Introduction to electrophysiology. Dr. Tóth András

Transcription

1 Introduction to electrophysiology Dr. Tóth András

2 Topics Transmembran transport Donnan equilibrium Resting potential Ion channels Local and action potentials Intra- and extracellular propagation of the stimulus

3 Level of significance Entry level (even under 6) Student level (for most of you) Gourmand level (only for the pros)

4 1. Transmembran transport

5 1 Major types of transmembran transport

6 2 dc J: net rate (flux) of diffusion J = DA dx A: area dc/dx: concentration gradient J = DA c x D: diffusion coefficient (D: cm 2 /s) J D = dc A dx Fick s first law of diffusion

7 3 Stokes Einstein equation D = 6 kt π r η Einstein relation ( x 2 ) = 2 Dt Diffusion of solutes as a consequence of the random thermal (Brownian) motion of the particles

8 4 Time required for diffusion as a function of diffusion distance

9 5 J J K = = Fick s law for membrane = DA DA D β x β c x c x β: partition coefficient K: permeability coefficient Diffusion across a semipermeable membrane

10 6 Osmotic motion across a semipermeable membrane

11 7 van t Hoff s Law π= irtm π= irtc π = RTΦic Φic = T f /1.86 Φ: osmotic coefficient Φic: osmotically effective concentration - osmolality I.e.: 154 mm NaCl solution π = 6.42 atm Φic = osmol/l Definition of the osmotic pressure

12 8 Mechanism of facilitated diffusion

13 9 Principle of transport of ions across ion channels

14 10 The principle of function of the Na /K ATPase

15 11 Secondary active transport processes

16 12 Michaelis-Menten equation V max : maximal rate of transport K m : concentration of the substrate for which the rate of transport is equal to V max /2 Transport via proteins shows saturation kinetics

17 2. Ionic equilibrium

18 13 µ = µ o RT ln C zfe µ = RT ln [ ] X [ ] X A B zf ( E E ) A B Electrochemical potential (difference)

19 14 Equilibrium 0 E = zf A [ ] X A ln [ ] zf A X B ( ) [ ] X EA EB = RT ln [ X ] RT [ X ] A EB = ln zf [ X ] B RT ( E E ) B A B For monovalent cations Z = 1 = 60mV lg [ ] X [ ] X E X A B Nernst equation

20 15 A B A B 0.1 M 0.01 M 1 M 0.1 M K K HCO 3 - HCO 3 - E A E B = -60 mv E A E B = 100 mv Is there equilibrium in any of the two cases? Examples of uses of the Nernst equation 1.

21 16 A B 0.1 M K 0.01 M K A B 1 M 0.1 M HCO - 3 HCO - 3 E A E B = 60 mv At 60 mv the K is in electrochemical equilibrium across the membran No electric force!!! Examples of uses of the Nernst equation 2.

22 17 A B 0.1 M K 0.01 M K E A E B = 60 mv At 60 mv the K is in electrochemical equilibrium across the membran No electric force A B 1 M HCO M HCO 3 - E A E B = 100 mv At the given membran potential the HCO 3- is not in electrochemical equilibrium Electric force: 40 mv Examples of uses of the Nernst equation 3.

23 18 A B A B [K ] = 0.1 M [P - ] = 0.1 M [K ] = 0.1 M [Cl - ] = 0.1 M [K ] = [Cl - ] = [P - ] = 0.1 M [K ] = [Cl - ] = Initial state Equilibrium? 1. The principle of electroneutrality should be preserved!!! 2. The electrochemical potential should be zero for each diffusible ion!!! (Not for the undiffusible ion!!!) Before Gibbs-Donnan equilibrium is established

24 19 A B A B [K ] = 0.1 M [P - ] = 0.1 M [K ] = 0.1 M [Cl - ] = 0.1 M [K ] = M* [Cl - ] = M* [P - ] = 0.1 M [K ] = M* [Cl - ] = M* Initial state Equilibrium state* (!?) 1. The principle of elektroneutrality is, indeed, valid!!! 2. The electrochemical potential is zero for K and Cl -!!! 3. * So, is there any problem??? Gibbs-Donnan equilibrium has been attained

