Membrane Potential (Em)

1. Membrane Potential (Em) What is it? How does the membrane potential arise? How do you estimate the Membrane Potential with the Nernst Equation? Why study membrane potential?= see next slide Page 191, 203 (ion channels), 208-209 (CF), 365-368 (Understanding mem pot, Nernst equa)

2. Why study the membrane potential Em? (Don’t memorize!!): • Our cells make ATP (possibly the most important reaction in the body) with use of the membrane potential. • Epilepsy is thought to be due to bad "voltage-gated" potassium channels (voltage-gated means that the channels are opened or closed by the membrane voltage Em). • Cystic fibrosis is due to bad chloride movement across membranes. • Heart drugs such as cardiotonic steroids (i.e., cardiac glycosides) are Na-K pump (active transport) inhibitors- this can effect Em • Neurotransmitters act by changing ion fluxes across membranes-this changes Em. • Ions move across membranes in the eye- changing Em, allowing us to see. • Ion movement across membranes and changes in Em is important for each muscle contraction and for each nerve impulse. • One of the most deadly poisons known (tetrodotoxin) is from the Puffer fish (a specialty in the Japanese diet); the poison acts by blocking sodium channels- this prevents action potentials (which are changes in Em). • Some anesthetics work by altering Em.

3. FIG. 5-2 The cell spends a tremendous amount of energy maintaining the Membrane Potential CHEMICAL WORK CHEMICAL WORK Note positive ion moving out Makes the membrane potential With an excess of + outside, - inside

4. The membrane potential: • VOLTAGE OF A MEMBRANE: Vm OR Em is the symbol • WHAT IS A VOLTAGE? SINCE MEMBRANES ARE HYDROPHOBIC AND PREVENT IONS FROM CROSSING, A POTENTIAL DIFFERENCE (IN UNITS OF VOLTAGE) CAN BUILD UP ACROSS THE MEMBRANE. • That is, + charges build up on one side of a Membrane, and – charges build up on the other • CELLS ARE REGULATED BY CHANGES IN THE MEMBRANE POTENTIAL (ESP. NERVE CELLS)

5. Membrane Voltage: IS THE INSIDE OF THE CELL POSITIVE OR NEGATIVE? FIG. 13-11 -4O MILLIVOLTS CELL HAS HI [K] AND LOW [Na] + We will measure membrane potential this way - CELL

6. If a Positive ion like K moves out of the cell, a membrane potential develops- where is the plus sign? Inside the cell or out? K+ K+ - + Voltage becomes very negative== -50 mV (or more)

7. For most cells (cells are “rest” or resting cells), as K moves out of the cell, this makes the membrane potential

8. Go through these web sites to review (see them on my web site for cell biology 3611) • http://carbon.cudenver.edu/~bstith/membrpotential.gif (D:\cell biol 3611\ch 7 8 9 membrane trans\membrpotential.gif) • http://carbon.cudenver.edu/~bstith/nernst.mov (D:\cell biol 3611\ch 7 8 9 membrane trans\nernst-short.mov) • http://distance.stcc.edu/AandP/AP/AP1pages/nervssys/unit10/resting.htm#what%20is%20RP • http://www.taumoda.com/web/nernstjava/ • For the computational biology students: view this paper: http://carbon.cudenver.edu/~bstith/koch.pdf

9. Then, if a Negative Ion like Chloride (Cl-) moves out of the cell, the membrane potential decreases toward zero Cl- Cl- + - Em changes from -50 mV to a less negative number (Cl- are leaving)--Such as -20 mV

10. How do Ions (like sodium or Na, potassium or K, Chloride or Cl) cross membranes? • IONS CROSS MEMBRANES THROUGH CHANNELS (A PROTEIN THAT CROSSES THE MEMBRANE) FIG 13-8

11. How does the membrane potential develop? • Most cells are negative inside, about – 40 to -60 mV • This potential is typically due to potassium moving out of the cell—there are more K channels open than channels for other ions • K moves out because K concentration is very high in the cell (and low outside)-so K moves from high to low conc. (due to the NaK pump) • What would happen to the membrane potential if negative Cl moved out of the cell? We will see...

12. ION CHANNELS allow ions to move across the membrane • ION CHANNELS ALLOW ONLY IONS TO CROSS, CHANGING MEMBRANE POTENTIAL OF CELL • CHANGE Em, TURN ON/OFF NEURON. • MANY MEDICINES AFFECT ION CHANNELS TO AFFECT NEURON (ANTIDEPRESSENT). • Typically, there are more K channels open, so K moves out of the cell and sets the membrane potential to negative inside. • In an action potential, sodium channels open up and sodium movement sets the membrane potential

13. In the Xenopus Oocyte • Potassium channels are more open, so K efflux sets the membrane potential to about -50 mV • However, if chloride channels open, chloride moves out of the cell and this reduces the membrane potential to about -20 mV.

