, Research Paper
Effects Of Solutions On Ion Transport Across Membranes
Concentration gradients exist between two different salt molarity solutions on opposite sides of a membrane. Movement of ions across these sides can be achieved by either active or passive diffusion. In our first three experiments we used these concentration gradients to determine the characteristics of selective permeability of an artificial membrane to potassium chloride (KCl), the potential difference across the skin of a frog, Rana pipens, and the effect of inhibitors in the same frog membrane epithelium.
We measured the potential difference between the two sides of the artificial membrane with a voltmeter. These measurements were then compared with the theoretical numbers, measured using the Nernst equation. The measured results were extremely close to the values measured by the Nernst equation, but even more accurate compared to the values from the Goldman equation. These results showed that as the difference in concentrations increased, the potential across the membrane increased steadily.
Using the membrane of frog s skin, the potential difference across the membrane was also measured as well as the current with a voltmeter and a short circuit current (S.C.C.). These measurements showed that as the sodium concentration was decreased, both the potential and the current decreased as well. In addition, another experiment was conducted in order to test the effects of inhibitors and hormones on the transport through the frog membrane. Results showed that when ouabain and cyanide were added to the epithelium, potential decreased steadily, but the addition of vassopressin increased potential due to the increase of sodium transport across the membrane. Because cyanide is an ATP inhibitor and ouabain is a physical barrier of the Na/K pump, this experiment concluded that active transport in the frog skin is indeed an active process and is not possible without ATP.
There exists a separation of electric charge across plasma membranes, known as a membrane potential, which provides an electric force that influences the movement of ions across the membrane (Vander1998). These potentials are formed due to changes from either electrical gradients, in which ions are transferred from one side of the membrane to the other by their opposite charged particle, or chemical gradients in which the differences in concentrations of solutions influence the transfer of ions across the membrane. Sodium potassium pumps cause a flow of 2 sodium ions to every 3 potassium ions, making the cell negatively charged, so a membrane potential must be created.
In the first experiment, concentrations of KCl were diluted and set in one side of an artificial membrane chamber along with a pure KCl solution in the other side. The membranes restrict the movement of either cations or anions by the presence of fixed charged groups along the water channels across the membrane. A cation that is in solution will be repelled by a channel lined with positive charges, and oppositely for an anion. Cation exchangers have fixed negative charges that permit cations to asociate or pass through it and anion exchangers have fixed positive charges and permit the passing of negative charges (Unpublished 5). Potential differences were then tested proving that an electrical gradient does indeed exist on both sides of the membrane.
Our values were compared to the Nernst equation which is:
Vm=58mV * log[C1]/[C2]
where Vm is the potential difference, and C1 and C2 are the concentration levels on each side of the membrane.
The measured results were also compared to the Goldman equation which is:
Vm=((RT/F) * ln((Pk[k]o + Pcl [Cl]I) / (Pk[K]I + Pcl[Cl]o))
Frogs must constantly use their environment in order to keep an equilibrium in their bodies. This process occurs by way of the sodium pump, which is a form of active transport because the process requires the use of energy in order to transport sodium against its concentration gradient. In order to do this, the frog must continually extract sodium from its environment to balance these losses. This is done by the active uptake of sodium by the frog s skin. By changing the concentrations of sodium on the mucosal, or outside of the frog skin, it was clear to see that as the concentration decreased, the potential did as well, therefore creating a chemical gradient across the membrane.
Membrane potentials can also occur electrochemically. Potentials can be adjusted by adding certain inhibitors or hormones with certain functions to the frog skin, and altering the usual environment of the frog skin. Cyanide ions inhibit cytochrome oxidase in electron transport, and Ouabain is a cardiac glycoside that blocks the sodium pump. Because ATP is necessary for the function of the sodium pump and functioning of frog skin equilibrium, it would be clear to see that these inhibitors would lower the potential difference across the membrane. However, when an antidiuretic hormone, vassopressin (ADH) was placed on the serosal side of the frog epithelium, the potential difference fluctuated. Such results are due to the fact that ADH is a hormone that modulates the activity in the sodium pump, therefore maintaining the sodium concentration inside the membrane necessary for equilibrium.
Materials and Methods
The materials and methods used in the labs using both the artificial and frog membrane were essentially the same taking into account the different chemical concentrations and solutions, as well as the actual membrane itself.
In the lab using the artificial membrane, the membrane itself was set up in between two sides of a solution chamber. The membrane was placed over the O ring on one half of the chamber, and then the other half of the chamber was placed on top of the membrane, thus exposing one side of the membrane to one chamber and the other side of the membrane to the other half of the chamber. Throughout the lab, one of the chambers contained only a completely pure solution of 100 mM KCl while the other side of the chamber was adjusted according to the different concentrations of serial dilutions ranging from 1-100mM that were needed to perform the experiment. A voltmeter was then set up using calomel electrodes. These electrodes were placed in two jars containing NaCl solution, with salt bridges connecting the jars to the two sides of the chamber. The solutions used were made by taking pure (100mM) KCl solution and diluting them in distilled water, and kept in separate flasks to make dilutions of:
100mM, 50mM, 25mM, 10mM, 5mM, 2.5mM, and 1mM. The actual lab procedure began with one side of the membrane chamber containing pure 100mM KCl and the other side of the chamber containing 1mM solution. The voltmeter was turned on and the potential was recorded until the highest possible voltage could be obtained. Once this was accomplished, the 1mM was removed. The chamber was washed with some of the 2.5mM solution, and then filled with the 2.5mM solution. The same potential was then recorded to the highest possible voltage. This process was repeated from the least concentrated solution to the highest.
