Introduction to cardiac electrophysiology 2. Dr. Tóth András 2018

Transcription

1 Introduction to cardiac electrophysiology 2. Dr. Tóth András 2018

2 Topics Ion channels Local and action potentials Intra- and extracellular propagation of the stimulus

3 4 Ion channels

4 4.1 Basic features

5 Ion channel Protein (or protein komplex) located within the plasma membrane, acting as a pore and facilitating selective transport of ions. Following its activation an ion channel does generate transmembrane electric current. This current may flow either inward or outward direction. Failure or abnormal function of an ion channel complex (most typically as a consequence of genetic disorders) may lead to severe malfunction at the organ level. Indeed, ion channel related disorders channelopathies are being identified as the background pathomechanisms of an increasing number of diverse human diseases.

6 Basic features of ion channels 1. Motto: For normal cell function precisely controlled and fully synchronized transport of a wide variety of positive and negative ions is essential. Ion channels are present within the plasma membrane of all cell types and intracellular organelles. Inside an ion channel there is a narrow tunnel that allows only ions of certain size and/or charge to pass through (selective permeability). A given ion channel may either be gated (regulated) or nongated (non-regulated). Compared to other types of ion transport proteins ion channels have two important, distinctive features: (1) The rate of ion transport through a channel is very high (often > 10 6 ion/s). (2) The direction of the passage of ions through the tunnel is determined by their electrochemical gradient (a function of the concentration gradient and membrane potential). For the "downhill" ion transport no metabolic energy (ATP, co-transport or active transport) is needed. Since they are able to regulate influx or outflux of ions, ion channels are often direct or many times indirect (!!!) targets for a wide variety of drugs.

7 Basic features of ion channels 2. Motto: Some ion channels are fully regulated (i.e. gated channels), others are pracically nonregulated (i.e. nongated or leakage channels). Gated ion channel the opening (and sometimes closing) of these channels is triggered by the income of a specific stimulus. This specific stimulus may be: (1) change of membran potential (voltage) (voltage gated ion channels) (2) binding of a specific ligand or signalling molecule (ligand gated channels) (neurotransmitter, hormon, local hormon) to the channel protein (3) mechanical stimulus (deformation, pressure, stretch); (4) light energy (photon); Since a gated ion channel is regulated it only opens following the income of the adequate trigger signal. The closing, however, may be both controlled or spontaneous. Nongated (or leakage) ion channel is always open (or leaking) and facilitates the passage of one or more ion types in direction of their electrochemical gradient. A nongated channel is nonregulated, as well, (i.e. further to the electrochemical potential the transport flux is solely a function of the temperature.

8 Principal structure of an ion channel 1 channel domains (typically four per channel) 2 outer vestibule 3 selectivity filter 4 diameter of the selectivity filter 5 site of phosphorylation 6 plasma membrane A typical channel pore is only one-two atoms wide at the narrowest point and is selective for its specific ion (Na +, H +, K + ). Nonetheless, several ion channels are less selective and let pass more that one type of ion, usually with the same type of charge: cations (positive charges) or anions (negative charges).

9 Biological roles Ion channels are involved in a huge variety of cellular functions, many of which has critical importance: e.g. chemical signaling, transcellular transport, ph regulation, regulation of cell volume, etc. Their significance is especially prominent in the nervous system (e.g. generation and propagation of the nerve impulses, signal propagation through the synapses via transmitter-activated ion channels, etc. (Most animal venoms and toxins produced by spiders, snakes, scorpions, sea snails, bees, etc. are acting by modulating ion channel conductance and/or kinetics.) In addition, ion channels are key components of a wide variety of physiological mechanisms: e.g. excitation-contraction coupling in cardiac, skeletal and smooth muscles, epithelial transport of nutrients and ions, activation of the immun system, insulin release from the beta-cells of the pancreas, etc. Not surprisingly, many ion channels are among the most favoured targets of the pharmaceutical research.

