Introduction to electrophysiology 1. Department of Pharmacology and Pharmacotherapy University of Szeged, Faculty of Medicine 12.09.2017

Topics Biophysical basis Resting membrane potential Basis of impulse propagation Electrotonic potentials Basic properties of ion channels Action potentials

Potential = Driving force (B1-A1)<(B2-A2)<(B3-A3) Potentials in general A3 B3 A2 B2 A1 B1 Potential = Driving force (B1-A1)<(B2-A2)<(B3-A3) A potential difference makes a possibility for a ball to roll down (or a charge is able to work in a given system). If there is no potential difference there is no (charge) movement The larger the potential difference the faster the movement of the ball (or the charge) The potential difference does not necessarily mean that a movement will happen. Other factors could be important!

Chemical potential Part ‘A’ Part ‘B’ The number of glucose molecules in part A is larger than in part B The membrane is permeable for glucose This system is not in equilibrium : a concentration gradient exists from A to B Chemical pot. The chemical potential is the driving force for the simple diffusion of the uncharged molecules. Essentially it is identical with the concentration difference The diffusion is effective only on short distances Eg.: O2-CO2 gas exchange in the lung 10 Part ‘A’ Part ‘B’ The number of glucose molecules in part A is equal with the number of part B The system is in dynamic equilibrium No concentration gradient, no chemical potential 5 5

The membrane is permeable only for K-ions 20 Cl- 0 K+ 0 Cl- K+ Cl- K+ Cl- K+ Cl- K+ Cl- Chemical (K+) K+ Cl- K+ Cl- K+ Cl- K+ Cl- K+ Cl- K+ Cl- K+ Cl- K+ Cl- The membrane is permeable only for K-ions K+ Cl- K+ Cl- Chemical (Cl-) K+ Cl- K+ Cl- K+ Cl- K+ Cl- K+ Cl- K+ Cl- K+ Cl- 20 Cl- 0 Cl- 14 K+ 6 K+ Chemic. - 20 mV Electrical - +

- + - + Δ µ= RTlg [cA] [cB] + zF(EA-EB) 0= RTlg Chemic. In equilibrium the chemical potential and electrical potential is equal and opposite THERE IS EQUILIBRIUM BUT The concentrations are not equal There is voltage difference between the two parts Cl- Cl- K+ K+ - + Cl- K+ Cl- K+ Cl- K+ - + Electrical Δ µ= RTlg [cA] [cB] + zF(EA-EB) 0= RTlg Chemical Electrical Electrochemical potential It is zero in equilibrium

- + - + [cA] EA-EB= -RT lg [cB] zF [XA] EX= -60mV lg [XB] Chemic. THERE IS EQUILIBRIUM BUT The concentrations are not equal Cl- Cl- K+ K+ - + There is voltage difference between the two parts Cl- K+ Cl- K+ Cl- In equilibrium the chemical potential and electrical potential is equal and opposite K+ - + Electrical [cA] EA-EB= -RT lg Equation of chemical potential [cB] zF [XA] Nernst-potential, For monovalent cations EX= -60mV lg [XB]

[XA] EX= -60mV lg [XB] [5] EK= -60mV lg [133] EK= - 85 mV The extracellular side of heart cells there are 5 mM, in the intracellular side there are 133 mM K+ EX= -60mV lg [XB] [5] EK= -60mV lg [133] Since the resting membrane potential is about – 80 mV, we can suggest the resting membrane potential is mainly defined by the concentration difference of K ions EK= - 85 mV The Nernst potential for any given ionic species is the membrane potential at which the ionic species is in equilibrium; i.e., there is no net movement of the ion across the membrane. Therefore, the Nernst potential for an ion is referred to as the equilibrium potential for that ion.

The Donnan-potential in all cases: ~ - 17 mV Donnan-equilibrium K+ K+ K+ K+ [K]A x [Cl]A = [K]B x [Cl]B K+ K+ K+ K+ K+ K+ K+ K+ K+ K+ K+ K+ There is no equilibrium for the pressure! Osmotic potential will develop A- A- A- A- Cl- Cl- Cl- Cl- A- A- A- A- Cl- Cl- Cl- Cl- The Donnan-potential in all cases: ~ - 17 mV K+ > K+ Cl- < Cl- Negative Positive

The Donnan-potential is the membrane potential of the death cell The Donnan-potential does not explain the development of the resting membrane potential 17 mV versus – 80 mV In a living cell, the continuous activity of the Na/K pump prevent the development of the Donnan-potential The Donnan-potential is the membrane potential of the death cell

