Medical knowledge | Higher education » Dr. Benyó Zoltán - A sejtfiziológia alapjai, a sejtmembrán szerkezete és funkciói

A SEJTFIZIOLÓGIA ALAPJAI BASICS OF CELL PHYSIOLOGY A SEJTMEMBRÁN SZERKEZETE ÉS FUNKCIÓI THE STRUCTURE AND FUNCTIONS OF CELL MEMBRANES Dr. Benyó Zoltán A SEJT ÁLTALÁNOS STRUKTÚRÁJA ULTRASTRUCTURE OF THE COMMON CELL ORGANELLES 1 HOL VANNAK MEMBRÁNOK A EUKARIÓTA SEJTEKBEN? WHERE ARE MEMBRANES IN THE EUKARIOTIC CELLS? A FOSZFOLIPID MOLEKULA ÉS A FOSZFOLIPID KETTİSRÉTEG THE STRUCTURE OF A PHOSPHOLIPID MOLECULE AND PHOSPHOLIPID BILAYER 2 LIPIDEK A SEJTMEMBRÁNBAN I. LIPIDS OF THE CELL MEMBRANE I. LIPIDEK A SEJTMEMBRÁNBAN II. LIPIDS OF THE CELL MEMBRANE II. 3 LIPIDEK A SEJTMEMBRÁNBAN III. LIPIDS OF THE CELL MEMBRANE III. A LIPIDEK LEHETSÉGES MOZGÁSFORMÁI A SEJTMEMBRÁNBAN TYPES OF LIPID MOVEMENTS WITHIN THE MEMBRANES 4 A KÜLÖNBÖZİ LIPIDEK ASZIMETRIKUS ELHELYEZKEDÉSE A MEMBRÁNBAN LIPID ASYMMETRY For many membranes, lipid composition of the two monolayers is different. This asymmetry is generated by phospholipid translocators & maintained

because of difficulty of flip-flop. FOSZFOLIPÁZOK METABOLOSM OF MEMBRANE LIPIDS: PHOSPHOLIPASES 5 A FOSZFOLIPIDEK BIOLÓGIAILAG AKTÍV MEDIÁTOROK ELİANYAGAI PHOSPHOLIPIDS ARE PRECURSORS OF BIOLOGICALLY ACTIVE MEDIATORS – PLA2: prostanoids leukotrienes platelet activating factor (PAF) – PLC: diacylglycerol (DAG) inositol 1,4,5-triphosphate (InsP3) – PLD: lysophosphatidic acid (LPA) sphinogosine-1-phosphate (S1P) MEMBRÁN PROTEINEK MEMBRANE PROTEINS Proteins confer specific functions to the membranes Distinctive features of different kinds of membranes Types of membrane proteins: 1. Integral (intrinsic) membrane proteins 2. Peripheral (extrinsic) membrane proteins 6 A membránfehérjék elhelyezkedése Different modes of association Peripheral proteins were once thought to interact only with integral proteins but are now thought to interact as much with lipids SZÉNHIDRÁTOK A MEMBRÁNBAN MEMBRANE CARDOHYDRATES 7 A FOLYADÉK-MOZAIK MEMBRÁN MODELL MEMBRANE

STRUCTURE LIPIDS + CARBOHYDRATE + PROTEINS Current model: Fluid-Mosaic Model The lipid bilayer is the core of the model which is present in fluid state. Individual lipid molecules move laterally. Proteins penetrate the entire bilayer and exist as a mosaic. A PLAZMAMEMBRÁN-LIPIDEK FUNKCIÓI FUNCTIONS OF PLASMA MEMBRANE-LIPIDS organelles 3 Precursors of biologically active mediators (prostanoids, leukotrienes, PAF, DAG, InsP3, LPA, S1P) 8 A PLAZMA MEMBRÁN FEHÉRJÉK FUNKCIÓI: FUNCTIONS OF PLASMA MEMBRANE PROTEINS: -Transzport -Enzimatikus aktivitás -Szignál transzdukció -Intercelluláris kapcsolódás -Sejt-sejt közötti felismerés -Kapcsolat a citoszkeletonnal és az extracelluláris matrixszal Membrántranszport Membrane-transport 9 VEZIKULÁRIS MEMBRÁNTRANSZPORT TRANSPORT ACROSS BUT NOT THROUGH THE MEMBRANES Endocytosis - phagocytosis - pinocytosis -receptor-mediated endocytosis Exocytosis Fusion of membrane vesicles ENDOCITÓZIS / ENDOCYTOSIS pinocytosis 10

