Purpose of experiment. Tasks of experiment: Theoretical topics. Equipment and materials. Theoretical part

Transcription

1 6.17. RESEARCH ON MEMBRANE RESTING POTENTIAL Purpose of experiment Measure the potential difference between two electrolyte solutions of different concentrations separated by a membrane. Tasks of experiment: Determine the membrane resting potential for five conbinations of different concentrations solutions. Calculate theoretical membrane difusion potentials. Theoretical topics Membrane structure, properties and function of membranes. Diffusion and active transport Factors that affect the net rate of diffusion Membrane resting potential. Equipment and materials Cobra3 Chem-Unit measurement system, Ussing chamber with membrane, ph measuring electrode, digital thermometer, bottles with different concentration solutions of HCl, and KCl, PC with software. Theoretical part For all known living organisms, the basic structural, functional and biological unit is the cell, consisting of protoplasm, enzymes, and chemicals. Within the cell there are also highly organized physical structures called intracellular organelles whose physical nature is as important as the cell s chemical constituents for cell function. Most of these organelles are enclosed in membranes composed primarily of phospholipids and proteins. The phospholipids act as a barrier to impede the movement of water and water-soluble substances from one cell compartment to another because water is not soluble in lipids. However, the protein molecules in the membrane frequently penetrate right through the membrane and provide specialized pathways, often forming actual pores, which allow specific substances to pass through the membrane. In addition, many other proteins in the membrane are enzymes that catalyze a multitude of different chemical reactions. For the purpose of discussing the transport of ions through the membrane, the membrane environment can be simplified into the system formed by the extracellular and the intracellular fluids separated by the membrane. The extracellular fluid contains a large amount of sodium but only a small amount of potassium whereas exactly the opposite is true of the intracellular fluid. In addition, unlike the extracellular fluid, the intracellular fluid contains only a very small quantity of chloride ions, but considerably greater concentrations of phosphates and proteins. Diffusion and Active Transport Passage through the cell membrane, whether through the phospholipid bilayer or through the proteins, occurs either by diffusion or active transport (Fig ). In the case of diffusion, RESEARCH ON MEMBRANE RESTING POTENTIAL 1

2 substances move randomly molecule by molecule, either through intermolecular spaces in the membrane or in combination with a carrier protein. By contrast, in the case of active transport, ions or other substances pass through the membrane in combination with a carrier protein that causes the substance to move against an energy gradient, such as passage from a low-concentration state to a high-concentration state. This movement requires an additional source of energy. Fig Transport pathways through the cell membrane, and the basic mechanisms of transport. Diffusion All molecules and ions in the body fluids, including water molecules and dissolved substances, have mean kinetic energy. When a moving molecule, A, approaches a stationary molecule, B, the electrostatic and other forces of molecule A repel molecule B, transferring some of the energy of motion of molecule A to molecule B. Consequently, molecule B gains kinetic energy, while molecule A slows down, losing some of its kinetic energy. This continual movement of molecules among one another in liquids or in gases is called diffusion. Ions diffuse in the same manner as whole molecules, and even suspended colloid particles diffuse in a similar manner, except that the large size of colloids prevents them from diffusing as rapidly as molecular substances. Where diffusion takes place through a cell membrane, it is categorized into one of two subtypes: simple diffusion or facilitated diffusion. In the case of Simple diffusion, the kinetic movement of molecules or ions occurs through a membrane opening or through intermolecular spaces and involves no interaction with carrier proteins in the membrane. The rate of diffusion is determined by the amount of the substance available, the velocity of kinetic motion, and the number and sizes of openings in the membrane through which the molecules or ions can move. Facilitated diffusion, on the other hand, requires interaction with a carrier protein. The carrier protein binds chemically with the molecules or ions and carries them through the membrane in this form. There is an important difference between facilitated diffusion and simple diffusion: whereas the rate of simple diffusion through an open channel increases proportionately with the concentration difference of the diffusing substance, in facilitated diffusion the rate of diffusion approaches a maximum, called V max, as the concentration of the diffusing substance increases. Why is the rate of facilitated diffusion limited? A carrier protein located in the membrane has a pore large enough to transport a specific molecule partway through and also has a binding receptor on the inside of the protein carrier. The molecule to be transported enters the pore and becomes bound. Then, in a fraction of a second, a conformational or chemical change occurs in the carrier protein, so that the pore now opens to the opposite side of the membrane. Because the binding force of the receptor is weak, the thermal motion of the attached molecule causes it to break away and to be released on the opposite side of the membrane. The rate at which molecules can be transported by this mechanism can never be greater than the rate at which the carrier protein molecule can undergo change back and forth between its two states. Note specifically, though, that this mechanism allows the transported molecule to move that is, to diffuse in either direction through the membrane RESEARCH ON MEMBRANE RESTING POTENTIAL 2

