Measurement of ion channel functions under in vitro conditions. Dr. Norbert Nagy Research Associate Department of Pharmacology and Pharmacotherapy

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1 Measurement of ion channel functions under in vitro conditions Dr. Norbert Nagy Research Associate Department of Pharmacology and Pharmacotherapy

2 Topics: -Electrophysiological techniques for basic research - Electrophysiological techniques for clinical diagnosis Dr. Norbert Nagy Research Fellow Department of Pharmacology and Pharmacotherapy

3 Part I. Electrophysiological techniques for basic research

Ohm s law I=U/R Measured biological signals in electrophysiology Current Produced by ion movements throughout channels or transporters The ion current changes the membrane potential Voltage The

4 Ohm s law I=U/R Measured biological signals in electrophysiology Current Produced by ion movements throughout channels or transporters The ion current changes the membrane potential Voltage The actual value of the membrane potential Can be action potential, or electrotonic potential (e.g.: EPSP, IPSP) Resistance (impedance) The cell membrane is an electric resistance The insulator property of the cell is strongly depends on the shape of the cell

5 Cardiac electrophysiology Some fields of electrophysiology Smoot muscle electrophyisiology Skeletal muscle electrophysiology Neurophysiology

6 The experiments can be performed Native cells: All ion Cells channels are presented Cell cultures: Only one type of ion channel is expressed Tissue Cardiac tissue, brain slices, aorta ring, skeletal muscle Organ Isolated heart In vivo Anaesthetized animals. Many types of species: mouse, rat, ferret, guineapig, rabbit, dog, monkey

Aim of the technique: To investigate the characteristic, function, and pharmacological properties of a special ion channel Sample: Isolated cells Patch clamp technique Rationale: The ion currents

7 Aim of the technique: To investigate the characteristic, function, and pharmacological properties of a special ion channel Sample: Isolated cells Patch clamp technique Rationale: The ion currents determine the shape of the action potential. Any disease, or pharmacological intervention which influence a macroscopic signal such as action potential, ECG etc. principally act on ion channel level. Classical patch clamp setup The patch clamp is the refinement of voltage clamp by Erwin Neher and Bert Sakmann. They received Nobel prize in physiology and medicine in Usually we use isolated cells Two solutions: the extracellular solution is the solution in the bath; the intracellular solution in the pipette solution (easy to modify the i.c. content) Theoretically, whichever ion current can be investigate by using appropriate voltage protocol and inhibitors

8 Patch clamp variations Whole cell: Recording of currents through multiple channels at same time. Inside-out: A patch of membrane is attached to the pipette, detached from the rest of the cell. The cytoplasmic surface is exposed to the external area. The advantage is the fast change of the intracellular milieu. Outside-out: After the whole-cell configuration is formed, the electrode is slowly withdrawn from the cell, allowing a bulb of membrane to bleb out from the cell. When the electrode is pulled far enough away, this bleb will detach from the cell and reform as a convex membrane on the end of the electrode. Examination of ion channels isolated from the cell, fast change of extracel. solutions. Perforated patch: Suction is not used to rupture the patch membrane. Instead, the electrode solution contains small amounts of an antifungal or antibiotic agent, such as amphotericin, which diffuses into the membrane patch and forms small pores in the membrane, providing electrical access to the cell interior. It preserves the intracel. milieu.

11 Aim of the technique: To measure action potentials Sample: Isolated cardiac cells Sharp microelectrode studies Rationale: Sharp microelectrodes mean electrodes with high impedance, which can be inserted inside the cell. The high impedance electrodes limit the ion exchange between the intracellular space and the solution in the electrode, causing minimal disturbance in the cell. Hodgkin & Huxley, Nobel prize in 1952

Extracellular measurements Aim of the technique: To measure various types of extracellular potentials such as ECG (electrocardiography), EEG (electroencephalography), ERG (electroretinography),

12 Extracellular measurements Aim of the technique: To measure various types of extracellular potentials such as ECG (electrocardiography), EEG (electroencephalography), ERG (electroretinography), electromyography (EMG), extracellular field potentials. Sample: Can be measured from the surface of the body, isolated cells, or brain slices Rationale: The excitable tissues constantly exchange ions with the extracellular fluid. Propagating waves of ion movement can generate small changes in the voltage across the tissue.

13 20. The measurement of field potential is often used in neurophysiology in vivo. The long term potentiation (LTP) the putative electrophysiological basis of the memory and learning was discovered by using this technique. Other type of field potential measurement is the ECG recording from isolated heart. The applied system is the Langendorff apparatus, where the heart is rhythmically beating when constantly perfused by physiological solution.

