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2019.09.03. 1 Membrane Membrane physiology physiology I. I. Biological Biological functions functions of of the the plasma plasma- membrane membrane. . Transmembrane Transmembrane transport transport processes processes (learning learning objectives objectives 2 & 3) 2 & 3) Péter SÁNTHA 4.9.2019. CELLS: morphological and functional units of the organism Plasma membrane: barrier between the intracellular (IC) and extracellular (EC) fluid compartments „Interface” – there is a continuous exchange of substances, energy and information across the plasma membrane Plasma membrane is a dynamic system Extreme importance in the medicine – „access to the cells” examples for drugs which influence the functions of the plasma membrane: Local and general anaesthetics, antiepileptic and antiarrhythmic drugs, diuretics, psychotropic drugs etc…

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Page 1: MembraneMembrane physiology physiology II.. · MembraneMembrane physiology physiology II.. BiologicalBiological functions functions of of thethe plasmaplasma- ... („Trafficking”)

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MembraneMembrane physiologyphysiology I.I.

BiologicalBiological functionsfunctions of of thethe plasmaplasma--

membranemembrane. . TransmembraneTransmembrane

transporttransport processesprocesses((learninglearning objectivesobjectives 2 & 3)2 & 3)

Péter SÁNTHA4.9.2019.

CELLS: morphological and functional units of the organism

Plasma membrane: barrier between the intracellular (IC) andextracellular (EC) fluid compartments

„Interface” – there is a continuous exchange of substances,energy and information across the plasma membrane

Plasma membrane is a dynamic system

Extreme importance in the medicine – „access to the cells”

examples for drugs which influence the functions of theplasma membrane: Local and general anaesthetics, antiepileptic and antiarrhythmic drugs, diuretics, psychotropic drugs etc…

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The „Fluid mosaic” model of the biological membranes

(Singer & Nicholson, 1972 )

Schmidt/Thews: Physiologie des Menschen 27. Auflage 1997

Structure of the plasma membrane I. - Lipids

Amphiphilic lipoid molecules form the lipid bilayer (ca. 2.5 nmØ)

Phospholipids: phosphatidylcholin, phosphatidylserin, etc.- saturated and unsaturated fatty acid chains

SphingomyelinGlycosphyngolipids: gangliosidesCholesterol

Membrane fluidity – depends on the chemical composition of the membrane Spontaneous membrane formation – artificial membranes and other structures+ micelles, liposome

Permeability of the phospholipid bilayer: hydrophobic >> hydrophylic subst.Plasticity: deformability, budding, fusion

„Lipid Rafts“: cholesterol and glycosphyngolipid rich islets in the membrane:„Detergent Resistant Lipid Microdomains“

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Artificial membrane and lipid structures

Detergents can destroy these structures, as well as the biological membranes!

A new trend: the lipid rafts

Significance:Spatial organization of the membrane components (sorting of protein and lipidmolecules, assembling of multi-molecular complexes)- Signal transduction, endo- or exocytotic processes, intercellular interactions, etc.

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Suggested definition of lipid rafts(Pike LJ. 2006. J. Lipid Research)

"Membrane rafts are small (10-200 nm),heterogeneous, highly dynamic, sterol- andsphingolipid-enriched domains thatcompartmentalize cellular (membrane)processes. Small rafts can sometimes bestabilized to form larger platforms throughprotein-protein and protein-lipid interactions."

Structure of the plasma membrane II. - Proteins (25-70% of the total weight)

Integral proteins are embedded in the hydrophobic central core of the lipid bilayer-Transmembrane domain(s) are rich in hydrophobic amino acid residues

(Val, Leu, Ile etc.)+ membrane associated proteins: e.g. GPI (glycophospholipid)-anchored proteins

(Re-)circulation of the protein (and lipid) components: secretory vesicles

Targeted transport into the membrane: intracellular transport („Trafficking”)

Lateral diffusion: relatively free movement in the horizontal plane of themembrane surface (2D diffusion). detection: „Single Particle Imaging/Tracking”but: lateral diffusion can be limited by the interaction of thesub membrane cyto- or membraneskeleton(„Confinement”)

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Example: structure of the Glucose Transporter - 1 molecule (GLUT-1)

12 transmembrane helices

Pore formation by the hydrophilic AA residues of transmembrane domains

3.6 AA / turns

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Structure of the membrane skeleton (erythrocytes)

Functions of the cyto- and membrane skeleton: •Stabilization of the shape and the structural polarity of the cells•Cell movements (cilia, intracellular transport, active contractions)•Transport of vesicles (exo-/endocytosis, trafficking, protein translocation)•Cell division

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Example for a stimulus-induced translocation of a transmembrane protein (TRPV1 Ion channel). Live cell imaging using confocal microscopy .