14. HOW DO YOU ESTIMATE THE MEMBRANE POTENTIAL?Nernst Equation: Vm or Em=(RT/ZF)ln([C]out/[C]in) (EQUATION 13.1 (old 9.1) IN TEXT) R= 1.987 cal/deg mole; Z is charge of the ion, F = 23,062 cal/volt equiv; T =temp in Kelvin (C +273), Ln is natural log base e= 2.718. • [C] = concentration of the ion that is most permeable (its channels are more open)- concentration outside the cell or inside the cell

15. outside the cell membrane: [Na]o =140 mM [K]o = 5 mM [Cl]o = 100 mM inside the cell [Na]in = 14 mM [K]in = 124 mM [Cl]in = 40 mM Example: Estimate Plasma Membrane Potential (Em)-write this down

16. Estimate the Membrane Potential for human cell (37C) Em = (RT/ZF) ln ([C]out/[C]in) Plug in what ion concentrations since it is the most permeable ion (more these ion channels are open so the ion’s concentrations determine the membrane potential) R= 1.987 cal/deg mole; Z is charge of the ion, F = 23,062 cal/volt equiv; T =temp in Kelvin (deg C +273), Ln is natural log base e= 2.718. • [C] = concentration of the ion that is most permeable (its channels are more open)- concentration outside the cell or inside the cell

17. Do the calculation

18. K is most permeable: Em = (RT/ZF) ln ([C]out/[C]in) = (1.987)(273+37)/ (+1)(23062) ln (5 mM/124 mM) = 0.0267 x -3.21 = -0.0857 Volts (note the concentrations must be in same units) Usually, scientists report answer in milliVolts: = - 85.7 mV is the estimate for the membrane potential of the liver cell For practice: questions 13-2, -4, and -5 in book

19. ACTION POTENTIAL in nerve cell IS DUE TO OPENING OF SODIUM ION CHANNELS, THEN CLOSING OF THESE ION CHANNELSRESTING CELL before ActPot: K IS MOST PERMEABLE (SOME K CHANNELS OPEN)PEAK OF ACTION POTENTIAL: Na IS MOST PERMEABLE

20. What if Na channels open? Em now set by Na Concentrations • Em = [(1.987x310)/(+1)23062]ln (140/14) = +61 mV (make sure that you can perform this calculation-on exam)

21. Before,K channels are more open and K determines the Em, then the Na channel open to determine Em (and closes) to make the action potential (turns on nerve cell) Na determines membrane potential S Na CH. open Na CH. closes K CH. open K CH. open Fig. 13-12

22. WHAT IF Chloride becomes the MOST PERMEABLE ION? • DOES THIS HAPPEN WITH ANIMAL CELLS? Yes. • Chloride channels open up to allow Cl- movement • The membrane potential changes from about -40 to -20 mV.

23. Estimate the Membrane Potential with Chloride channels open • [Cl]in = 40 mM [Cl]out = 100 mM • Cl has a negative charge (z=-1) Em = (RT/ZF) ln ([C]out/[C]in) = HORMONES RELEASE CALCIUM INTO THE CYTOPLASM AND CALCIUM OPENS CHLORIDE CHANNELS TO CHANGE THE Em • WE WILL MEASURE THIS CHANGE IN Em…

24. Chloride Channels in human disease- cystic fibrosis • Chloride channels need to be present and functional for cells to function • In cystic fibrosis, chloride channels do not make it to the plasma membrane AND CHOLORIDE DOES NOT MOVE ACROSS THE MEMBRANE • So, the symptoms of cystic fibrosis develop (high salt in sweat, destruction of organs, thick mucus in lungs that causes infections). • Pgs. 208-209 in 6th edition of our text

25. Chloride Channel Fig. 13-8a

26. We will study the Chloride Channel in Xenopus frog oocytes • Acetylcholine binds to a membrane receptor • To increase the concentration of Calcium in the cytoplasm • Calcium binds to and opens the Chloride channel • Chloride moves across the membrane and out of the cell • This Cl movement causes the membrane potential to change from -50 mV to a lower value: -25 mV

27. PA (new hormone?) Acetylcholine Calcium Ach receptor Cl- Note that chloride Efflux reduces the Membrane potential (less negative inside, Less positive outside) - - - + + + +

28. Today, we will use the NeuroLab program (later, maybe the Neuroscience Program from HHMI)

29. end