10 Diversity There are over 300 types of functional ion channels in a living cell. Their classification can be accomplished in several different ways: by the nature of the gating mechanism, the species of ion passing through the pore, or the structure and/or localization of the channel proteins. A further heterogeneity arises in the cases when channels with different constitutive subunits give rise to a single, specific current. Absence or mutation of one or more of the contributing types of channel or regulatory subunits may result in loss of function, and potentially underlie multiple diseases.

11 Q: What is the principal difference between membrane receptor and ion channel?

12 Q: Are there membrane receptors, which also serve as ion channels?

13 4.2 Experimental techniques

14 Bert Sakmann Nobel laureate developer of the patch clamp technique

15 Principles of the patch clamp technique Needed: Electrode + pipette Special pipette solution Gigaseal (R > 1GOhm) Voltage command Current measurement

16 Steps of patch clamp recording

17 Patch clamp configurations Common investigations: Single channel current Current of a channel type or a group of channels Special pipette solution modification of the IC milieau

18 Automated patch clamp measurement

19 4.3 Principles of regulation

20 Simple, dual-state ion channel background channels spontaneously oscillate between open and closed states

21 State diagram of a komplex multistate ion channel

22 4.4 Biophysical properties

23 Determination of single channel current 1. A single channels is 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 & latency of the open state 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.

24 Integrated (macroscopic) current of two groups of voltage dependent channels following a voltage command: 1) Na + ; 2) delayed K + Sum of 300 recordings macroscopic Na + current The Na + channel spontaneously inactivates, the K + channel not

25 Determination of the mean open time Modulation of the channel can be followed in change of the mean open time

26 Current-voltage relationship characteristic for the inward and outward rectifying channels

27 Current-voltage functions of two groups of ion channels (Na + & K + ) and their combination The K + current shows linear relationship no voltage-inactivation The Na + current is nonlinear because of the voltage inactivation

28 Current-voltage relationship of two types of ion channels - determined in isolated atrial cardiomyocytes

29 Principles of activation/inactivation kinetics Working structural model of slowly inactivating Kv4 type K + channels

30 4.5 Classification of ion channels

31 Classification by gating mechanism 1 Voltage-gated ion channels Their opening (and often closing) is driven by changes in membrane potential, i.e the equilibrium state of their spontaneous oscillation (probability of the open/closed states) is voltage dependent. Major subfamilies - Voltage-gated Na + channels (Nav) - Voltage-gated K + channels (Kv) - Voltage-gated Ca 2+ channels (Cav) - Voltage-gated H + channels (Hv) - Hyperpolarization-activated channels - Transient receptor potential (TRP) channels Functions They have a crucial role in excitable tissues (neurons, muscles, glands) by ensuring excitability and stimulus-propagation. Depending on circumstances voltage-gated ion channels allow rapid and synchronized depolarization in response to voltage stimuli, but can also contribute to repolarization or hiperpolarization.

32 Classification by gating mechanism 2 Ligand-gated ion channels Also known as ionotropic receptors - open in response to specific ligand molecules binding to a channel protein: e.g. neurotransmittergated, G-protein-dependent, modulated (kovalent modification phosphorilation, etc.) channels. Binding of the ligand causes a conformation change and subsequent ion flux across the channel. These channels are typically involved in initialization of impulses (i.e. early phase of depolarization of excitable cells nerves, muscles, glands). Depending on circumstances they may initiate depolarization, repolarization or hyperpolarization, as well. Examples: cation-permeable nicotinic acetylcholine receptor, ATP-gated P2X receptors, ionotropic glutamate-gated receptors, anion-permeable GABA receptor. Sometimes 2-nd messenger-activated channels are also mentioned here, though ligand and 2-nd messenger are different categories.