Intracellular transport of membrane proteins(Trafficking)

Functions of the plasma membrane

Diffusion barrier – controlled exchange of substances (transmembrane transport)

Electric insulation (resistor and capacitor)

Communication – signal transduction (receptors, ion channels, second messenger systems)

Cell identity: cell-specific macromolecules (MHC antigens, blood group ags. etc.)

Intercellular interactions: adhesion molecules, immune mechanisms, gap-junctions

Metabolism: lipid mediators originate from the membrane lipoids :Phosphatidil Inositol (IP3) – diacylglycerol and inositol triphosphateArachidonic acid: prostaglandins, leucotriens, endogenous cannabinoids

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Transmembrane transport processes

Net flux of substances through the plasma membrane Exchange between the extracellular and intracellular fluid compartments

transmembrane transport mechanisms:

Free diffusion

Diffusion through ion channels (and pores)

Facilitated diffusion (carrier mediated transport)Active transport (pumps)

Exo-/Endocytosis (vesicular transport)

+Transepithelial transport: directed transport of substances across a continuous layer of epithelial cells (2 membranes – 3 compartments model)

See later – renal physiology

Free diffusion

Passive movement of solutes or gas particles in fluid (or gas) compartments

driving force: differences in local concentrations (gradient)+in case of charged particles: electrostatic field (electrical potential diff.)

Uncharged particles:

driving force is proportional to the ratio of the concentrations (RT x ln[Xi]/[X0])direction of the driving force determines the direction of the net flux

(sum of the inward and the outward fluxes)

The transport kinetic (rate) is described by the Fick’s diffusion law

(model: two compartments separated by a permeable barrier)

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Fick’s diffusion law applied on biological membranes

dm/dt = -D x ∆c x A/d

Applications:

Cell physiology+alveolar gas transport (lungs)microcirculation (capillary endothel)absortption in the GI tract

Schmidt/Thews: Physiologie des Menschen 27. Auflage 1997

dm/dt: rate of the diffusion D: diffusion constantA: diffusion surfaced: thickness of the barrier∆c: concentration difference

The diffusion constant is determined by:

Temperature

Chemical characteristics of the substances:lipid vs. water solubility(gas molecules, ethanol, urea, lipids etc.)

Schmidt/Thews: Physiologie des Menschen 27. Auflage 1997

permeability

Urea

glycerine

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Osmotic pressure

Posm=R x T x n/V

Osmolarity of the blood plasma:

~300 mosmol/L

660 KPa (7x atmospheric pressure)

Colloidosmotic pressure:

Osmotic pressure elicited by the

macromolecules (colloids)

ΠPlasma ~ 25 Hgmm

Ion channels:

Unitary conductivity: 106-108 ions/s (Siemens (S): pS equals 10-12 S)

Functional parts of the ion channels: pore region, selectivity filter and gate regions

Selectivity: selective (e.g.: Cl-; K+) and non-selective ion channels (e.g.:Na++Ca2+

Rectification: conductivity is dependent on the direction of the ion flux

opening (probability) is controlled by the gating mechanisms – activation/inactivation voltage (transmembrane potential)ligand bindingstretch (e.g.: mechanoreceptors, hair cells (cochlea))temperature (e.g: thermo receptors, nociceptors)intracellular signals (second messenger systems)

„Leaky channels” – these are constantly open (set the resting potential)Pores: high conductance, low selectivity, diverse functions (perforine, complement)Water channels: aquaporin family – constant or inducible water permeabilityof the membranes (e.g.: kidney collecting duct epithelium - effect of ADH)

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Functional model of the ion channels – interaction between theions and the surface of the pore region

Spontaneous oscillation (conformationchange) of the channel molecule allows

the guided transition of the ion– selectivity filter!

potential energy conformation

IC EC

ECIC

Filter mechanism of a calcium channel:Carboxy residues of 4 glutamate molecules

Example I: ligand-gated ion channel-nicotinic acetylcholine receptor

(motor endplate, autonomic nervous system)Ionotropic receptor: the transmitter receptor is an

ion channel

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Example II.: voltage gated Na+-channel (Axons – node of Ranvier, muscle cellsmyocardium)

-Gating: voltage sensor (charged residues)-other regulatory elements (inactivation gate)

Voltage gated ion channels

Example III: receptor (G-protein) coupled ion channel: muscarinic Ach receptor(heart SA/AV nodes, smooth muscle cells, secretory epithelial cells, etc.)

metabotropic receptors – indirect activation (second messengers)

Schmidt/Thews: Physiologie des Menschen 27. Auflage 1997

Ach receptor – G-protein – G-protein-gated K+ channel

Ach-receptor(7 TM protein)

G-protein

G-protein regulated K+ channel

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Example IV: Temperature sensitive ion channels

Change in the temperature results in opening of theion channels

Warm (hot) receptors (e.g.: capsaicin rec. – „paprika”)

cold receptors (e.g.: menthol rec. – peppermint)