Transepithelial electrical measurements Aim of the technique: A specialized technique of extracellular measurements providing information on net ion transport across polarized epithelial cell

14 Transepithelial electrical measurements Aim of the technique: A specialized technique of extracellular measurements providing information on net ion transport across polarized epithelial cell monolayers. Sample: Epithelial cells from skin, gastrointestinal tract, or from airway samples Rationale: Epithelial cells are polarized having a basolateral and an apical side. Transepithelial resistance or transepithelial voltage can be measured to characterize the physiology and pharmacology of ion transport.

Cellular impedance measurement Aim of the technique: To measure electrical impedance of the cell Sample: Cell culture Rationale: Cells are grown over electrodes located in the bottom of the cell

15 Cellular impedance measurement Aim of the technique: To measure electrical impedance of the cell Sample: Cell culture Rationale: Cells are grown over electrodes located in the bottom of the cell culture dish. Impedance is increasing with increasing coverage until a confluent layer of the cell is established. Any changes to the cell shape or to the current pathways changes the measured impedance. Thus cellular functions can be monitored: migration, chemotaxis, contraction

16 Fluorescent optical technique Aim of the technique: Monitoring the changes of the intracellular Ca, Na, voltage, ph by applying a specific fluorescent dye. Often combined with action potential or current measurement. Sample: Isolated cells, isolated tissue, or whole heart on Langendorff apparatus Rationale: The actual levels of Ca, Na, H+ have crucial role in arrhythmogenesis. Many pathological conditions, drugs are able to alter this factors. The change of these factors can influence several ion currents. Cell chamber Excitation (488 nm) Enzymatic dissociation ΔV LAMP Free Ca 2+ Fluo-4 +AM Fluo-4 Acetomethyl-ester + - Electrodes Emission (506 nm) P/V conv. Photon multiplier

17 Part II. Electrophysiological techniques for clinical diagnosis

Electroencephalography (EEG) Aim of the technique: Monitoring the electrical activity of the brain. Electrodes are placed in the scalp, the technique is non-invasive.

It is used to diagnose epilepsy, sleep disorders, coma, encephalopathy, brain death.

18 Electroencephalography (EEG) Aim of the technique: Monitoring the electrical activity of the brain. Electrodes are placed in the scalp, the technique is non-invasive. It measures field potentials generated by ion movements within the neurons. Rationale: Several diseases changes the normal ionic activity of a brain. It is used to diagnose epilepsy, sleep disorders, coma, encephalopathy, brain death. Special variation of the EEG is the magnetoencephalography (MEG) which measures the magnetic field generated by the ionic currents of the neurons. Non-invasive. The electrocorticography (ECoG) is similar to the EEG but the electrodes are place directly to the surface of the brain. Better resolution, and it avoids from the distortion of the skull. Invasive method

It measures field potentials generated by ion movements within the heart.

19 Electrocardiography (ECG) Aim of the technique: Monitoring the electrical activity of the heart. Electrodes are placed in the skin, the technique is non-invasive. It measures field potentials generated by ion movements within the heart. Rationale: The cardiac diseases influences the ion currents and action potentials in the heart. Since the ECG is the summation of the action potentials of the heart ECG also changes during pathophysiological condition. Wide use in cardiology. William Einthoven, 1901

Electromyography (EMG) Aim of the technique: A diagnostic procedure to assess the health of muscles and the nerve cells that control them (motor neurons).

20 Electromyography (EMG) Aim of the technique: A diagnostic procedure to assess the health of muscles and the nerve cells that control them (motor neurons). Rationale: Motor neurons transmit electrical signals that cause muscles to contract. An EMG translates these signals into graphs, sounds or numerical values that a specialist interprets.. During a needle EMG, a needle electrode inserted directly into a muscle records the electrical activity in that muscle. A nerve conduction study, another part of an EMG, uses surface electrodes to measure the speed and strength of signals traveling between two or more points. EMG results can reveal nerve dysfunction, muscle dysfunction or problems with nerveto-muscle signal transmission.

21 Thank you for your attention!

Voltage clamp and patch-clamp techniques

Voltage clamp and patch-clamp techniques Dr. Nilofar Khan Objectives Historical background Voltage Clamp Theory Variations of voltage clamp Patch-clamp Principal Patch-clamp configurations Applications