastoma cells, HEK293 cells, and primary rat dorsal root ganglion (DRG) neurons were used to assess cell-type dependence of the permeabilization protocol.
How did muscarinic ligands affect mitochondrial respiration in this study?
Muscarinic agonists and antagonists produced distinct effects on oxygen consumption in coupling assays, but minimal effects were observed in electron flow assays under the tested conditions.
What are the implications of this method for pharmacologic studies?
This approach enables detailed analysis of how drugs modulate mitochondrial respiration in a cellular context, which is valuable for drug discovery and mechanistic studies.