The increase in the membrane current (conductance)is larger than that would be expected due tothermodynamic increase of the passive ion fluxes

sensory neurons, temperature and pain sensation

Nagy und Rang J.Neusci. 2002

Medical importance of ion channels

• Ion channels are targets of many different drugs:voltage dependent sodium channels: local anaesthetics, anti-arrhythmic and antiepileptic drugsIonotrop acetylcholine receptors: muscle relaxantsATP-sensitive K+ channels: oral antidiabetic drugsGABA-A receptors (ionotrop Cl- channels): hypnotic, sedative drugs

• Congenital malfunctions of ion channels (or carriers): „Channelopathies”

Congenital arrhythmias of the heart (e.g.: long QT syndrom) – K+ channelsMyotony (delayed relaxation of the muscles): - Cl- channels

Not strictly ion channels:Renal type diabetes insipidus (failure of water conservation, polyuria): failure of the aquaporin-2 functionCyistic fibrosis: CFTR Protein (Cystic Fibrosis Transepithelial conductance Regulator – dysfunctional Cl- transport)

The electric (and other functional) properties of excitable and non-excitable

cells are determined by the expression pattern of the ion channels

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Water channels – Aquaporin family

Unitary conductance: 109 H20 molecules/sSome aquaporins are also pereamble for urea, glycerol, ammonium ion

http://www.ks.uiuc.edu/Publications/Papers/abstract.cgi?tbcode=WANG2005

AqpZ (E. Coli)GlpF (E. Coli)

Increased (and in part controlled) water permeability of cellsNobel price of Chemistry 2003: Peter AGRE – discovery of the aquaporinsMajor function in renal physiology (Antidiuretic hormone)Other secretory epithelia (plexus choroideus)

Pore formation in the cell membrane – immune defense effector mechanisms

Lysis of the targeted cells via the insertion of high conductance non selectivepore complexes

Activation of the plasma complement system – final step: formation of the Membrane-Attack ComplexCytotoxic T lymphocytes and Natural Killer cells (Perforin complex)

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Transporter-mediated transport - Carrier molecules:

Enzyme analogy: S(in)⇔S+Carrier⇔S(out)Transport rate: <104 (Pumps 102) particles/s

Passive (facilitated diffusion): downhill transport according to the concentrationdifference or electro-chemical gradient of the transported substrate

Active (primary, secondary, tertiary): uphill transport against the concentration difference/electro-chemical gradient of the transpoted substrate

Primary active trp.: pumps, ATPase-esSecondary/tertiary active trp.: functional coupling of active and passive transporters

Uniporter: transport of a single particle (GLUT1-5: glucose transporter family)Symporter: Transport of two or more different particles in one direction Antiporter: Transport of two or more different particles in opposite directions

Stoichiometric ratio of transported particles: e.g.: 3 Na+ out and 2 K+ inTransport of ions: electrogenic (e.g.: Na+/K+ ATPase) or electro neutral (K+/H+ ATPase

Activity of the active transporters is dependent on the energy state (ATP cc.) of the cells

Protein-assisted transmembrane transport

(Ion) channels Transporters

Uniporters Cotransporters(symporters, antiporters)

Passive (Primary) active

PumpsATP-ases

Nav1.1 (Voltage-gated Na channel)

GLUT 2(glucosetransporters)

Na-K-Cl-symporterCl-HCO3-antiporter

Na-K-ATPaseCa-ATPaseMDRP

Channels Transporters

SLC TransportersSolute Carrier superfamily (48)

F, P,V type ATP-asesABC transporters

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Transport kinetic: analogy with the enzyme kinetic of biochemical reactions(see Michaelis-Menten equation)

Vmax is proportional to thenumber of active carriers

Schmidt/Thews: Physiologie des Menschen 27. Auflage 1997

Diffusion through

the membrane or channel

Transport by a carrier

saturation

Transport rate

Concentration (gradient) of the substrate

It is also dependent on the velocityof the elementary cycle

(e.g.: temperature, regulators)

Example I.: facilitated diffusion – glucose transporter molecule

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Example II.: primary active transport – Na+-K+ ATPase (pump)electrogenic antiport

Jens C Skou – Nobel price for Chemistry 1997

Effect of the inhibition ofATP synthesis

(lack of O2 or intoxication:DNP - Dinitrophenol)

Selective blockers:Heart glycosides

(Digoxin, Ouabain)

Digitalis lanata- foxgloveSchmidt/Thews: Physiologie des Menschen 27. Auflage 1997

ECF

ICF

Example III.: symport and antiport mechanisms in the secondary

active transport processes

Schmidt/Thews: Physiologie des Menschen 27. Auflage 1997

NCX: Na-Ca Exchanger SGLT: Na-Glucose Linked (Luminal) Transporter (Robert K. CRANE, 1960)