Regulation of Respiration in Permeabilized Muscle Cells: Apparent KM for ADP Shows the Mitochondrial Outer Membrane Permeability

Abstract

The aim of this work was to study the regulation of respiration and energy fluxes in permeabilized oxidative and glycolytic skeletal muscle fibers. Despite certain similarities of mitochondrial organization into Intracellular Energetic Units (ICEUs), different muscle types have distinct metabolic pattern with specific regulatory mechanisms of mitochondrial respiration. In this work we measured ADP fluxes through mitochondrial outer membrane (MOM) in permeabilized fibers oxygraphically using pyruvate kinase - phosphoenolpyruvate system for trapping ADP produced in mitochondrial creatine kinase localized behind MOM. These fluxes were high in permeabilized fibers from glycolytic muscles and very low or absent in soleus muscle fibers and cardiac myocytes. These results indicate that the permeability of voltage-dependent anion channel (VDAC) in MOM correlates with the apparent Km for ADP in regulation of respiration. In cardiac cells, the MOM permeability seems to be regulated by the interaction of VDAC with cytoskeletal protein βII tubulin. To ascertain the role of this protein in skeletal muscles we visualized the localization pattern of βII tubulin together with mitochondrial protein VDAC immunocytochemically. We used the Metabolic Control Analysis to evaluate the Flux Control Coefficients of the respiratory chain complexes (I,III, IV), ANT, ATP synthase, and MtCK in permeabilized soleus muscle fibers under conditions of respiration stimulated by exogenous ADP and by endogenous ADP produced in activated MtCK reaction.