membranemembrane physiology physiology ii.. · membranemembrane physiology physiology ii.....
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2019.09.03.
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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
Fun
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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)