2. 1 LIU Chuan Yong 刘传勇 Institute of Physiology Medical School of SDU Tel 88381175 (lab) 88382098 (office) Email: liucy@sdu.edu.cnliucy@sdu.edu.cn Website: www.physiology.sdu.edu.cn

3. 2 Section 2 Bioelectrical Phenomena of the Cell

4. 3 Basic Concepts  Volt  A charge difference between two points in space

5. 4 Basic Concepts  Ions – charged particles  Anions – Negatively charged particles  Cations – Positively charged particles

6. 5 Basic Concepts Forces that determine ionic movement  Electrostatic forces  Opposite charges attract  Identical charges repel  Concentration forces  Diffusion – movement of ions through semipermeable membrane  Osmosis – movement of water from region of high concentration to low

7. 6 Selective Permeability of Membranes  Some ions permitted to cross more easily than others  Neuronal membranes contain ion channels  Protein tubes that span the membrane  Some stay open all the time (nongated)  Some open on the occasion of an action potential, causing a change in the permeability of the membrane (gated)

8. 7 I. Membrane Resting Potential  A constant potential difference across the resting cell membrane  Cell’s ability to fire an action potential is due to the cell’s ability to maintain the cellular resting potential at approximately –70 mV (-.07 volt)  The basic signaling properties of neurons are determined by changes in the resting potential

9. 8 Membrane Resting Potential  Every neuron has a separation of electrical charge across its cell membrane.  The membrane potential results from a separation of positive and negative charges across the cell membrane.

10. 9 Membrane Resting Potential  excess of positive charges outside and negative charges inside the membrane  maintained because the lipid bilayer acts as a barrier to the diffusion of ions  gives rise to an electrical potential difference, which ranges from about 60 to 70 mV.  (Microelectrode)

11. 10 Concept of Resting Potential (RP)  A potential difference across the cell membrane at the rest stage or when the cell is not stimulated.  Property:  It is constant or stable  It is negative inside relative to the outside  Resting potentials are different in different cells.

12. 11 Ion Channels Two Types of Ion Channels  Gated  Non-Gated

13. 12 Resting Membrane Potential  Na + and Cl - are more concentrated outside the cell  K + and organic anions (organic acids and proteins) are more concentrated inside.

14. 13 Intracellular vs extracellular ion concentrations Ion Intracellular Extracellular Na + 5-15 mM 145 mM K + 140 mM 5 mM Mg 2+ 0.5 mM 1-2 mM Ca 2+ 10 -7 mM 1-2 mM H + 10 -7.2 M (pH 7.2) 10 -7.4 M (pH 7.4) Cl - 5-15 mM 110 mM

15. 14 Resting Membrane Potential  Potassium ions, concentrated inside the cell tend to move outward down their concentration gradient through nongated potassium channels  But the relative excess of negative charge inside the membrane tend to push potassium ions out of the cell

16. 15 Potassium equilibrium -90 mV

17. 16 Resting Membrane Potential But what about sodium? Electrostatic and Chemical forces act together on Na + ions to drive them into the cell The Na + channel close during the resting state Na + is more concentrated outside than inside and therefore tends to flow into the cell down its concentration gradient Na + is driven into the cell by the electrical potential difference across the membrane.

18. 17 Na + electrochemical gradient

19. 18 Equilibrium Potentials  Theoretical voltage produced across the membrane if only 1 ion could diffuse through the membrane.  If membrane only permeable to K +, K + diffuses until [K + ] is at equilibrium.  Force of electrical attraction and diffusion are = opposite.

20. 19 Calculating equilibrium potential Nernst Equation  Allows theoretical membrane potential to be calculated for particular ion.  Membrane potential that would exactly balance the diffusion gradient and prevent the net movement of a particular ion.  Value depends on the ratio of [ion] on the 2 sides of the membrane.

21. 20 Nernst equation Equilibrium potential (mV), E ion = ln RT zF [C] o [C] i where, [C] o and [C] i = extra and intracellular [ion] R = Universal gas constant (8.3 joules.K -1.mol -1 ) T = Absolute temperature (°K) F = Faraday constant (96,500 coulombs.mol -1 ) z = Charge of ion (Na + = +1, Ca 2+ = +2, Cl - = -1) For K +, with [K + ] o = 4 mmol.l -1 and [K + ] i = 150 mmol.l -1 At 37°C, E K = -97mV E Na = +60mv

22. 21 [K + ] o = 4 mmol.l -1

23. 22 Resting Membrane Potential  Resting membrane potential is less than E k because some Na + can also enter the cell.  The slow rate of Na + influx is accompanied by slow rate of K + outflux.  Depends upon 2 factors:  Ratio of the concentrations of each ion on the 2 sides of the plasma membrane.  Specific permeability of membrane to each different ion.  Resting membrane potential of most cells ranges from - 65 to – 85 mV.

