Membrane potentials estimate

Shapiro HM. 2000. Membrane potential estimation by flow cytometry. Methods 21 271-279. 

The sodium channels are very selective for Na+ over K+, allowing Na+ influx down the electrochemical gradient to generate positive membrane potentials. The sodium channels are also permeable to Li+ and NH4+. The narrowest portion of the channel pore is estimated to be rectangular (3.1 x 5.2 A). 

In rat liver mitochondria, in state 4, the AP was estimated to be about 220 mV, with the membrane potential representing about 90% of this (Nicholls, 1974 Appendix 3). Similar values have been reported for human and rat skeletal muscle mitochondria in state 4 (Stumpf et al., 1982). The control of the rate of electron transport is not only determined by the availability of ADP, but also of Pj oxidizable substrates, and oxygen. There is evidence for futile cycling of protons in intact normal rat hepatocytes (Brand et al., 1993). Recently, Porter and Brand (1993) found a correlation between the proton permeability of the inner membrane of liver mitochondria and body size in animals from the mouse (20 g) to horses (150 kg) with a decrease in permeability with increasing weight of several-fold at a constant... 

Staverman [165, 166) has shown how a number of transport phenomena across membranes, such as ion migration, hydraulic permeability, and membrane potentials are related to the Lilt s. In general, if there are n components, n [n— I) independent measurements are required to estimate all Lik s. So for a system consisting of 3 components (e.g. sodium ions, chlorine ions and water) 6 independent measurements are necessary. 

Transport numbers can be measured by different methods. The small mono- and divalent inorganic ions have been used to demonstrate skin permselectivity, and have been determined from membrane potential measurements, or by the Hittorf method [10,25,77,79]. The latter method has been frequently used for drugs alternatively, the transport number can be estimated from the slope of a plot of drug flux as a function of current intensity (Figure 14.4) [18,66]. 

The normal potential difference between the inner and outer parts of nerve cells is about -70 mv as estimated above. Transmission of a nerve impulse is initiated by a lowering of this potential difference to about -20 mv. This has the effect of temporarily opening the Na+ channel the influx of these ions causes the membrane potential of the adjacent portion of the nerve to collapse, leading to an effect that is transmitted along the length of the nerve. As this pulse passes, K+ and Na+ pumps restore the nerve to its resting condition. 

Note that this equilibrium involves only the membrane potential, and is independent of the pH gradient. Estimation of membrane potential from this Nemst equation involves the determination of the equilibrium concentration gradient of the indicator ion across the membrane, either by the use of isotopes, or by using an electrode in the medium responsive to the decrease in external concentration as the ion is accumulated by the organelle [13,24]. 

The ions which are most frequently employed to estimate mitochondrial membrane potential are Rb in the presence of valinomycin, and lipophilic phosphonium cations. The ionophore valinomycin creates a uniport pathway for K or Rb in a variety of membranes [25]. The use of Rb enables the concentration gradient to be determined iso topically after rapid separation of the mitochondria from the medium Rb is usually considered to behave ideally in the matrix (i.e., not to be bound). Indeed, other methods of membrane potential measurement are usually referred back to the K or Rb gradient in the presence of valinomycin for their calibration. However, a disadvantage is that the ionophore will also transport K, which means that when the ionophore is added the membrane potential will tend to shift towards the value given by any K-gradient which happens to be pre-existing across the membrane. 

The lipophilic phosphonium cations, TPP (tetraphenyl phosphonium) and TPMP" (triphenylmethyl phosphonium) can cross bilayer membranes as charged species and distribute according to the membrane potential [26]. Since they can be used at very low concentration they tend to disturb the potential less than the use of valinomycin. The gradients established can be estimated either iso topically, or by the use of an electrode specific to the cation [27]. The latter allows the membrane potential in mitochondrial incubations to be monitored continuously. However, a disadvantage of these cations, particularly in the case of TPP, is that they do not behave ideally but bind to components in the matrix. A number of techniques have been described for correcting the accumulation ratios of the cations [28,29]. 

The transfer of the information described in the preceding sections of this chapter to the in vivo situation is a matter where opinions are sharply divided, even if more than 20 years have elapsed since the discovery by Vasington and Murphy [4]. One key problem, naturally, is the impossibility of reproducing the composition and the conditions of the cytosol in in vitro experiments. The above mentioned effect of Mg on the rate of Ca influx into mitochondria is but one striking example of the difficulties inherent to the extrapolation to the in situ conditions. Of interest in this respect are recent experiments [124,125] in which methods have been devised to estimate simultaneously the membrane potential across the plasma membrane and the mitochondria of nerve endings in situ. The conclusion of this work has been that the concentration of free Ca in the cytosol correlates directly to the membrane potential across the mitochondrial membrane, and is maintained at a steady-state level below 1 jaM. Simulation of the in situ conditions has also been the aim of studies [126] in which isolated liver endoplasmic reticulum has been added to media in which isolated liver mitochondria were made to take up Ca, or in which liver cells have been treated with digitonin to abolish the permeability barrier of the plasma membrane. It was found that respiring mitochondria lower the external Ca " concentration to about 0.5 /iM. The addition of endoplasmic reticulum vesicles produces a further decrease of the external Ca " to about 0.2 jaM. Thus, mitochondria... 

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