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The calcium conductance of the inner membrane of rat liver mitochondria and the determination of the calcium electrochemical gradient.

ABSTRACT: 1. A method is described for establishing steady-state conditions of calcium transport across the inner membrane of rat liver mitochondria and for determining the current of Ca2+ flowing across the membrane, together with the Ca2+ electrochemical gradient across the native Ca2+ carrier. These parameters were used to quantify the apparent Ca2+ conductance of the native carrier. 2. At 23 degrees C and pH7.0, the apparent Ca2+ conductance of the carrier is close to 1 nmol of Ca2+-min-1-mg of protein-1 mV-1. Proton extrusion by the respiratory chain, rather than the Ca2+ carrier itself, may often be rate-limiting in studies of initial rates of Ca2+ uptake. 3. Under parallel conditions, the endogenous H+ conductance of the membrane is 0.3 nmol of H+-min-1-mg of protein-1-mV-1. 4. Ruthenium Red and La3+ both strongly inhibit the Ca2+ conductance of the carrier, but are without effect on the H+ conductance of the membrane. 5. The apparent Ca2+ conductance of the carrier shows a sigmoidal dependence on the activity of Ca2+ in the medium. At 23 degrees C and pH7.2, half-maximum conductance is obtained at a Ca2+ activity of 4.7 muM. 6. The apparent Ca2+ conductance and the H+ conductance of the inner membrane increase fourfold from 23 degrees to 38 degrees C. The apparent Arrhenius activation energy for Ca2+ transport is 69kJ/mol. The H+ electrochemical gradient maintained in the absence of Ca2+ transport does not vary significantly with temperature. 7. The apparent Ca2+ conductance increases fivefold on increasing the pH of the medium from 6.8 to 8.0. The H+ conductance of the membrane does not vary significantly with pH over this range. 8. Mg2+ has no effect on the apparent Ca2+ conductance when added at concentration up to 1 mM. 9. Results are compared with classical methods of studying Ca2+ transport across the mitochondrial inner membrane.

SUBMITTER: Heaton GM

PROVIDER: S-EPMC1163798 | biostudies-other | 1976 Jun

REPOSITORIES: biostudies-other

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Project description:1. Marker enzymes for the mitochondrial matrix, inner membrane, inter-membrane space and outer membrane were measured in mitochondria isolated from control and regenerating rat liver. The specific activity of these enzymes was then followed for up to 30 days after operation. 2. The specific activity of marker enzymes for the matrix, inner membrane and inter-membrane space remained constant during liver regeneration. 3. However, the specific activities of monoamine oxidase and kynurenine hydroxylase, both outer-membrane markers, fell by 67% and 49% respectively from their control values at 4 days after operation, and returned to normal by about 3 weeks. 4. The repression of kynurenine hydroxylase activity was shown to be unrelated to any independent variation in tryptophan catabolism, based on tryptophan pyrrolase assays. 5. These results are considered to indicate that enzymes of the inner and outer mitochondrial membranes are synthesized asynchronously during morphogenesis. 6. The enzyme complement of purified outer membrane at 4 days after operation was about 50% of that of the appropriate control. Thus the composition of the outer membrane itself may vary dramatically, and supports the concept that constitutive enzymes may turn over independently of a membrane's existence. 7. The behaviour of the rotenone-insensitive, NADH cytochrome c reductase did not parallel the other outer-membrane enzymes for intact mitochondria, but did so when assayed in highly purified fractions of outer membrane. This suggests a labile binding to the outer membrane during the early stages of morphogenesis. 8. Electrophoresis of inner- and outer-membrane proteins revealed little difference between control and experimental mitochondria at 4 days, except for an increase in several, high-molecular-weight components of the outer membrane. These bands closely correspond to similar bands derived from smooth endoplasmic reticulum. 9. The results are discussed in relation to the biogenesis and turnover of mitochondria, and are considered to provide evidence for turnover as a unit, at least for the matrix, inner membrane, inter-membrane space and possibly some form of primary outer membrane.

Project description:Cell membrane capacitance and conductance are key pieces of intrinsic information correlated with the cellular dielectric parameters and morphology of the plasma membrane; these parameters have been used as electrophysiological biomarkers to characterize cellular phenotype and state, and they have many associated clinical applications. Here, we present our work on the non-invasive determination of cell membrane capacitance and conductance by an optically activated microfluidics chip. The model for determining the cell membrane capacitance and conductance was established by a single layer of the shell-core polarization model. Three-dimensional finite-element analyses of the positive and negative optically induced dielectrophoresis forces generated by the projected light arrays of spots were performed, thus providing a theoretical validation of the feasibility of this approach. Then, the crossover frequency spectra for four typical types of cells (Raji cells, MCF-7 cells, HEK293 cells, and K562 cells) were experimentally investigated by using a micro-vision based motion-tracking technique. The different responses of these cells to the positive and negative ODEP forces were studied under four different liquid conductivities by automatic observation and tracking of the cellular trajectory and texture during the cells' translation. The cell membrane capacitance and conductance were determined from the curve-fitted spectra, which were 11.1 ± 0.9 mF/m2 and 782 ± 32 S/m2, respectively, for Raji cells, 11.5 ± 0.8 mF/m2 and 114 ± 28 S/m2 for MCF-7 cells, 9.0 ± 0.9 mF/m2 and 187 ± 22 S/m2 for HEK293 cells, and 10.2 ± 0.7 mF/m2 and 879 ± 24 S/m2 for K562 cells. Furthermore, as an application of this technique, the membrane capacitances of MCF-7 cells treated with four different concentrations of drugs were acquired. This technique introduces a determination of cell membrane capacitance and conductance that yields statistically significant data while allowing information from individual cells to be obtained in a non-invasive manner.

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The calcium conductance of the inner membrane of rat liver mitochondria and the determination of the calcium electrochemical gradient.

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