RECEPTOR-MEDIÁLTA ENDOCITÓZIS RECEPTOR-MEDIATED ENDOCYTOSIS EXOCITÓZIS / EXOCYTOSIS 11 HÍZÓSEJT HISZTAMINT SZABADÍT FEL HISTAMINE RELEASE FROM MAST CELL MOLEKULÁK MEMBRÁNON KERESZTÜL TÖRTÉNİ SZÁLLÍTÁSA Protein-mediated transport Active transport Cotransport―indirect/ secondary active transport (direct/primary) 12 I. DIFFÚZIÓ / DIFFUSION Az oldott anyag részecskéinek random hımozgása következtében jön létre. Random movement of solute due to Brownian motion. A diffúzióhoz szükséges idı a távolság függvényében Time Required for Diffusion to Occur over Various Diffusion Distances 13 A tiszta foszfolipid kettısréteg áteresztıképessége szelektív A pure phospholipid bilayer acts as a selectively permeable barrier Fick elsı diffúziós törvénye J = -DmA (∆ ∆c/∆ ∆x) J: Anyagáramlás egységnyi idı alatt Dm: Molekuláris diffúziós állandó A: Membránfelület ∆c: Koncentráció-különbség ∆x:

Membrán-vastagság (∆ ∆c/∆ ∆x: Koncentráció-grádiens) 14 Fick’s First Law of Diffusion J = -DmA (∆ ∆c/∆ ∆x) J: Net rate of diffusion in moles or grams per unit time Dm: Coefficient of molecular diffusion A: Area of the membrane ∆c: Concentration difference across the membrane ∆x: Thickness of the membrane (∆ ∆c/∆ ∆x: Concentration gradient) A Dm-et befolyásoló tényezık Factors influencing Dm 15 MEMBRÁN-FEHÉRJÉK ÁLTAL KÖZVETÍTETT TRANSZPORTFOLYAMATOK 1. Facilitált diffúzió: nem igényel energiát 2. Aktív transzport: energiát igényel - direkt v. primer aktív transzport (pumpa) - indirekt v. másodlagos aktív transzport (kotranszport) PROTEIN-MEDIATED MEMBRANE TRANSPORT 1. Facilitated transport (facilitated diffusion) is NOT linked to metabolic energy. 2. Active transport must be linked to energy metabolism in some way. - direct/primary active transport - cotransport (indirect/secondary active transport) 16 II.

FACILITÁLT DIFFÚZIÓ II. FACILITATED DIFFUSION 1. Rapid transport 2. Chemical specificity 3. Competition between structurally related molecules 4. Regulation by compounds that are not structurally related to the substrates 5. Saturation kinetics 6. Compounds go down their own concentration (electrochemical) gradient Transzport kinetika / Transport Kinetics 17 FACILITÁLT DIFFÚZIÓ CSATORNA ÚTJÁN FACILITATED DIFFUSION VIA CHANNEL FACILITÁLT DIFFÚZIÓ UNIPORTER ÚTJÁN FACILITATED DIFFUSION VIA UNIPORTER 18 Different uniporters have different transport kinetics Direct/primary active transport: Na+/K+-ATPase 19 Direct/primary active transport: Ca2+-ATPase Kotranszport: indirekt/másodlagos aktív transzport Cotransport: indirect/secondary active transport Forms: Concept: 20 Na+/glükóz kotranszport Mechanism of Na+/glucose cotransport Komplex transzepitheliális transzport: a glükóz felszívódása a bélben Complex transepithelial transport: glucose

