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Mitochondrial membrane protein thiol reactivity with N-ethylmaleimide or mersalyl is modified by Ca2+: correlation
A J Kowaltowski1, A E Vercesi, R F Castilho
1Departamento de Bioquímica, Universidade Estadual de Campinas, SP, Brazil.
Abstract:
The content of mitochondrial membrane protein thiol groups accessible to react with the monofunctional thiol reagents mersalyl or N-ethylmaleimide (NEM) was determined using Ellman's reagent. Deenergized mitochondria incubated in the presence of Ca2+ (0-500 microM) undergo a very significant decrease in the content of membrane protein thiols accessible to NEM, and an increase in the content of thiols accessible to mersalyl. This process is time-dependent and inhibited by Mg2+, ruthenium red and ADP, but not by cyclosporin A. This suggests that Ca2+ binding to the inner mitochondrial membrane promotes extensive alterations in the conformation of membrane proteins that result in location changes of thiol groups. The relationship between these alterations and mitochondrial membrane permeability transition was studied through the effect of NEM and mersalyl on mitochondrial swelling induced by Ca2+ plus t-butyl hydroperoxide (t-bOOH) or Ca2+ plus the thiol cross-linkers 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) or phenylarsine oxide (PhAsO). We observed that the hydrophobic thiol reagent NEM inhibits the effects of t-bOOH, DIDS and PhAsO, while the hydrophilic thiol reagent mersalyl inhibits only the effect of DIDS. Permeability transition in all the situations studied is accompanied by a significant decrease in the total membrane protein thiol content. In addition, mitochondrial membrane permeabilization induced by PhAsO is inhibited by EGTA, but not by ruthenium red. This result suggests that PhAsO leads to permeability transition through a mechanism independent of intramitochondrial Ca2(+)-induced alterations of thiol group reactivity, but dependent on Ca2+ binding to an extramitochondrial site. This site is sensitive to extramitochondrial Ca2+ concentrations in range of 1-50 microM.
Insights
Calcium ions alter mitochondrial membrane protein thiol accessibility, influencing protein conformation and membrane permeability. These changes are modulated by specific reagents and ions, revealing distinct mechanisms of mitochondrial dysfunction.
Area of Science:
- Mitochondrial biochemistry
- Membrane protein dynamics
- Cellular signaling
Background:
- Mitochondrial membrane protein thiols play a crucial role in regulating mitochondrial function.
- Calcium ions (Ca2+) are known to influence mitochondrial processes, but their precise effects on membrane protein thiol accessibility remain incompletely understood.
- Understanding these interactions is key to elucidating mechanisms of mitochondrial permeability transition.
Purpose of the Study:
- To investigate the impact of Ca2+ on the accessibility of mitochondrial membrane protein thiol groups to different thiol reagents.
- To explore the relationship between Ca2+ induced alterations in thiol accessibility and mitochondrial membrane permeability transition.
- To differentiate the roles of intramitochondrial and extramitochondrial Ca2+ binding sites in these processes.
Main Methods:
- Quantification of accessible mitochondrial membrane protein thiols using Ellman's reagent.
- Incubation of deenergized mitochondria with varying concentrations of Ca2+ in the presence or absence of other ions (Mg2+) and inhibitors (ruthenium red, ADP, cyclosporin A).
- Assessment of mitochondrial swelling induced by Ca2+ combined with various agents (t-butyl hydroperoxide, DIDS, phenylarsine oxide) and the inhibitory effects of thiol reagents (NEM, mersalyl).
Main Results:
- Ca2+ significantly decreases NEM-accessible thiols and increases mersalyl-accessible thiols in mitochondrial membranes, a process inhibited by Mg2+, ruthenium red, and ADP.
- The hydrophobic reagent N-ethylmaleimide (NEM) inhibited Ca2+ plus t-butyl hydroperoxide, DIDS, and phenylarsine oxide-induced mitochondrial swelling.
- The hydrophilic reagent mersalyl inhibited only DIDS-induced swelling, while permeability transition consistently decreased total membrane protein thiols.
- Phenylarsine oxide-induced permeabilization is sensitive to extramitochondrial Ca2+ (1-50 microM) and EGTA, suggesting a distinct mechanism independent of intramitochondrial Ca2+ alterations.
Conclusions:
- Ca2+ binding to the inner mitochondrial membrane induces conformational changes in membrane proteins, altering thiol group accessibility.
- Distinct mechanisms underlie Ca2+ induced mitochondrial permeability transition, involving both intramitochondrial and extramitochondrial Ca2+ binding sites.
- The differential effects of NEM and mersalyl highlight the importance of thiol accessibility and location in regulating mitochondrial membrane permeability.