24. 23 The Sodium-Potassium Pump Dissipation of ionic gradients is ultimately prevented by Na + -K + pumps extrudes Na + from the cell while taking in K

25. 24 Resting Potential

26. 25 The formation of resting potential depends on:  Concentration difference of K + across the membrane  Permeability of Na + and K + during the resting state  Na + -K + pump

27. 26 Factors that affect resting potential  Difference of K + ion concentration across the membrane  Permeability of the membrane to Na + and K +.  Action of Na + pump

28. 27 Basic Electrophysiological Terms I:  Polarization: a state in which membrane is polarized at rest, negative inside and positive outside.  Depolarization: the membrane potential becomes less negative than the resting potential (close to zero).  Hyperpolarization: the membrane potential is more negative than the resting level.

29. 28 Basic Electrophysiological Terms I:  Reverspolarization: a reversal of membrane potential polarity.  The inside of a cell becomes positive relative to the outside.  Repolarization: restoration of normal polarization state of membrane.  a process in which the membrane potential returns toward from depolarized level to the normal resting membrane value.

30. 29 II Action Potential Successive Stages: (1)Resting Stage (2)Depolarization stage (3)Repolarization stage (4)After-potential stage (1) (2)(3) (4)

31. 30 Concept  Action potential is a rapid, reversible, and conductive change of the membrane potential after the cell is stimulated.  Nerve signals are transmitted by action potentials.

32. 31 Action Potential Sequence Voltage-gated Na + Channels open and Na + rushes into the cell

33. 32 Action Potential Sequence At about +30 mV, Sodium channels close, but now, voltage-gated potassium channels open, causing an outflow of potassium, down its electrochemical gradient

34. 33 Action Potential Sequence equilibrium potential of the cell is restored

35. 34 Action Potential Sequence The Sodium – Potassium Pump is left to clean up the mess…

36. 35 Ion Permeability during the AP Figure 8-12: Refractory periods

37. 36 Basic Electrophysiological Terms II (1)  Excitability: The ability of the cell to generate the action potential  Excitable cells: Cells that generate action potential during excitation.  in excitable cells (muscle, nerve, secretery cells), the action potential is the marker of excitation.  Some scholars even suggest that in excitable cells, action potential is identical to the excitation.

38. 37 Basic Electrophysiological Terms II (2)  Stimulus: a sudden change of the (internal or external) environmental condition of the cell.  includes physical and chemical stimulus.  The electrical stimulus is often used for the physiological research.  Threshold (intensity): the lowest or minimal intensity of stimulus to elicit an action potential  (Three factors of the stimulation: intensity, duration, rate of intensity change)

39. 38 Basic Electrophysiological Terms II (3)  Types of stimulus:  Threshold stimulus: The stimulus with the intensity equal to threshold  Subthreshold stimulus: The stimulus with the intensity weaker than the threshold  Suprathreshold stimulus: The stimulus with the intensity greater than the threshold.

40. 39 Action Potential Summary  Reduction in membrane potential (depolarization) to "threshold" level leads to opening of Na + channels, allowing Na + to enter the cell  Interior becomes positive  The Na + channels then close automatically followed by a period of inactivation.  K + channels open, K + leaves the cell and the interior again becomes negative.  Process lasts about 1/1000th of a second.

41. 40 Properties of the Action Potential  “All or none” phenomenon  A threshold or suprathreshold stimulus applied to a single nerve fiber always initiate the same action potential with constant amplitude, time course and propagation velocity.  Propagation  Transmitted in both direction in a nerve fiber

42. 41 III Initiation of Action Potential

43. 42 Squid giant axon

44. 43 Gated channel states

45. 44 CO 2 H Outside NH 2 ++++++ ++++++ ++++++ ++++++ Inside Na + Channel   -Subunit Structure IIIIIIIV RVIRLARIGRILRLIKGAKGIR + + + + I F M I F M - Inactivation “Gate” IVS4 Voltage Sensor NH 2 CO 2 H 

46. 45 Voltage gated But “ready”Not “ready”

47. 46 Activation & Fast Inactivation

48. 47 Sodium Activation and Inactivation Variable vs Voltage Activation Gate Inactivation gate If resting potential depolarized by 15 – 20 mV, then activation gate opened with 5000x increase in Na + permeability followed by inactivation gate close 1 ms later

49. 48 Positive feedback loop Reach “threshold”? If YES, then... Stimulation

50. 49 Action potential initiation S.I.Z.