absorption in the intestine 21 Komplex transzepitheliális transzport: HCl szekréció a gyomorban Complex transepithelial transport: HCl secretion in the stomach Ozmózis / Osmosis Osmosis is the selective passage of solvent molecules through a porous membrane from a dilute solution to a more concentrated one. A semipermeable membrane allows the passage of solvent molecules but blocks the passage of solute molecules. Osmotic pressure (π) is the pressure required to stop osmosis. 22 van’t Hoff’s Law π = RT(ϕ ϕic) Jacobus π : Osmotic pressure Henricus R: Ideal gas constant vant Hoff Nobel prize 1901 T: Absolute temperature ϕ: Osmotic coefficient i: Van’t Hoff factor: number of ions formed by dissociation of a solute molecule c: Molar concentration of solute Osmolarity: The sum of the molarities of all dissolved particles in a solution:Φ ic! Isotonic ~300 mosmol/l Hypotonic < 300 mosmol/l Hypertonic > 300 mosmol/l Vörösvértestek ozmotikus

alakváltozásai Osmosis in erythrocytes 23 Nyugalmi és akciós membrán potenciál Membrane Resting and Action Potentials Dr. Benyó Zoltán A nyugalmi membránpotenciál mérése Measurement of Resting Membrane Potencial 24 A nyugalmi membránpotenciál meghatározása Definition of Resting Membrane Potential Dinamikus egyensúlyi állapot, melyben az egyes ionok sejtmembránon keresztüli kiés beáramlása kiegyenlítıdik. An equilibrium condition, in which there is no net movement of ions across the plasma membrane, is known as the resting membrane potential. Az ionmozgásokat befolyásoló erık: alapfogalmak • • • • Kémiai v. koncentrációs erı/grádiens Elektromos erı/grádiens Elektrokémiai grádiens Elektrokémiai egyensúly 25 Two forces influence ion movement across membrane • Diffusion force (determined by the concentration difference of ions across the membrane) and • Electrical force (determined by the voltage difference across the

membrane). Concentration and Electrical Gradients Ions flow along their concentration gradient when they move from an area of high concentration to an area of low concentration. Ions flow along their electrical gradient when they move toward an area of opposite charge. Together, the electrical and chemical gradients constitute the electrochemical gradient. Ion movement (flow) along electrochemical gradients underlies all the electrical phenomena in neurons. 26 Electrochemical equilibrium When an ion shows no further net flux across a membrane even if the membrane is freely permeable to that ion. Nernst egyenlet Nernst equation Equilibrium potential (Ex) for single ions The equilibrium potential depends on: • Absolute temperature (T) • Valence of the diffusible ion (z) • Ratio of concentrations on the two sides of the membrane [x]inside / [x]outside 27 Nernst egyenlet / Nernst equation Koncentrációs erı Elektromos erı R * T ln ([Xo] /[Xi]) + z F (Eo - Ei) = 0

Diffusion force Electrical force Eo - Ei = -(R * T / z F) ln ([Xo] / [Xi]) For practical use: Eo - Ei = -(60 / z) * log ([Xi] /[Xo]) Eo - Ei = -(60 / z) * log ([Xi] /[Xo]) 28 Különbözı ionok egyensúlyi potenciálja Equilibrium potentials of different ions Typical resting membrane potential Elektromos és kémiai grádiensek a nyugalmi membránpotenciálon Electrochemical Gradients at Resting Membrane Potential Cl- cc cc Na+ E E -70 mV cc E K+ 29 A nyugalmi membránpotenciál fenntartásáért felelıs tényezık The resting membrane potential is developerd and maintained by ionic differences which are the consequences of: different resting membrane permeabilities of ions (leaky channels, impermeability for protein anions) operation of the sodium-potassium pump A (nyugalmi) membránpotenciál meghatározása: Goldman-Hodgkin-Katz egyenlet Determination of the (Resting) Membrane Potential: Goldman-Hodgkin-Katz Equation Generalization of the Nernst equation