33 Classification by gating mechanism 3 Other gating mechanisms Include activation/inactivation from the inside of the membrane by e.g. second messengers. Certain ions may also be considered as second messengers causing direct activation of the channel (e.g.: inward rectifying K + channels, Ca 2+ -activated K + channels, two-pore-domain K + channels Probability of the open and closed states of G-protein gated channels is modulated via binding of a G-protein, activated by activation of a receptor (e.g. muscarinic Ach receptor) Modulated channels may also be voltagegated, however, covalent modification (e.g. phosphorylation) also effectively modulates the probability of the open/closed states.

34 Classification by gating mechanism 4 Mechanosensitive ion channels (1) 1 Opening of these channels is triggered by mechanical stimulus (stretch, shear, pressure, displacement, vibration) Light-gated channels (2) These channels (e.g. channelrhodopsin) are directly opened by the action of light. Temperature gated channels Some TRP receptors (TRPV1, TRPM8) are opened either by hot (e.g. the capsaicine receptor) or cold temperatures. Cyclic nucleotide-gated channels Two families: 1) the cyclic nucleotide-gated (CNG) channels (camp and cgmp binding), and 2) the hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels. 2

35 Classification by transported ion type 1 Sodium channels Voltage-gated Na + channels (Nav) Epithelial Na + channels (ENaC) Potassium channels Voltage-gated K + channels (Kv) Ca 2+ -activated K + channels (BKCa, SK, etc.) Inward-rectifier K + channels (Kir) Two-pore-domain K + channels ( leak channels) Calcium channels Voltage-gated Ca 2+ channels (CaV)

36 Classification by transported ion type 2 Proton channels Voltage-gated H + channels (Hv), with strong ph dependence and tiny conductance Cloride channels Poorly-understood superfamily of anion channels with at least 13 members. (pl. ClC, CLIC, Bestrophin, CFTR). Nonselective for small anions. Since cloride is the most abundant transported anion, they are called cloride channels. Non-selective cation channels These channels are permeable to several cations (mainly Na +, K + and Ca 2+ ). A number of TRP (transient receptor potential) channels are also classified into this group.

37 Further ways of classifications Based on less common features, e.g number of transmembrane pores or transient behaviour of response. Two pore channels Small (2 members) cation-selective ion channel family, containing two K V -style 6-TM domains, probably forming a dimer in the membrane. Tranzient receptor potential (TRP) channels This family, containing at least 28 members has extremely diverse activation properties. Some are probably constitutively open, others show voltage, [Ca 2+ ], ph, redox state, osmolarity or stretch dependence. Their selectivity is also greatly variable, some are Ca 2+ -selective, others are less selective cation channels. 6 subfamilies: canonical (TRPC), vanilloid (TRPV), melastatin (TRPM), polycystin (TRPP), mucolipin (TRPML), and ankyrin transmembrane protein 1 (TRPA).

38 Example: superfamilies of K + channels

39 Q: How is that possible, that Na + ions may pass an ion channel, but K + ions not? How is that possible, that K + ions may pass an ion channel, but Na + ions not? Help Ion Atomic radius (Angstöm) Energy of hydration (kcal/mol ) Na K

40 4.6 Structure

41 Representative examples of ion channel superfamilies

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

43 2D model for the voltage dependent Na + channel

44 2D model for the voltage dependent Ca 2+ channel

45 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 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.

46 Voltage gated sodium channels (Nav) Contains at least 9 members. Is largely responsible for creation and propagation of action potentials. The pore-forming α subunits are very large (up to 4,000 amino acids) and consist of four homologous repeat domains (I-IV) comprising 6-TM segments (S1-S6) each. This α subunits also coassemble with auxiliary β subunits, each spanning the membrane once.

47 Voltage-gated Ca 2+ channels (Cav) The Cav family contains 10 members. They are known to play a crucial role in excitation-contraction coupling in the muscles, as well, as their fast neuronal excitation with transmitter release. The structure of the α 1 subunits have a resemblance to α subunits of the Na + channels. They are also known to coassemble with other (α 2 δ, β, γ) subunits.

