We use an SLM Aminco spectrofluorometer with a cell holder provided with continuous magnetic stirrer and a constant temperature system. Different factors affecting the values obtained are the following:. Salts that may be taken up by the yeast cells such as phosphate, sodium, or potassium salts should not be used to avoid their interference with the functioning of the cells. Amounts of cells and dye.
With different monitors, different amounts of cells and dye concentrations should be assayed to find those in which the best tracings are obtained. External binding.
When fluorescence is followed incubating the cells in the presence of an adequate buffer, even in the absence of a substrate, an increase of fluorescence is observed, which appears to be due to the simple binding of the dyes to the negative external charges of the membrane. Accumulation in the mitochondria. Uncouplers in yeast, at least S. This indicates that low concentrations can uncouple the mitochondria, without affecting the PMP.
Binding to the internal components. This factor is impossible to eliminate, and it is the actual basis of the analysis of the PMP by fluorescence, since as already mentioned, the monitors used in these studies show very low fluorescence values in water and require their interaction with the internal components of the cells. However, as it will be seen ahead, binding values can be measured. Changes of fluorescence of different monitors are an excellent way to follow not only the qualitative variations of the PMP in yeast, depending on the dye and conditions used.
This has been shown with ethidium bromide [ 13 , 27 ]. Regarding the claims that follow the displacement of the maximal fluorescence, peaks can provide the way to determine the actual value of the PMP and should be taken with caution [ 19 ]. When using acridine yellow [ 15 ], the fluorescence changes observed when deenergizing the mitochondria are particularly large.
This means that this dye may be particularly useful to monitor the changes of the electric potential difference of mitochondria. Initial attempts to measure the PMP based on the accumulation of cationic agents [ 6 , 7 ] were unreliable because of the slow entrance of the cationic agents used and the apparently incomplete equilibrium reached between the inside and outside of the cells. Previous calculations with the simple accumulation of different dyes, either ethidium bromide [ 13 ], or DiSC 3 3 [ 14 , 16 ], gave results that appeared too high, and the variations under the different conditions too small.
In summary, its activity was typical of that observed with other dyes, such as ethidium bromide or DiSC 3 3. Following a similar procedure to that used with other dyes, its accumulation was measured to calculate the PMP under different conditions. From these readings and the linear part of a standard curve, the external concentrations were calculated. The internal amount of dye was obtained by subtracting that amount from that originally added.
Main articles: Ion channel and Passive transport. On the other hand, the inhibition of K 2P channels leads to the depolarisation of the membrane potential and the contraction of smooth muscle cells Nelson and Quayle, Maintenance of the resting potential can be metabolically costly for a cell because of its requirement for active pumping of ions to counteract losses due to leakage channels. Science , — This channel is defective in patients with cystic fibrosis. Recovery from an action potential is partly dependent on a type of voltage-gated potassium channel that is closed at the resting voltage level but opens as a consequence of the large voltage change produced during the action potential. Gao BX,Ziskind-Conhaim L, Development of ionic currents underlying changes in action potential waveforms in rat spinal motoneurons.
Its concentration was calculated considering the value of internal water of yeast, which has been measured and equivalent to 0. These results were already encouraging and different to those obtained before with DiSC 3 3 [ 16 ], but we still had to consider that at least part of the dye was not free, but bound to the internal components of the cells. Values were also interesting regarding their magnitude and reproducibility. With glucose alone, the highest accumulation was observed present in both the cytoplasm and the mitochondria.
CCCP addition produced a decrease because the dye was no longer concentrated in the mitochondria, reaching a new equilibrium with the external concentration. It is also important to emphasize that the addition of NaCl produced only a small change. Considering that we had no way to produce the efflux of the dye bound to the internal components of the cell, we decided to use another approach, trying to measure it. Chitosan, a cationic polymer mostly composed of glucosamine is very effective to permeabilize the plasma membrane of Candida albicans [ 30 ], but actually this effect was found before on S.
After centrifuging the cells, it was possible to calculate the amount of the dye leaking out, and of course, that remaining inside bound to the internal components.
In this way the amount of dye that remained in the cells after the addition of chitosan could be subtracted from that remaining under the different conditions. Results of one typical of those experiments are the following:. Chitosan produces the permeabilization of the plasma membrane of the cells, producing the efflux of the dye, but still part of it remains inside, bound by its cationic nature.
However, still one problem exists: the values of the dye remaining inside the cells when KCl was added were found to be lower than those obtained with chitosan.
Microscope images show that after the uncoupler, the dye is no longer in the mitochondria and distributes in the cytoplasm, but it is absent from the vacuole [ 15 ]. This implies that the distribution volume of the dye is smaller than that in the total cell water. Considering this, the internal water in which the dye distributes is not the total value of 0. Values of the accumulation of acridine yellow obtained in a representative experiment under different conditions.