extended to include the relative permeability of each species of ion. Eions = -60 log PK[K+]i + PNa[Na+]i + PCl[Cl-]o PK[K+]o + PNa[Na+]o + PCl[Cl-]i PK, PNa, PCl = the permeability constants [K+]i, [Na+]i, [Cl-]i, [K+]o, [Na+]o, [Cl-]o = the intracellular and extracellular ion concentrations 30 Membránpotenciál: Alapfogalmak • • • • • Depolarizáció Repolarizáció Hyperpolarizáció Elektrotónusos potenciál Akciós potenciál Membrane Potentials: Signals Neurons use changes in membrane potential to receive, integrate, and send information Membrane potential changes are produced by: Changes in membrane permeability to ions (Alterations of ion concentrations across the membrane) Two types of signals are produced by a change in membrane potential: graded potentials (short-distance) action potentials (long-distance) 31 Polarization Definitions Depolarization Inside of the membrane becomes less negative (or even reverses) – a reduction in potential

(Depolarization increases the probability of producing nerve impulses.) Repolarization The membrane returns to its resting membrane potential Hyperpolarization Inside of the membrane becomes more negative than the resting potential –an increase in potential (Hyperpolarization reduces the probability of producing nerve impulses.) Ioncsatornák fajtái • • • • Szivárgó Ligand-függı Feszültség-függı Mechanoszenzitív 32 Types of Ion Channels Depolarization and hyperpolarization Passive or leakage channels Always open: resting MP and repolarization Chemically-gated channels Open with binding of a specific neurotransmitter (the ligand) Voltage-gated channels Open and close in response to changes in the membrane potential Mechanically-gated channels Open and close in response to physical deformation of receptors Ligand-függı ioncsatornák mőködése Ligand Gated Channels (Chemically Gated) Closed when a neurotransmitter is not bound to the extracellular

receptor Open when a neurotransmitter is attached to the receptor Na+ enters the cell K+ exits the cell 33 Feszültség-függı ioncsatornák mőködése Voltage-Gated Channel Example: Na+ channel Closed when the intracellular environment is negative Open when the intracellular environment is positive - Na+ can enter the cell Mechanoszenzitív ioncsatornák mőködése Mechanically-gated Channel 34 Ioncsatornákra ható gyógyszerek I: „Ca2+-antagonisták” Drugs acting on ion channels I: „Ca2+-antagonists” Sandmann S, Unger T; J Clin Basic Cardiol 1999; 2: 187–201. L-típusú feszültségfüggı Ca2+-csatorna L-type voltage gated Ca2+-channel Ioncsatornákra ható gyógyszerek II: Sulfonylurea Drugs acting on ion channels II: Sulfonylurea 35 Ioncsatornákra ható gyógyszerek III: GABAA-receptor (Cl--csatorna) agonisták és antagonisták Drugs acting on ion channels III: Agonists and antagonists of GABAA-receptor (Cl--channel) Patch clamp technika Patch

Clamp Technique • It allows to measure transmembrane ion currents and voltages as well as changes in membrane capacitance. • Lehetıvé teszi a membránon keresztüli ionáramok és feszültség, valamint a membrán kapacitás mérését. 36 A patch clamp technika Patch Clamp Technique Microelectrodes Mikroelektródák 37 Single Channel Recording Egyes ioncsatorák vizsgálata Whole Cell Recording Teljes sejt mérés 38 AKCIÓS POTENCIÁL ACTION POTENTIAL Gyors membrán potenciál változás, melyet a nyugalmi potenciálhoz való visszatérés követ Rapid change in the membrane potential followed by a return to the resting membrane potential Különbözı típusú akcióspotenciálok Different Types of Action Potentials 39 Az idegsejt akcióspotenciál fázisai Phases of the Action Potential in Nerves Az idegsejtek akciós potenciáljának kialakulásában a feszültség-függı Na+ és K+ csatornák játsszák a fıszerepet Voltage-gated Na+ and K+

channels are the major players in generating nerve action potentials A TEA és a TTX hatásai az akciós potenciálra Effects of TEA and TTX on the action potential TEA: Tetraethylammonium TTX: Tetrodotoxin 40 Feszültség-függı Na+ csatornák Voltage-gated Na+ channels • Very few types • Mostly one role – Initiate and propagate action potentials • Structure well known • Three types of conformational state controlled by membrane voltage (resting, activated and inactivated) Feszültség-függı K+ csatornák Voltage-gated K+ channels • Many types – E.g nerve K+ channels • Many roles – E.g action potential repolarization • Structure is known – Four subunits form homotetramer • Two types of conformational states controlled by membrane potential (closed and open) 41 4 Phases of the Action Potential Küszöbinger, a „minden vagy semmi” törvénye Threshold of Action Potentials Threshold Voltage– membrane is depolarized by 15 to 20 mV