For the labs involving frog skin, the same chamber was used involving the voltmeter, salt bridges, and electrodes. However we also recorded the current running through the membrane using a short circuit current (S.C.C), which only shows active transport because it is recorded at a potential difference of 0, when ion transfer should be stopped. The skin of the frog was cut off of the underbelly of the frog, and the serosal, or inside of the skin was placed over the O ring, with a negative electrode on the side with the mucosal, or outside layer of skin. A positive electrode was placed on the side with the inside layer of skin.
The solutions used for this particuar lab were:
Normal Ringer: .117M NaCl, .01M TriS, .0025M KCl, .0015M CaCl2
Tris Sulfate (TSR): .127M TriS, .00125M K2SO4, .00015M CaSO4
Potassium Sulfate (KSR): .01M TriS, .0125M K2SO4, .00015M CaSO4, .0475M Na2SO4
Sodium Sulfate (NaSR): .0585M Na2SO4, .01M TriS, .00125M K2SO4, .00015M CaSO4
These ringers were used on both sides of the membrane, with different combinations of each, as well as varying concentrations. Among the pure concentrations of solutions, we also used fractioned concentrations of NaSR. These were accomplished by first creating a 1/3 sample. This was done by adding 10mL of NaSR to 20mL of TSR. Once this was accomplished, a solution of 1/9, 1/27, and 1/81 of NaSR was prepared in the same fashion as the previous lab. Different combinations of solutions were placed on the two sides of the chamber throughout the lab and the potential difference was recorded until the highest possible number was reached. Once this was achieved, the current was recorded. This was done by placing the S.C.C. electrode in the mucosal side of the chamber and the S.C.C was then manipulated until the voltmeter read 0 and then the current was recorded.
To demonstrate active transport with the effect of inhibitors and hormones, a frog skin was taken from the underbelly of the amphibian and the same setup as the other lab was used. However, we used a normal ringer (NR), 5mM Cyanide (KCN), 1mM Ouabain, and 0.2 I. u. / mL of the Antidiuretic Hormone (ADH). The lab began with soaking the frog skin with NR in both chambers for approximately 5-15 minutes. Once this was done, the solutions were removed, a fresh volume of NR was placed in the mucosal chamber, and either ouabain or ADH in the serosal side. The KCN solution was placed on the mucosal side, with the NR on the serosal side. The potential difference and S.C.C. were both recorded every 2 minutes for a total of 40 minutes for each solution.
For the lab regarding the artificial membrane, the combined results were very similar to the theoretical results calculated by the Nernst equation. These results showed that as the ratio of mM concentrations increased, the potential difference did as well. These results are displayed in Table 1 and Graph 1.
In the first lab using frog skin, the results displayed that there is indeed transport in the skin, regardless of the presence of a gradient. The potential difference and current display the amount of energy used to keep the Na/K pump going, and by changing the concentrations of sodium on the mucosal, or outside of the frog skin, it was clear to see that as the concentration decreased, the potential did as well, creating a chemical gradient across the membrane. These results are shown in Table 2 and 3, and Graph 2 and 3.
Finally, the results in the third lab show the effect of different inhibitors and hormones on active transport in the frog skin. A decrease in potential difference and current show that active transport is either blocked, or at least slowed down by a specific inhibitor, and vice versa. The results for this experiment are found in Tables 4 and 5, and Graphs 4 and 5.
In the lab, we have determined the effect of outside sources on either passive diffusion or active transport. In the experiment involving the artificial membrane, the values demonstrated by the Nernst equation should display that ions should pass through the membrane according to the whether or not the membrane is an anion or cation exchanger. Although our values were extremely close to those of the Nernst equation, they were even closer to the Goldman predictions because the Goldman equation allows for the transfer of more than one ion, whereas the Nernst only permits the transfer of one ion at a time. These results display that as the difference between the concentrations increased, the potential difference across the artificial membrane did as well or in other words, the higher the difference in concentration levels, the more work was done by the Na/K pump to transfer the charges across the membrane, thus creating a greater potential difference.
During the lab regarding the active transport in the frog skin, the main goal was to determine whether or not active transport was occurring without a concentration gradient present. This goal was achieved when there was a potential difference across the membrane. This was also true of the current within the skin. Because there is a potential difference and current across the membrane without a difference in concentration, proves that active transport, no matter how large or small, was indeed present in the membrane used.
Finally, the purpose of the third lab was to decide whether or not ATP was needed to perform active transport in the frog skin membrane. The results showed that because the addition of cyanide, which inhibits ATP use, and ouabain, which blocks the Na/K pump all together, decreased the potential difference and current across the membrane, that ATP was very much needed in the functioning of the Na/K pump.
Unpublshed. Bio411 Systemic Physiology Lab Manual.
Vander, Arthur, Sherman, James, and Luciano, Dorothy. 1998. Human Physiology, The
Mechanisms of Body Function, 7th ed. NY, NY: McGraw Hill.