Introduction to electrophysiology 1. Dr. Tóth András

Transcription

1 Introduction to electrophysiology 1. 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 Time required for diffusion as a function of diffusion distance

8 4 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

9 5 Osmotic motion across a semipermeable membrane

10 6 Mechanism of facilitated diffusion

11 7 Principle of transport of ions across ion channels

12 8 The principle of function of the Na + /K + ATPase

13 9 Secondary active transport processes

14 1 0 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

15 Q: What are the principal differences between the following iontransporters? 1. Sodium-calcium exchanger 2. Sodium-hidrogen exchanger 3. Calcium pump of the sarcolemma

16 2. Ionic equilibrium

17 1 1 µ = µ o + RT ln C + zfe µ = RT ln [ ] X + [ ] X + A B + zf ( E E ) A B Electrochemical potential (difference)

18 1 2 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

19 Q: What does equilibrium potential mean for a given ion???

20 How the Nernst equation can be used to analyze ion movements in case of diffusible ions???

21 1 3 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 use of the Nernst equation 1.

22 1 4 A B 0.1 M K 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 use of the Nernst equation 2.

23 1 5 A B 0.1 M K M K + A B 1 M HCO M HCO 3 - E A E B = 60 mv At 60 mv the K + is in electrochemical equilibrium across the membran No electric force 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.

24 What will happen, if the membrane is NOT permeable for at least one ion???

25 1 6 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 Donnan equilibrium is established

26 1 7 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

27 1 8 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

28 Q: When is Gibbs-Donnan equilibrium present across a living cell membrane?

29 3. Resting potential

30 1 9 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

31 2 0 Na + A B 0.1 M NaCl M NaCl In case of electrochemical equilibrium E A E B = - 60 mv The concentration battery

32 Q: In Fig. 22 how much Na + has to pass the membrane to reach equilibrium?

33 Living cells can well be modelled as multi-ion concentration batteries

34 2 1 Measured intra- and extracellular ionconcentrations

35 2 2 Cl - Na + E E 1) Na + IC(mM) 12 EC(mM) 145 E eq + 65mV cc cc K ,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

36 = = = = = 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

37 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

38 2 5 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

39 2 6 C Major factors affecting resting potential

40 Q: Which are the primary conditions for establishing and maintaining steady resting potential?

41 A: 1. Separated ion compartments 2. Selective permeability of the membrane 3. Ionic concentration gradients 4. Energy supply and ion transporters

42 Cardiac cells!!!

43 2 7 Also in cardiac cells the resting potential is supposed to be [K + ] dependent

44 2 8 In cardiac cells the resting potential is, indeed, primarily [K + ] dependent

45 Q: 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?

46 Q: What are the major factors determining the actual value of membrane potential?

47 A: 1. Concentration gradients of the monovalent cations 2. Selective permeability of the membrane for cations 3. Concentration of non-permeable intracellular anions

48 4. Ion channels

49 Q: What is the difference between a membrane receptor and an ion channel?

50 Q: Are there membrane receptors, which are also ion channels?

51 4.1 Experimental techniques

52 Bert Sakmann Nobel prize for the patch clamp technique

53 2 9 Principles of the patch clamp technique Electrode Special pipette solution Gigaseal (R > 1GOhm) Voltage command Current measurement

54 3 0 Patch clamp configurations Investigations: Single channel current Current of a channel type or a group of channels Special pipette solution modification of the intracellular milieau

55 3 1 Steps of patch clamp recording

56 3 2 Determination of single channel current 1. The channels are in either open or closed state. 2. The open state of a channel is short lived compared to the time course of the macroscopic current. 3. The length and latency of the open state of the also channel is highly variable, it may happen that the channel does not open, at all. 4. The probability function of channel opening correlates with the shape of the macroscopic current curve. 5. A channel may open several times during a single cycle if there is no inactivation. A single ion channel has only two functional states: open or closed

57 3 3 Integrated (macroscopic) current of two groups voltage dependent channels, 1) Na + and 2) delayed K + following a voltage command Of the two channel types only the Na + channel shows spontaneous inactivation

58 3 4 Determination of the mean open time

59 3 5 Current-voltage relationship of the inward and outward rectifying channels

60 3 6 Current-voltage functions of two channel groups (Na + and K + ) The K + current shows linear relationship no voltage-inactivation The Na + current is nonlinear because of the voltage inactivation

61 3 7 Current-voltage relationship of two types of ion channels - determined in cardiac atrial cardiomyocytes

62 3 8 Activation/inactivation kinetics Working structural model of slowly inactivating Kv4 type K + channels

63 4.2 Principles of regulation

64 3 9 State diagram of a simple, dual-state ion channel

65 4 0 State diagram of a multiple-state ion channel

66 4 1 Basic regulatory mechanisms of ion channels

67 4 2 Background channels spontaneously oscillate between open and closed states

68 4 3 Voltage-gated channels also oscillate between the two states, but voltage shifts the equilibrium

69 4 4 The open state of neurotransmitter-gated channels is altered by the binding of a neurotransmitter to the channel (e.g. nicotinic receptor)

70 4 5 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)

71 4 6 Modulated channels may be voltage-gated, but the ability of voltage to open the channel may be influenced by covalent modification (e.g. phosphorylation)

72 4.3 Structure

73 4 7 Ion channel superfamilies

74 4 8 2D model for the voltage dependent Na + channel

75 4 9 2D model for the voltage dependent Ca 2+ channel

76 5 0 Superfamilies of K + channels

77 Q: How is that possible, that Na + ions can pass an ion channel, but K + ions cannot?

78 Q: How is that possible, that K + ions can pass an ion channel, but Na + ions cannot?

79 4.4 Structure-function relation

80 5 1 S 4 helices are the voltage-sensors of voltage-gated channels amino acid homology is extensive

81 5 2 Suggested models of activation mechanism of voltage dependent ion channels a Conventional gating model: The voltage sensor (S4 segment) moves across the plasma membrane b Novel paddle model: The voltage sensor S4 and the S3 segments compose a hairpin outside of the channel. Activation of the channels is induced by the rotation of these paddles. c The transporter model: This new model is based on the assumption that the charges present on the voltage sensor (S4) do not move accross the membrane, insteed they rotate around their longitudinal axes, shifting the gating charges from extacellular to intracellular position.