3 Factors that Affect the Net Rate of Diffusion Effect of concentration difference on net diffusion through a membrane. In the case of a cell membrane with a fixed substance in high concentration on the outside (C o ) and low concentration on the inside (C i ) (Fig A), the rate at which the substance diffuses inward is proportional to the concentration of molecules on the outside, because this concentration determines how many molecules strike the outside of the membrane each second. Conversely, the rate at which molecules diffuse outward is proportional to their concentration inside the membrane. Therefore, the rate of net diffusion into the cell is proportional to the concentration difference: C o C i. Effect of membrane electrical potential on diffusion of ions the Nernst potential. If an electrical potential is applied across the membrane (Fig B), the electrical charges of the ions cause them to move through the membrane. Thus, in the left panel of Fig B, the concentration of negative ions is the same on both sides of the membrane, but a positive charge has been applied to the right side of the membrane and a negative charge to the left, creating an electrical field gradient across the membrane. Therefore, net diffusion of positive charge occurs from left to right. After some time, large quantities of negative ions have moved to the right, creating the condition shown in the right panel of Fig. 2 B, where a concentration difference of the ions has developed. The concentration difference now tends to move the ions to the left, while the electrical field tends to move them to the right. When the concentration difference rises high enough, the two effects balance each other. In geberal, the electrical difference ΔE that will balance a given concentration difference of univalent ions such as sodium (Na + ) ions can be determined from the Nernst equation:, (6.17.1) where R - the universal gas constant, T the absolute temperature, z the number of moles of electrons transferred in the cell reaction, F the Faraday constant, C 1 is the concentration on side 1, and C 2 is the concentration on side 2. Effect of a pressure difference across the membrane. At times, a considerable partial pressure difference develops between the two sides of a diffusible membrane (Fig C). This occurs, for instance, at the blood capillary membrane in all tissues of the body. Pressure actually means the sum of all the forces of the different molecules striking a unit surface area at a given instant. Therefore, when the pressure is higher on one side of a membrane than on the other, the sum of all the forces of the molecules striking the channels on that side of the membrane is greater than on the other side. In most instances, this is caused by greater numbers of molecules striking the membrane per second on one side than on the other side. The result is that increased amounts of energy are available to cause net movement of molecules from the high-pressure side toward the low-pressure side. Membrane Potentials Caused by Diffusion Diffusion potential caused by an ion concentration difference on each side of the membrane. In Fig A, the potassium concentration is high inside a nerve fiber membrane but very low outside the membrane. Assuming that the membrane in this instance is permeable to the potassium ions, there is a strong tendency for extra numbers of potassium ions to diffuse outward through the membrane due to the large potassium concentration gradient from inside to outside. As they diffuse outwards, they carry positive electrical charges to the outside, thus creating electropositivity outside the membrane and electronegativity inside because of negative anions that remain behind when the potassium diffuses outward. Within a millisecond or so, the potential difference between the inside RESEARCH ON MEMBRANE RESTING POTENTIAL 3