The amount of the dye that had not entered the cells, subtracted from the total added, allowed to get the amount that entered the cells. To obtain the internal concentration of the dye, this latter amount was divided by the internal water content of the cells, which was estimated before for this yeast strain of 0. Values in parenthesis were obtained considering that if the dye does not enter the vacuole, the total water in which the dye was distributed was only 0. Question marks indicate that the values were not calculated because the efflux of the dye with KCl was larger than that with chitosan.
Our work is a constant attempt to clarify a long time controversy, first about the estimation of the fluorescence changes of different monitors with different methods. We could add to the list acridine yellow, long known, but also quite inexpensive, which can be used to that purpose. With this dye larger concentrations are required, which may be probably due to the fact that it has a lower binding affinity to the internal components of the cells. With these corrections, larger differences under the conditions tested could be obtained with acridine yellow, as compared to those obtained with other agents, but much higher than those reported by other authors [ 19 ].
The simple accumulation in the presence of glucose already resulted in values lower than those reported before. Then, the addition of CCCP produced an apparent large decrease of the calculated PMP, because of the release of the large accumulation by the mitochondria and then outside the cells. From previous and recent work, we can summarize the basic conditions needed to obtain reliable results, such as the buffer used, which must not contain cations or organic molecules that may interfere or modify the PMP of the cells.
When using different monitors, different conditions should be tested, mainly the concentration of the dye to adjust it depending on the yeast used. Finally, corrections can be applied by using the correct volumes for the distribution of the dye, as well as its efflux with a permeabilizing agent. There is another factor influencing the results obtained. When the dye concentration after the addition of the monovalent cation is subtracted from that obtained after the addition of chitosan, a negative net accumulation results, meaning that the cells would have a positive PMP value, which is hard to accept.
The most probable explanation is that when chitosan is present, the dye goes out of the cells, leaving inside that bound, and the dye binds inside because of its hydrophobic and also its cationic nature. The conclusion then is that, although the accumulation of acridine yellow provides an adequate method to measure the PMP of yeast, the values obtained after the addition of positively charged ions that accumulate in large concentrations within the cell are distorted because of the displacement of the dye from what most probably are anionic sites inside the cell.
This potential is the resting membrane potential ; its magnitude depends on the type of cell, but usually ranges between and mV.
By convention the polarity positive or negative of the membrane potential is stated in terms of the sign of the excess charge on the inside of the cell The membrane potential can be accounted for by the fact that there is a slightly greater number of negative charges than positive charges inside the cell and a slightly greater number of positive charges than negative charge outside. The excess negative charges inside the cell are electrically attracted to the excess positive charges outside the cell, and vice versa. Thus, these excess ions collect along a thin shell on the inner and outer surfaces of the plasma membrane, whereas the bulk of the intracellular and extracellular fluid is electrically neutral.
The total number of positive and negative charges that have to be separated across the membrane to account for the potential is an insignificant fraction of the total number of charges actually in the cell.
The resting membrane potential is determined mainly by two factors:. The resting membrane potential is determined mainly by two factors: the differences in ion concentration of the intracellular and extracellular fluids and the relative permeabilities of the plasma membrane to different ion species.
Sodium, potassium, and chloride ions are present in the highest concentrations and therefore generally play the most important roles in the generation of the resting membrane potential. These concentration differences for sodium and potassium are due to the action of a membrane active transport system which pumps sodium out of the cell and potassium into it. The membrane potential moves from negative seventy millivolts to positive thirty millivolts, which in turn, opens K voltage-gated channels, having the opposite repolarizing effect on the cell membrane returning the membrane to its natural resting membrane potential.
In the heart, a high or low level of potassium changes the transmembrane potential, which in and of itself promotes cardiac arrhythmia, but also indirectly promotes cardiac arrhythmia by altering the electrical gradient of other ions, namely Na, Cl, and calcium.
At the neuronal level change in mental status with or without seizure can happen due to an altered transmembrane potential which can occur due to increased or decreased levels of Na, K and Ca.
Understanding the basic physiology of the cell membrane and how ion channels control it has lead to a better understanding of so many disease processes and accordingly developing effective medical treatments for them. By altering the resting membrane, potential hypokalemia can lead to tachyarrythmias and hyperkalemia to bradyarrhythmias. Magnesium has been successfully used in severe exacerbations of asthma and in eclampsia where it alters the neuromuscular transmissions by changing calcium entry into the cellular membrane.
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Physiology, Membrane. Article Author: Tom Herrmann. Sandeep Sharma. Introduction The human cell membrane is the cornerstone of an elaborate interplay between the extracellular and the intracellular worlds. Cellular Membrane Composition At the basic level, the cell membrane is a collection of lipid namely phospholipid and protein components. Membrane Transportation Transport across the cell membrane takes the form of simple diffusion, facilitated diffusion, and active transport.
Journal of supramolecular structure. How to break open the black box?