Cell resting membrane potential measurement method.

Experiments show that the ratio of large cell surface area to volume is so large that the decline of ion gradient is extremely slow.
even if metabolic poisons block the energy metabolism that rely on ATP, the rest membrane potential can be maintained for a considerable period of time, indicating that the ATP-dependent pump is not a direct energy source for maintaining the membrane potential.
, for example, the cell membrane potential drifts by only 1.4mV when the application of cardiac glycolside inhibits the Na-K-pump on the giant axial membrane of the gun-bird thief.
this indicates that in most cases, the contribution of the Na-K-plus pump to the resting film potential is very small.
in order to verify that the formation of the static membrane potential is due to the existence of the ion concentration gradient on both sides of the membrane and the selective penetration of the membrane on these ions, the following two methods to verify the above theory.
optical methods to record the static membrane potential of the static membrane amphibian skeletal amphibian and lactating skeletal muscle cells are generally -90mV.
smooth muscle cells are -55mV and human red blood cells are only -9mV.
however, the transmembrane potential of certain bacteria and plant cells can reach -200mV.
for some particularly small cells and organelles, such as red blood cells, mitochondria, neurons synaptic end-of-life extremely fine protrusions, can not be used microelectrodes to measure their membrane potential, then can be measured using a spectroscopic technique, as shown in Figure 1. Figure 1 of
shows this indirect approach.
this technique first labels the cell or celler with an appropriate fluorescent dye molecule, and then measures the fluorescence absorption spectrum of the dye, where the optical signal can be indirectly converted into potential differences inside and outside the cell membrane.
showed in Figure 1, different potentials are recorded when the thin protrusions of neurons and distant cells are stimulated.
the potential pattern recorded in the cell at this time is basically the same as it is obtained in real cells, indicating that this indirect measurement method is feasible.
the relationship between the internal and external potential difference of the cell membrane and the electric field (E) is as follows: E-Vm/d, vm here for the internal and external potential difference, d is the thickness of the cell membrane.
assuming a potential difference of 0.IV inside and outside the membrane and 4nm (i.e., 40x10-8cm) of the film, the trans-membrane electric field strength will reach 250,000V/cm.
can be seen, although the membrane potential is very small, but it maintains a large electric field.
this electric field is bound to have a great impact on special types of membrane signaling proteins (voltage-sensitive channels).
many classical experiments have proved the relationship between membrane potential and ion concentration gradient.
Paul Horowicz and Alan Hodgkin measured the membrane potential of frog muscle fibers, which were bathed in an improved physiological solution and replaced with CL-, soons to eliminate the effects of anions on membrane potentials.
at a normal K-concentration gradient (amphibian skeletal muscle fibers (K-0) s. 2.5mmol and sna-0-120mmol), its membrane potential is -94mV.
the membrane potential drifts in the positive direction when the film potential is more than 2.5mmol (with K plus 0 instead of Na-plus), and the in-film potential becomes more negative when the smh.com.au decreases below 2.5mmol (Figure 2).
a large number of experiments show that the direct source of the static membrane potential energy is not the ion pump, but the potential energy stored by the ion concentration difference itself.
for most cells, the contribution of the concentration gradient of intra- and outer K-plus to the resting membrane potential is the most important.
of course, as a secondary transporter, the Na-K-pump is important for the production and maintenance of this concentration gradient of ions, the direct contribution of the ion pump to the membrane potential is about 20%, and the remaining 80% comes mainly from the passive diffusion of K-plus and Na-plus.
the ionmembrane measurement restmembrane potential application isomatic state system can also be used to study the relationship between ion concentration gradient and cell trans membrane potential.
scientists have developed a similar experiment called a flat lipid double-layer model (Figure 3).
experimental device is composed of 2 separated small chambers filled with aqueous solution, the middle of the chamber is separated by a membrane with a diameter of 200 m, the thickness of the small hole is about 4 nm, the thickness of the equivalent of lipid double molecular layer, equivalent to the two small chambers into the inner and outer part of the cell membrane.
adding synthetic membrane proteins and other molecules to the flat liposome system, the complex metabolic function of living cells can be studied in an outlier-like state.
access to the Ag/AgCl electrode and the voltage meter in this system, which are connected via a salt bridge to a solution on both sides of the membrane for measuring the transmembrane potential of the system.
researchers can manually adjust the concentration gradient of ions in 2 small chambers.
if you put 4 mmolKCl in the left chamber, place 155 mmolKCl in the right chamber to simulate the concentration of mammalian myoblast membrane, outer K plus.
to eliminate the permeable flow of water between the small chambers on both sides, add a sufficient amount of non-electrolytic glycool in the left chamber at the same time.
assume that the flat double-layer film cannot separate the solution on both sides, and that due to the unequal KCl concentration in the small chambers on both sides, the K-plus will spread from the high concentration side to the lower side under the drive of the difference.
However, if a pure lipid bimolecular layer is added to the small holes in the membrane that are isolated in 2 small chambers, the permeable diffusion of Cl-and K-plus can be prevented.
now, by adding a purified K-channel or a K-carrier (uibiogires), the membrane's penetration of k-plus is selectively regulated.
assuming that at this time the K-channel is in an open state, but not to Cl-permeable, the right side (as determined as membrane) and the left side (as if not outside the membrane), should be electronegative, obviously this is due to the right k-plus concentration (155mmol) is much higher than the left side (4mmol), the right K-plus diffusion force is much greater than the left side.
as the right potential becomes more negative, the increasing negative potential prevents further flow of the right K-plus, eventually stopping the net flow of k-plus.
the system is at a equilibrium point at this time, with a trans-membrane voltage of 92.4 mV and a negative right side.
the concentration gradient of K-plus in the two small chambers of the system is similar to that of skeletal muscle cells, and the voltage difference recorded is equivalent to its resting membrane potential, which is actually the diffusion potential of this ion.
when in equilibrium, the potential can also be derived from the Nernst equation.
Source: Zuo Ming Xue.