4 and outside, called the diffusion potential, becomes great enough to block further net potassium diffusion to the exterior, despite the high potassium ion concentration gradient. In the normal mammalian nerve cell, the potential difference required is about 94 millivolts, with negativity inside the cell membrane. Fig Effect of concentration difference (A), electrical potential difference affecting negative ions (B), and pressure difference (C) to cause diffusion of molecules and ions through a cell membrane. Fig , Establishment of a diffusion potential across a nerve fiber membrane, caused by diffusion of potassium ions from inside the cell to outside through a membrane that is selectively permeable only to potassium. B, Establishment of a diffusion potential when the nerve fiber membrane is permeable only to sodium ions. Fig B shows the same phenomenon as in Fig A, but this time with high concentration of sodium ions outside the membrane and low sodium inside. This time, the membrane is highly permeable to the sodium ions but impermeable to all other ions. Diffusion of the positively charged sodium ions to the inside creates a membrane potential of opposite polarity to that in Figure 5 1A, RESEARCH ON MEMBRANE RESTING POTENTIAL 4

5 negativity being outside and positivity inside. As before, the membrane potential rises high enough within milliseconds to block further net diffusion of sodium ions to the inside. Resting Membrane Potential The resting membrane potential of large nerve fibers when not transmitting nerve signals is about 90 millivolts which means that inside the fiber the potential is 90 millivolts more negative than the potential in the extracellular fluid on the outside of the fiber. This potential is determined by the movement of Na + and K + ions through the nerve membrane: sodium ions are pumped to the outside of the cell and potassium ions to the inside. This is an electrogenic pump, because more positive charges are pumped to the outside than to the inside leaving a net deficit of positive ions on the inside and causing a negative potential inside the cell membrane. Assuming that the diffusion of potassium ions is the only movement of ions through the membrane, due to the high ratio of potassium ions inside to outside, 35:1, the Nernst potential corresponding to this ratio is 94 millivolts. In this case, if potassium ions were the only factor causing the resting potential, the resting potential inside the fiber would be equal to 94 millivolts. However, nerve membrane is slightly permeable to the sodium ions, caused by the minute diffusion of sodium ions through the K+-Na+ leak channels. The ratio of sodium ions from inside to outside the membrane is 0.1, and this gives a calculated Nernst potential for the inside of the membrane of +61 millivolts. Intuitively, one can see that, if the membrane is highly permeable to potassium but only slightly permeable to sodium, the diffusion of potassium contributes far more to the membrane potential than does the diffusion of sodium. In the normal nerve fiber, the permeability of the membrane to potassium is about 100 times as great as its permeability to sodium. Using this value in the Goldman equation gives a potential inside the membrane of 86 millivolts. The Na+-K+ pump provides an additional contribution to the resting potential. The fact that more sodium ions are being pumped to the outside than potassium to the inside causes continual loss of positive charges from inside the membrane creating an additional degree of negativity (about 4 millivolts additional) on the inside beyond that which can be accounted for by diffusion alone. Therefore, the net membrane potential with all these factors operative at the same time is about 90 millivolts. In summary, the diffusion potentials alone caused by potassium and sodium diffusion would give a membrane potential of about 86 millivolts, almost all of this being determined by potassium diffusion. An additional 4 millivolts is contributed to the membrane potential by the continuously acting electrogenic Na+-K+ pump, giving a net membrane resting potential of 90 millivolts. Methodology The membrane resting potential experimental setup consists of Ussing chamber vessel and two electrodes connected to the Cobra3 Chem-Unit measurement system. Ussing chamber vessel is a system of two closed vessels separated by the membrane. Two vessels are filled with different concentration solutions ( or KCl). With one membrane it is recommended to measure just one type of solution. In order to measure the conductivity of different type of solution it is necessary to change the membrane in the Ussing chamber vessel. Students are not allowed to change membrane by themselves. It is strongly recommended to consult about measurements of different solutions with worker of the laboratory. During the resting potential measurement two silver chloride electrodes are immersed into vessels and Cobra3 Chem-Unit measurement system measures the potential difference between those two electrodes RESEARCH ON MEMBRANE RESTING POTENTIAL 5