51. 50 Action potential termination

52. 51

53. 52 Threshold Potential  Threshold potential plays a key role in the genesis of action potential.  Threshold potential is a critical membrane potential level at which an action potential can occur.  Why can all the Na + channel open at the threshold potential? It is dependent on the gating property of the voltage-gated Na+ channels.  The value of threshold potential of most excitable cell membrane is about 15 to 20 mV less negative than the resting potential.  The threshold stimulus is just strong enough to depolarize the membrane to the threshold potential level, therefore it can cause an action potential.

54. 53 Electrophysiological Method to Record Membrane Potential I Voltage Clamp

55. 54 The Nobel Prize in Physiology or Medicine (1963) “for their discoveries concerning the ionic mechanisms involved in excitation and inhibition in the peripheral and central portions of the nerve cell membrane” Alan Lloyd Hodgkin Andrew Fielding Huxley

56. 55  Cole and colleagues developed a method for maintaining V m at any desired voltage level (FBA, Feedback Amplifier)  Required monitoring voltage changes, feeding it through an amplifier to drive current into or out of the cell to dynamically maintain the voltage while recording the current required to do so The voltage clamp

57. 56 The Hodgkin-Huxley Model of Action Potential Generation

58. 57

59. 58 Triphasic response

60. 59 Evidence for a Sodium Current Remove extracellular sodium

61. 60 Modern proof of nature of currents Use ion selective agents

62. 61  Removing Na + from the bathing medium, I Na becomes negligible so I K can be measured directly.  Subtracting this current from the total current yielded I Na.

63. 62

64. 63 Conductance of Na + and K + channels

65. 64 Voltage-Dependence of Conductance

66. 65 An action potential  g Na increases quickly, but then inactivation kicks in and it decreases again.  g K increases more slowly, and only decreases once the voltage has decreased.  The Na + current is autocatalytic. An increase in V increases g Na, which increases the Na + current, and increases V, etc.  Hence, the threshold for action potential initiation is where the inward Na + current exactly balances the outward K + current.

67. 66 "for their discoveries concerning the function of single ion channels in cells" The Nobel Prize in Physiology or Medicine (1991) Erwin NeherBert Sakmann

68. 67 CytoplasmIon channels "Giga-seal" Glass microelectrode Suction 1 µm Patch clamp recording Cell Membrane

69. 68

70. 69 100 ms 4 pA Closed Open Single channel record

71. 70 One result from patch clamp studies was the finding that ion channels conduct currents in an all or nothing fashion

72. 71 Voltage-dependent Channel Conductance

73. 72 How channel conductances accumulate Next page shows an idealized version

74. 73

75. 74 Inactivating Na + channel currents

76. 75 IV Local Response

77. 76 Graded (local) potential changes 2 x more chemical= 2 x more potential change

78. 77 Local Response  Definition:  Local response is a small change in membrane potential caused by a subthreshold stimulus  Properties:  It s a graded potential  Its propagation is electronic conduction  It can be summed by two ways  Spatial summation  Temporal summation

79. 78 Excitatory Inhibitory Time Membrane Potential (mV) Spatial Summation Spatial Summation a b c d a b c d

80. 79 Excitatory Inhibitory Time Membrane Potential (mV) Temporal & Spatial Summation Temporal Summation a b c d a b c d

81. 80 Distribution of channels Leak channels everywhere Axon Hillock (Trigger Zone)

82. 81 Role of the Local Potential  Facilitate the cell.  This means it increase excitability of the stimulated cell  Cause the cell to excite once it is summed to reach the threshold potential

83. 82

84. 83 V. Propagation of the Action Potential

85. 84

86. 85

87. 86 Myelinated neuron of the central nervous system

88. 87 Saltatory conduction: The action potential jumps from node to node

89. 88

90. 89 Saltatory Conduction

91. 90 Saltatory Conduction  The pattern of conduction in the myelinated nerve fiber from node to node  It is of value for two reasons:  very fast  conserves energy.

92. 91

93. 92 Factors that affect the propagation  Bioelectric properties of the membrane  Velocity and amplitude of membrane depolarization

94. 93 V Excitation and Excitability of the Tissue

95. 94 Excitation and Excitability of the Tissue  Review: Excitation and Excitable Cell  Review: Threshold Stimulation and Excitability  Change of Excitability after the Excitation

96. 95

97. 96 Slide 3 of 28

98. 97 4. Factors that Determine the Excitability  Resting potential  Threshold potential  Concentration of extracellular Ca 2+

99. 98 If resting potential depolarized by 15 – 20 mV, then activation gate opened with 5000x increase in Na + permeability followed by inactivation gate close 1 ms later Activation Gate Inactivation gate

100. 99 the threshold for action potential initiation is where the inward Na + current exactly balances the outward K + current.

101. 100 Concentration of extracellular Ca 2+