48 Voltage gated potassium channels (Kv) This family contains almost 40 members, which are further divided into 12 subfamilies. These channels are known mainly for their role in repolarizing the cell membrane following action potentials. The α subunits (homologous to the sodium channels) have 6-TM segments, and also assemble as tetramers to produce a functioning channel.

49 Functional model of a voltage-gated K + channel

50 "Birth of an Idea", m x 0.90 m x 0.90 m Steel, glass, wood Sculpture by Julian Voss- Andreae based on potassium channel Photo by Dan Kvitka Sculpture commissioned and owned by Roderick MacKinnon Please use only with link to

51 4.7 Structure-function relation

52 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

53 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

54 A few cardiac ion channels

55 Cardiac action potentials and major ion currents involved

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

57 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 than in other direction Gating - oscillation between conducting and nonconducting states Regulation - gating is influenced by voltage, or binding a ligand, or covalent modification Pharmacological modulation!!! Sites of action of clinically important drogs!!!

58 5 Local & action potentials

59 5.1 Local response

60 Q: What is the difference between electrochemical and membrane potentials?

61 Local (subthreshold) response

62 Q: Which are the most important features of the local response?

63 Temporal summation

64 Spatial summation

65 Q: Special forms of local response?

66 5.2 Action potential

67 Responses in the membrane potential to increasing pulses of depolarizing current

68 Action potentials from three vertebrate cell types

69 Q: What are the major differences between local response and action potential?

70 5.3 Action potentials in the heart

71 Ion concentrations in mammalian heart

72 Fast and slow response in the heart

73 Regional variability in the morphology of the action potential in cardiac cells

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

75 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 in cardiac AP-s

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

77 The effect of tetrodotoxin on the fast response

78 Q: What is the effect of tetrodotoxin on fast response in cardiomyocytes?

79 6 Propagation of the stimulus

80 6.1 Basic principles of propagation

81 Q: Why is it feasible to maintain a large resting potential in our body cells, if it costs us a large amount of energy (ATP)?

82 Potential changes recorded (measured) by an extracellular electrode located at different distances from the current electrode

83 Maximum change in recorded (measured) membrane potential plott-ed versus distance from the point of current passage

84 Potencial changes calculated for a model RC-circuit

85 Electric model of the axon membrane

86 R m R i C Time constant determined in a membrane

87 Model of the decremental propagation (voltage divider - resistance ratio)

88 R R m i Length constant determined in the axon membrane

89 Model of conduction of the local (subthreshold) response

90 Electric model for the propagation of potential changes

91 Q: What is the explanation for the well known fact, that postsynaptic action potentials are always generated at the axon hillock?

92 Model of conduction of the AP in nonmyelinated fibers

93 Saltatory conduction of the action potential in myelinated fibers

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

95 Q: Which factors determine the conduction velocity of the action potential in myelinated fibers? And in unmyelinated fibers?

96 Q: Why is conduction velocity significantly higher in myelinated then in unmyelinated fibers?

97 6.2 AP propagation in heart

98 Q: How would you explain the common statement that cardiac muscle is functional syncytium?

99 MW 1500 Ca 2+ ph E m Structure of the electric synapse (gap junction)

100 Q: Where are electric synapses (gap junctions) located in the mammalian body?

101 Q: Which are the major functional differences between electric & chemical synapses?

102 Electric model of the cardiac cells

103 Computer simulation of impulse propagation at the microscopic level

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

105 Q: Why is the transmission of stimulus through the AV node dramatically slower than its propagation in other regions of the heart?

106 Q: Are there fast & slow velocities of action potential propagation in the heart? What may be the reason?

107 Bonus 4 U!!! The significance of the gap junction system in physiological stimulus propagation in the heart

108 Subcellular stimulus propagation

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

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

111 Impulse propagation (isochron lines) in case of physiological gap junction coupling (homogenious AP-population)

112 Impulse propagation (isochron lines) in severe uncoupling of the gap junctions (heterogenous AP-population)

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

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

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

116 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

117 THE END