6 Procedures 1. A cellophane sheet is moistened and stretched over the large opening of a Ussing chamber vessel without the screws in place. It is fixed in place with an elastic band and left to dry for about 5 minutes. 2. The second vessel is then put in place, the screws are pressed through the cellophane sheet and both vessels are screwed together (Fig ). 3. After removing the protective caps, two silver chloride reference electrodes are placed in a cylinder with 0.1 mol/l KCI and are connected to the BNC plug / 4 mm socket adapter. The adapter is plugged into the Cobra3 Chem-Unit measurement system BNC socket (8, Fig ). The Cobra3 BASIC-Unit measurement system is connected to the PC using a USB port (11, Fig ) and to the power supply (14, Fig ). 4. As shown in Table 1, for the first test the two vessels of the Ussing chamber are filled with NaCI 0.01 mol/l and mol/l concentration solutions. The two vessels should always be filled simultaneously (with the aid of two 100-ml graduated cylinders). Table 1. Test combinations for solutions Fig Ussing chamber vessel and two electrodes. Vessel with concentrated solution Vessel with dilute solution Test 1 Test 2 Test 3 Test 4 Test mol/l 0.1 mol/l 1 mol/l 1 mol/l 0.01 mol/l mol/l mol/l 0.01 mol/l 0.01 mol/l 0.1 mol/l 5. Measure the temperatures of the experimental solutions with the digital thermometer. 6. The electrodes are removed from the storage cylinder and are dipped into the experimental solutions (Fig ). 7. Run the measure Cobra3 software. 8. Run the new experiment in the software: in the menu bar choose File New measurement. In the newly opened window set the parameters of the experiment. The parameters that are displayed in Fig should be set as follows: in the window Get value choose on key press, in the window X data choose Number, in the window Channels choose Voltage. 9. In the New measurement window press the Continue button. 10. Measure several (5-10) potential values repeatedly pressing the Save value button (Fig ). 11. Press Close button. 12. Calculate the average value of the resting potential: in the measure Cobra3 software menu bar choose Analysis Show average value. 13. Replace the electrodes in the storage cylinder with 0.1 mol/l KCI. 14. The measurements are repeated twice at intervals of about 2 minutes. The electrodes should always be replaced in 0.1 mol/l KCI between measurements and be tested for equivalence. If a potential is measurable between the two electrodes in 0.1 mol/l KCI, this asymmetry potential must be subtracted from the measured diffusion potential RESEARCH ON MEMBRANE RESTING POTENTIAL 6

7 15. Repeat the measurements (4-14 procedures) for each combination of experimental solutions (tests 1-5 in Table 1). The vessels of the Ussing chamber should be rinsed out several times with distilled water when changing the experimental solutions. A new membrane must be introduced when changing the ion (from NaCI to HCI or KCI). 16. Calculate the mean values of the potential difference for each experimental combination of solutions taking asymmetry potential into account. 17. Calculate the theoretical diffusion potential on the cation-permeable membrane according to the following formula, which is derived from the Nernst-Planck equation: ; (6.17.2) here T absolute temperature of solution, µ - diffusion rate of the cation, υ - diffusion rate of the anion, a 1 activity of more concentrated solution, a 2 activity of more dilute solution. Activity is equal to the concentration multiplied by the activity coefficient ( ). 18. Compare the measured and theoretical diffusion potentials. Table 2. Diffusion rates µ, cation Rate, cm/s υ, anion Rate, cm/s H OH Na Cl K Table 3. Activity coefficient Fig measure Cobra3 software New measurement window. Fig measure Cobra3 software measuring window mol/l 0.01 mol/l 0.1 mol/l 1 mol/l KCl RESEARCH ON MEMBRANE RESTING POTENTIAL 7