82 5 3 Model of the function of the S 4 helix as voltage sensor A total of 6 charges should relocate in the membrane to open the channel

83 5 4 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

84 5 5 Functional model of a K + channel

85

86

87 5 7 Cardiac ion channels

88 5 8 Cardiac action potentials and major ion currents involved

89 Q: Which are the most important properties of the ion channels?

90 A: Integral membrane protein Aqueous pore through the membrane Selective permeability to one or several small ions Single channel conductance to ion flow Rectification current passes more easily in one direction than another Gating oscillate between conducting and nonconducting states Regulation gating is influenced by voltage, binding a ligand, or covalent modification Pharmacological targets - sites of action of clinically important drogs!!!

91 To be continued!

92 Introduction to electrophysiology 2. Dr. Tóth András

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

94 5. Local and action potentials

95 5.1 Local response

96 Q: What is the difference between electrochemical potential and membrane potential?

97 4 6 Local (subthreshold) response

98 3 1 Q: Which are the most important features of the local response?

99 4 7 Temporal summation

100 4 8 Spatial summation

101 Q: Special forms of local response?

102 5.2 Action potential

103 4 9 Responses in the membrane potential to increasing pulses of depolarizing current

104 5 0 Action potentials from three vertebrate cell types

105 3 1 Q: What are the major differences between local response and action potential?

106 5.3 Action potentials in the heart

107 5 1 Ion concentrations in mammalian heart

108 5 2 Fast and slow response in the heart

109 5 3 Regional variations in the shape of the action potential of the heart cells

110 Q: What is the reason for the very different kinetic properties of the action potentials recorded in different cell types?

111 5 4 Ion currents! Fast sodium Funny Delayed rectifier Calcium I L T+L 0 0 Tranzient outward Background Sodium Inward rectifier Explanation of the kinetic differences

112 Q: How could you change the shape of the action potential?

113 5 5 The effect of tetrodotoxin on the fast response

114 Q: What is the effect of tetrodotoxin on fast response?

115 6. Propagation of the stimulus

116 6.1 Basic principles of propagation

117 Q: Why is that good for us to maintain a resting potential in the cells of our body, if it costs such a substantial amount of energy (ATP)?

118 5 6 Potential changes recorded by an extracellular electrode located at different distances from the current electrode

119 5 7 Maximum change in recorded membrane potential plotted versus distance from the point of current passage

120 5 8 Potencial changes in a model RC-circuit

121 5 9 Electric model of the axon membrane

122 6 0 R m R i C Time constant determined in a membrane

123 6 1 Model of the decremental propagation (voltage divider - resistance ratio)

124 6 2 R R m i Length constant determined in the axon membrane

125 6 3 Model of conduction of the local (subthreshold) response

126 6 4 Electric model for the propagation of potential changes

127 Q: What is the explanation for the fact, that postsynaptic action potentials are generated at the axon hillock?

128 6 5 Model of conduction of the AP in nonmyelinated fibers

129 6 6 Saltatory conduction of the action potential in myelinated fibers

130 6 7 Conduction velocity of the action potential determined in unmyelinated and myelinated fibers

131 Q: Which factors determine action potential conduction velocity in myelinated fibers? And in unmyelinated fibers?

132 Q: Why is conduction velocity significantly higher in myelinated than in unmyelinated fibers?

133 6.2 AP propagation in heart

134 Q: How would you explain the expression that cardiac muscle is a functional syncytium?

135 6 8 MW < 1500 Ca 2+ ph E m Structure of the electric synapse (gap junction)

136 Q: Where are the electric synapses (i.e. gap junctions) located in the mammalian body?

137 Q: Which are the major functional differences between electric and chemical synapses?

138 6 9 Electric model of the cardiac cells

139 7 0 Computer simulation of impulse propagation at the microscopic level

140 Q: What is the prime factor determining direction of impulse propagation in the three dimensional cardiac muscle?

141 Q: Why is the transmission of stimulus through AV node dramatically slower than in other parts of the heart?

142 Q: Is there fast and slow action potential propagation? What may be the reason?

143 Bonus 4 U!!! The significance of gap junctions in normal stimulus propagation in the heart

144 7 1 Subcellular stimulus propagation

145 7 2 Differences in delays of intra- and intercellular activation single cell wide network

146 7 3 Differences in delays of intra- and intercellular activation multi-cell wide network

147 7 4 Impulse propagation (isochron lines) in case of normal gap junction coupling (homogenious AP-population)

148 7 5 Impulse propagation (isochron lines) in severe gap junction uncoupling (heterogenous AP-population)

149 7 6 In severe gap junction uncoupling propagation velocity may decrease 2 orders of magnitude (!!!) (from 36.7 cm/s to 0.31 cm/s)

150 7 7 In case of normal gap junction coupling isochron lines are relatively regularly placed, AP-population is homogenous

151 7 8 In case of critical gap junction uncoupling action potentials form clusters with significant delays

152 7 9 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

153 THE END