Development of the resting membrane potential The membrane is an electric barrier and the ion transport through the ion channels is regulated The unequal ion distribution causes potential difference. The inner side is negative In macroscopic level both part is electroneutral Factors maintaining the resting membrane potential Unequal ion distribution Selective permeability of the membrane Operation of the Na/K pump Donnan-potential All living cells has any resting membrane potential

- + [X+]A [X+]B [X+]A [X+]B A&B: Unequal ion distribution + selective permeability Unequal ion distribution + semipermeable membrane = leads to development of equilibrium potential - In the resting membrane potential the K ion distribution is important EX=-RT lg [X+]A [X+]B zF ~ -90 mV 135 mM KCl 5 mM KCl [X+]A EX=-60mV lg - + [X+]B R: standard gas constant: 8,31 J/mol/K T: absolute temperature (K) Z: valency of the ion (K+ = 1) F: Faraday constant 96500 C/mol E(K)= -90 mV E(Na) = +70 mV E(Ca) = +120 mV

C) Na/K pump Electrogenic operation: 3 Na+ out, 2 K+ in (net 1 + charge out, moves the membrane potential to the negative direction Contributes directly to the resting membrane potential only with 10-20 mV However it supports unequal distribution for Na and K ions Its full inhibition causes death however certain drugs inhibit this pump (in heart failure)

D) Donnan-potential It is developed by the large, negatively charged, impermeable proteins in the cell The Donnan-potential is only about 15-17 mV, therefore it has marginal role in the setting of the resting membrane potential The Donnan-potential is the potential of the death cells

In glial cells and in Purkinje cells of the heart the resting membrane potential is nearly equal with K-equilibrium potential = the cell in rest is permeable only to K-ions In neurons the resting membrane potential is a bit positive than the K-equilibrium potential = The cell has resting conductance for Na ions In the sinus-node and in the AV-node there is no stable resting potential Cell type Rest.memb.pot. Hair cell, cochlea -15 to -40mV Skeletal muscle −95 mV Smooth muscle –60 mV Astroglia –80 to –90 mV Neuron –60 to –70 mV Erythrocita –8.4 mV Chondorcita -8 mV Aorta smooth muscle cell -45 mV Cardiac ventricular cell -80 mV Photoreceptor cell –40 mV

What is the purpose of the resting membrane potential? In neurons the basis of the impulse propagation In smooth muscle and skeletal muscle cells the action potential generation and contraction In heart muscle cells the action potential generation, contraction and impulse propagation In non-excitable cells it is important in the secondary active transport systems (e.g.: in Na-glucose symporter) 40% of the energy consumption of the cells is expended to the maintenance of the unequal ion distribution Without resting membrane potential there is no heart beating, or neuron function, or muscle contraction…

Change of the resting membrane potential External stimuli (light, sound, smell, mechanic, termic…etc) The actual value of resting mebrane potential is changing… Ligands (neurotransmitters, signal molecules, ions, stb), voltage change Local potential change happens (electrotonic potential) Becomes more negative (hyperpolarization) It is abolished Becomes more positive (depolarization) Propagates, evokes new action potentials Action potential developed

X Electrotonic potential changes + + + + + + + + + + + + + + + + + + + Ligand-activated Na channels + + + + + + + + + + + + + + + + + + + + + + + + + + + + - - - - - - - - - - - - - - - - - - - - - - - - - Axon Ligand-activated Na channels Negative charges - X - + + Axon + + + + + + + + Positive charges + + + + + + + + + + + + + + + + + +

The potential change declines in the function of the distance Reasons: Na-channel Axon + - The potential change declines in the function of the distance Reasons: 1.: The potential change cannot activate new ion channels 2.: The Na/K pump is restoring the initial Na and K values Time constant: The time required during the potential decreases to the 37% percent of the initial potential Length constant: The distance required during the potential decreases to the 37% of the initial potential

The larger the stimulus the larger the evoked potential The larger the stimulus the larger the evoked potential. It has no treshold potential Depolarization or hyperpolarization The potentials can be strengthen or weaken each other (spatial and temporal summation) Cannot be inhibited There is no refractory period When the amplitude reaches the threshold it evokes action potential Voltage-dependent fast Na channels or Ca channels are not involved

It has important physiological relevance Excitatory postsynaptic potential (EPSP) Inhibitory postsynaptic potential (IPSP) In the retina caused by light stimulus Its function is the signal propagation, cell-cell communication but it is effective only in small distance In larger distance action potentials required

Action potentials After meeting with appropriate stimuli, the membrane of the excitable cells respond with a special potential change which is called action potential The morphology of the action potentials is changing in the different tissues. Furthermore a given tissue has different types of action potentials (e.g.: heart tissue) Function: Neurons: Impulse propagation Skeletal and smooth muscle: development of contraction Heart muscle: Impulse propagation and contraction

The action potentials have different morphology even within the given tissue It means that the underlying ion channels are largely different in different cell types It has important physiological consequences!