8 References 1. B H Brown, R H Smallwood, D C Barber, P V Lawford and D R Hose, Medical Physics and Biomedical Engineering, New York N.Y.; London: Taylor and Francis (1999). 2. Arthur C. Guyton, John E. Hall, Textbook of medical physiology, Philadelphia Pa.: Saunders/Elsevier (2011). 3. Irving P. Herman, Physics of the human body, Berlin; Heidelberg: Springer (2007) RESEARCH ON MEMBRANE RESTING POTENTIAL 8

9 Cobra3 Chem-UNIT measure system Appendix 1 Fig Cobra3 Chem-UNIT measure system operating elements. 1. Extension connector for Units. 48 pin plug in the side wall for docking a further Unit. 2. Connecting element with positioning foot. The immovable connecting element on the left-hand side is for fixing a further unit to this side. The swing-out foot enables the CHEM-UNIT to be set to an inclined position. The gray colour of the foot signalizes the immovable connecting element. 3. Input for conductivity measuring cells with 4 mm plugs 4. Input for conductivity-temperature probes or Pt1000 temperature probes with diode plug. When the conductivity-temperature electrode is plugged in here, then a further conductivity probe cannot be used at input (3). 5. Rubber feet. 6. Temperature input NiCr-Ni to which thermocouples with DIN plugs (type K) can be connected. Temperature inputs T1 and T2 are galvanic separated from analog input (7) as well as from the input ph/potential (8). Temperature input T3 is galvanic separated from the two inputs for conductivity electrodes (3/4) and from the two temperature inputs T1 and T2. 7. Analog input. Earth-related analog input with two 4 mm safety sockets for the measurement of direct voltages, with the three measuring ranges: ±3.2 V, ±32 V, ±81 V. This analog input is galvanic separated from the two inputs for conductivity electrodes (3/4) and from the two temperature inputs T1 and T2. 8. Input for ph electrodes and redox electrodes. BNC socket for the connection of ph electrodes or redox electrodes with BNC plugs. This high ohm input allows ph values and redox potentials to be measured. This ph/potential input is galvanic separated from the two inputs for conductivity electrodes (3/4) and from the two temperature inputs T1 and T2. 9. TTL input with 4 mm safety sockets (and the same earth socket as TTL output 10) for the acquisition of TTL pulses. 10. TTL output with 4 mm safety sockets (and the same earth socket as TTL input 9) for regulating with TTL pulses. 11. RS232 connection. A 9-pin SUB-D socket on the right-hand side of the unit for connection to the serial interface of a computer by means of a data cable. 12. Extension connection for a further unit. A further unit can be docked onto this 48-pin plug in the right-hand side of the unit. The plug connection can be secured with a special connecting element (13). 13. Connecting element with positioning foot. The connecting slide element on the right-hand side is for fixing a further unit to this side. The swing-out foot enables the CHEM-UNIT to be set to an inclined position. The yellow colour of the foot signalizes the connecting slide element RESEARCH ON MEMBRANE RESTING POTENTIAL 9

10 14. Side voltage supply. A low voltage socket (inner positive pole) in the right hand side of the unit for connection of a Cobra3 power supply with 12 V direct voltage. This is an alternative to connecting the power supply to input (16) on the front of the unit. 15. Control lamp. This green light-emitting diode acts as indicator to show when the unit is switched on. 16. Front voltage supply. A low voltage socket (inner positive pole) at the front of the unit for connection of a Cobra3 power supply with 12 V direct voltage. This is an alternative to connecting the power supply to input (14) on the right-hand side of the unit. 17. Fixed voltage output. A pair of 4 mm safety sockets for withdrawal of a direct voltage of 5 V with maximum 0.2 A. 18. Dovetail connections. Dovetail connections that enable units to be firmly fixed one on top of the other. This mechanical connection does not include any electrical connection of the units to each other. An appropriate plug is additionally required to make such an electrical connection. 19. Connecting thread for a clamp RESEARCH ON MEMBRANE RESTING POTENTIAL 10