25 20 PH = 2.99 atm!!! A B A B [K ] = 0.1 M [P - ] = 0.1 M [K ] = 0.1 M [Cl - ] = 0.1 M [K ] = M [Cl - ] = M [P - ] = 0.1 M [K ] = M [Cl - ] = M Starting state Equilibrium state (There is no equilibrium between pressures!!!) In Gibbs-Donnan equilibrium a transmembrane hydrostatic pressure gradient is present

26 3. Resting potential

27 21 A B 0.1 M NaCl 0.01 M NaCl If the membrane is permeable for cations, but unpermeable for anions, cation current is needed to reach equilibrium!!! The concentration battery

28 22 Na A B 0.1 M NaCl 0.01 M NaCl In case of electrochemical equilibrium E A E B = - 60 mv The concentration battery

29 23 Measured intra- and extracellular ionconcentrations

30 24 Cl - Na E E 1) Na IC(mM) 12 EC(mM) 145 E eq 65mV cc cc K 160 3,5-100mV - Cl 3, mV -90 mv - Prot E 2) P K P 100 Na cc K 3) Prot= 0 4) E m = 90mV A simplified model of the resting membrane potential in the human skeletal muscle

31 = = = = = K K m K Na Na m Na Cl Cl m Cl g E E I g E E I g E E I R g R U I ) ( ) ( 0 ) ( 1 Conditions for the chord conductance equation Theoretical estimation for the resting potential 1. 25

32 Na I Na ( E E m m I E = g K K Na = 0 g ) g K g Na Na = ( E E K m g E K g K Na g ) g K Na E Na E E m K m 100 = E E Na K g Na = 1 g K = The chord conductance equation

33 27 Theoretical estimation for the resting potential 2. E m = RT F ln k k pk pk [ K [ K ] ] o i k k pna pna [ Na [ Na ] ] o i k k pcl pcl [ Cl [ Cl ] ] i o The constant field (Goldman-Hodgkin-Katz) equation

34 28 C Major factors affecting resting potential

35 29 Also in cardiac cells the resting potential is supposed to be [K ] dependent

36 30 In cardiac cells the resting potential is, indeed, primarily [K ] dependent

37 4. Ion channels

38 4.1 Experimental techniques

39 31 Major configurations of the patch clamp technique

40 32 Single channel current

41 33 Determination of the mean open time

42 34 Current-voltage relationship of the inward and outward rectifying channels

43 4.2 Principles of regulation

44 35 State diagram of a simple, dual-state ion channel

45 36 State diagram of a multiple-state ion channel

46 37 Basic regulatory mechanisms of ion channels

47 37 a Background channels spontaneously oscillate between open and closed states

48 37 b Voltage-gated channels also oscillate between the two states, but voltage shifts the equilibrium

49 37 c The open state of neurotransmitter-gated channels is altered by the binding of a neurotransmitter to the channel (e.g. nicotinic receptor)

50 37 d The open state of G-protein gated channels is altered by binding of activated G-protein subunits to the channel (following receptor activation e.g. muscarinic receptor)

51 37 e Modulated channels may be voltage-gated, but the ability of voltage to open the channel may be influenced by covalent modification (e.g. phosphorilation)

52 4.3 Structure

53 38 Ion channel superfamilies

54 39 2D model of the Na channel 1.

55 40 2D model of the Na channel 2.

56 4.4 Structure-function relation

57 41 S 4 helices are the voltage-sensors of voltage-gated channels amino acid homology is extensive

58 42 Model of the function of the S 4 helix as voltage sensor A total of 6 charges should relocare in the membrane to open the channel

59 43 Top view of the Na channel showing how the central ion channel is proposed to be lined by one of the helices from each domain

60 44 Functional model of a K channel

61 45 Cardiac ion channels

62 5. Local and action potentials

63 5.1 Local response

64 46 Local (subthreshold) response

65 47 Temporal summation

66 48 Spatial summation

67 5.2 Action potential

68 49 Responses in the membrane potential to increasing pulses of depolarizing current

69 50 Action potentials from three vertebrate cell types

70 5.3 Action potentials in the heart

71 51 Ion concentrations in mammalian heart

72 52 Fast and slow response in the heart

73 53 Regional variations in the shape of the action potential of the heart cells