Sections of the action potentials Depolarization: Initial phase of the AP. All APs has depolarization. The underlying ion current mostly the Na+ current (In sinus and AV node the Ca2+ current) Repolarization: The AP declines to the value of the resting potential. All AP has repolarization. The underlying ion current is K-current Plateau phase: Exist in heart muscle and smooth muscle. The underlying current is the Ca2+ current, it has important role in the contraction Dep. Rep. Plat. Resting membrane potential: Nearly isoelectric section between two action potentials. The majority of APs have resting potential except of sinus node and AV node, heart Purkinje cells

Ion conductances during the action potential The membrane permeability is not constant The ion channels are able to open and close during an action potential

Goldmann-Hodgkin-Katz equation The Nernst-equation provides the equilibrium potential of an ion in the presence of given external and internal concentrations, when the membrane permeability is constant In the presence of multiple ions and changing permeability we use the GHK-equation Em=-RT lg F PK[K+]out+PNa[Na+]out+PCl[Cl-]in PK[K+]in+PNa[Na+]in+PCl[Cl-]out

Nernst-equation vs. GHK-equation There is only 1 diffusible ion in the system More diffusible ion can be in the system Does not take into account the changing permeability of the channels Counts with the changing permeability It is cannot applicable to calculate the actual value of the action potential The actual value of the membrane potential during an AP can be calculated in any time

Types of action potentials I. Neuronal action potential Resting potential: ~ 70 mV Duration: ~ 1-2 ms Amplitude: 100-110 mV Overshoot: 30-35 mV Afterpotential: hyperpolarization Ion channels: fast, voltage dependent Na channel, slow voltage dependent K channel The refractory period is very short

Types of action potentials II. Skeletal muscle action potential Resting potential: ~ 90 mV Duration: ~ 2-3 ms Amplitude: 110-120 mV Overshoot: 20-25 mV Afterpotential: Depolarization Ion channels: fast, voltage dependent Na channel, slow voltage dependent K channel

Types of action potentials III. Smooth muscle action potential The morphology is very variable Resting potential: ~ -50 és -60 mV Duration : ~ 50-300 ms Amplitude: 60-70 mV Overshot: Marginal Afterpotential: (Depolarization) Ion channels: Mainly slow Ca2+, slow voltage dependent K-channels, oscillating background K-conductance (slow wave)

Types of action potentials IV. Heart muscle slow action potentials SINUS-NODE ACTION POTENTIAL Amplitude Slow diastolic depolarization Maximal diastolic potential Sinus node, AV-node Resting membrane potential: No (most negative point kb -50 mV) Duration: 250-350 ms Amplitude: 50-60 mV Overshoot: Marginal Afterpotential: No Ion channels: Depolarization by Ca2+ current, there is no voltage dependent Na current

Types of action potential IV. Heart muscle action potential Atrial, ventricular, Purkinje cells Resting potential: -70 -90 mV Duration: 250-350 ms Amplitude: 105-130 mV Phases: 0. Depolarization (INa) Early repolarization (Ito) Plateau (ICaL) Delayed repolarization (IKr, IKs, IK1) Resting potential (IK1) Overshoot: 20-30 mV Afterpotential: No Ion channels: Fast voltage dependent Na-current, slow Ca2+ current, slow K+-current

Electrotonic potentials Action potentials Stimuli larger than threshold evoke action potential Depolarization or hyperpolarization depending on the stimulus. No threshold The amplitude is a all-or-nothing phenomenon. If it is evoked, always has the same magnitude. It depends on the properties of the ion channels (Na, Ca) The amplitude is proportional with the stimulus The amplitude is few milivolts. < 20 mV The magnitude of the amplitude depends on the tissue type, but mainly larger than 100 mV It is evoked by the opening of ligand-activated, mechanosensitive, thermosensitive ion channels. No voltage dependent Na channels are involved It is evoked by voltage-gated Na and Ca channels No refractory period It has absolute and relative refractory period Spatial and temporal summation Summation is not possible because of the refractory period and the all-or-nothing phenomenon Propagates with decrement Propagates without decrement