Subthreshold stimuli produce subthreshold depolarizations and are not translated into APs Stronger stimuli produce depolarizing currents that are translated into action potentials All-or-None phenomenon – action potentials either happen completely, or not at all 42 Az akciós potenciál fázisai I: Nyugalmi állapot Action Potential: (1) Resting State Na+ and K+ channels are closed Each Na+ channel has two voltage-regulated gates – Activation gates – closed in the resting state – Inactivation gates – open in the resting state Az akciós potenciál fázisai II: Depolarizáció Action Potential: (2) Depolarization Depolarization opens the activation gate (rapid) and closes the inactivation gate (slower) of the Na+ channel. The gate for the K+ is slowly opened during depolarization. Na+ activation gates open quickly and Na+ enters causing local depolarization. This opens more activation gates and cell interior becomes progressively less negative. Rapid depolarization and

polarity reversal 43 Az akciós potenciál fázisai III: Repolarizáció Action Potential: (3) Repolarization Positive intracellular charge opposes further Na+ entry. Inactivation gates of Na+ channels close. As sodium gates close, the slow voltage-sensitive K+ gates open and K+ leaves the cell following its electrochemical gradient and the internal negativity of the neuron is restored Az akciós potenciál fázisai IV: Hyperpolarizáció Action Potential: (4) Hyperpolarization The slow K+ gates remain open longer than is needed to restore the resting state. This excessive efflux causes hyperpolarization of the membrane. The neuron is insensitive to stimulus and depolarization during this time. 44 4 Phases of the Action Potential Refrakter fázisok Refractoriness 45 A küszöbinger változása a refrakter fázisok alatt Changes in Threshold During Refractory Periods Abszolút refrakter fázis Absolute Refractory Period When a section of membrane is generating an AP

and Na+ channels are open, the neuron cannot respond to another stimulus The absolute refractory period is the time from the opening of the Na+ activation gates until the closing of inactivation gates 46 Relatív refrakter fázis Relative Refractory Period The relative refractory period is the interval following the absolute refractory period when: Na+ gates are closed K+ gates are open Repolarization is occurring During this period, the threshold level is elevated, allowing only strong stimuli to generate an AP (a strong stimulus can cause more frequent AP generation) Az akciós potenciál terjedése Conduction of Action Potential (The action potential is a self-reinforcing signal spreading with no decrement.) • Unidirectional (because of refractoriness) • Large range of conduction velocities (1 m/s to 120 m/sec or 432 km/h) • Continuous (in non-myelinated axons): slow, impact of axon diameter • Saltatory (in myelinated axons): fast, less costly in energy 47 Az

akciós potenciál folyamatos terjedése Continous propagation of an action potential The action potential is self-propagating and moves away from the stimulus (point of origin) A mielinhüvelyes rost The Myelin Sheet 48 Saltatorikus ingerületvezetés Saltatory Conduction Current passes through a myelinated axon only at the nodes of Ranvier (Na+ channels concentrated at nodes) Action potentials occur only at the nodes and jump from node to node fast and economic The Nerve Action Potential – Summary I. • is a transient reversal of the polarity of the membrane potential • has a rising phase (depolarization) caused by the opening of Na+ channels • has an overshoot that approaches ENa • has a falling phase (repolarization) caused by opening of K+ channels and inactivation of Na+ channels 49 The Nerve Action Potential – Summary II. • has an absolute refractory period because most Na+ channels are first rapidly opening and then rapidly becoming inactivated. •

has a relative refractory period because some Na+ channels are inactivated and some K+ channels are open. • propagates in one direction along axons through the sequential action of Na+ channels (“unidirectional”). 50