74 54 Ion currents! Fast sodium Funny Delayed rectifier Calcium I L TL 0 0 Tranzient outward Background Sodium Inward rectifier Explanation of the kinetic differences

75 55 The effect of tetrodotoxin on the fast response

76 6. Propagation of the stimulus

77 6.1 Basic principles of propagation

78 56 Potential changes recorded by an extracellular electrode located at different distances from the current electrode

79 57 Maximum change in recorded membrane potential plotted versus distance from the point of current passage

80 58 Potencial changes in a model RC-circuit

81 59 Electric model of the axon membrane

82 60 R m R i C Time constant determined in a membrane

83 61 Model of decremental propagation (voltage divider - resistance ratio)

84 62 R R m i Length constant determined in the membrane

85 63 Model of conduction of the local (subthreshold) response

86 64 Electric model for the propagation of potential changes

87 65 Model of conduction of the AP in nonmyelinated fibers

88 66 Saltatory conduction of the action potential in myelinated fibers

89 67 Conduction velocity of the action potential determined in unmyelinated and myelinated fibers

90 6.2 AP propagation in heart

91 68 MW < 1500 Ca 2 ph E m Structure of the electric synapse (gap junction)

92 69 Electric model of the cardiac cells

93 70 Computer simulation of impulse propagation at the microscopic level

94 The significance of gap junctions in normal stimulus propagation in the heart

95 71 Subcellular stimulus propagation

96 72 Differences in delays of intra- and intercellular activation single cell wide network

97 73 Differences in delays of intra- and intercellular activation multi-cell wide network

98 74 Impulse propagation (isochron lines) in case of normal gap junction coupling (homogenious AP-population)

99 75 Impulse propagation (isochron lines) in severe gap junction uncoupling (heterogenous AP-population)

100 76 In severe gap junction uncoupling propagation velocity may decrease TWO orders of magnitude (!!!) (from 36.7 cm/s to only 0.31 cm/s)

101 77 In case of normal gap junction coupling isochron lines are relatively regularly placed, AP-population is homogenous

102 78 In case of critical gap junction uncoupling action potentials form clusters with significant delays

103 79 Distribution of the cells forming the different clusters in case of critical uncoupling turn back behaviour of the stimulus easily leading to reentry can be observed

104 Questions What are the principal differences between the following iontransporters? Sodium-calcium exchanger Sodium-hidrogen exchanger Calcium pump of the sarcolemma What does equilibrium potential mean for a given ion? How the Nernst equation can be used to analyze ion movements in case of diffusible ions? What will happen, if the membrane is not permeable for at least one ion? When is Gibbs-Donnan equilibrium present across a living cell membrane? In Fig. 22 how much Na has to pass the membrane to reach equilibrium? Which are the primary conditions for establishing and maintaining steady resting potential? What is the reason, for in one cell type (rbc) the resting potential equals 30 mv, while in an other (cardiac) cell type it equals 90 mv? What are the major factors determining the actual value of the membrane potential?

105 Questions What is the difference between a membrane receptor and an ion channel? Are there membrane receptors, which are also ion channels? How is possible, that Na ions can pass an ion channel, but K ions don t? How is possible, that K ions can pass an ion channel, but Na ions don t? Which are the most important properties of the ion channels? What is the difference between electrochemical potential and membrane potential? Which are the most important features of the local response? Special forms of local response? What are the major differences between local response and action potential? What is the reason for the very different kinetic properties of the action potentials recorded in different cell types? How could you change the shape of the action potential? What is the effect of tetrodotoxin on fast response? Why is good for us to maintain a resting potential in the cells of our body, if it costs such a substantial amount of energy (ATP)?

106 Questions What is the explanation for the fact, that postsynaptic action potentials are generated at the axon hillock? Which factors determine action potential conduction velocity in myelinated fibers? And in unmyelinated fibers? Why is conduction velocity significantly higher in myelinated than in unmyelinated fibers? How would you explain the expression that cardiac muscle is functional syncytium? Where are electric synapses (i.e. gap junctions) located in the mammalian body? Which are the major functional differences between electric and chemical synapses? What is the prime factor determining direction of impulse propagation in the three dimensional cardiac muscle? Why is the transmission of stimulus through AV node dramatically slower than in other parts of the heart? Is there fast and slow action potential propagation? What may be the reason?

107 THE END