membrane. Transmembrane Transmembrane

2016.09.07.
Membrane physiology I.
Biological functions of the plasma
plasma-membrane.. Transmembrane
membrane
transport processes
Péter SÁNTHA
8.9.2016.
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 anaesthetics, general anesthetics, antiepileptic and
antiarrhythmic drugs, diuretics, psychotropic drugs etc…
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2016.09.07.
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
Sphingomyelin
Glycosphyngolipids: gangliosides
Cholesterol
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!
Asymmetric distribution of lipid components of the plasma membrane
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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 the
membrane surface (2D diffusion).
detection: „Single Particle Imaging/Tracking”
but: lateral diffusion can be limited by the interaction of the
sub membrane cyto- or membraneskeleton
(„Confinement”)
Example: structure of the Glucose Transporter - 1 molecule (GLUT-1)
12 transmembrane helices
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Structure of the membrane skeleton (erythrocytes)
Functions of the cyto- and membrane skeleton:
•Stabilization of the shape and polarity of the cells
•Cell movements (cilia, intracellular transport, active contractions)
•Transport of vesicles (exo-/endocytosis, trafficking, protein translocation)
•Cell division
Intracellular transport of membrane proteins
(Trafficking)
Example for a stimulus-induced translocation
of a transmembrane protein (TRPV1
Ion channel). Live cell imaging using
confocal microscopy .
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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 triphosphate
Arachidonic acid: prostaglandins, leucotriens, endogenous cannabinoids
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
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Free diffusion
Passive movement of solutes or gas particles in fluid (or gas) compartments
driving force: differences in local concentrations
+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)
Fick’s diffusion law applied on biological membranes
dm/dt = -D x ∆c x A/d
dm/dt: rate of the diffusion
D: diffusion constant
A: diffusion surface
d: thickness of the barrier
∆c: concentration difference
Applications:
Cell physiology
+
alveolar gas transport (lungs)
microcirculation
(capillary endothel)
absortption in the GI tract
Schmidt/Thews: Physiologie des Menschen 27. Auflage 1997
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2016.09.07.
Permeability of the
lipid bilayer
The diffusion constant is determined by:
Temperature
Chemical characteristics of the substances:
lipid vs. water solubility
Urea
glycerine
The lipid bilayer is highly permeable for
gas molecules (O2, CO2, NO, N2 etc.)
lipids (free fatty acids, cholesterol, steroids)
small (apolar) molecules (ethanol, urea, iodothyronin)
Non permeable for:
Ions,
proteins and other macromulecules,
Small molecular polar solutes
Schmidt/Thews: Physiologie des Menschen 27. Auflage 1997
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 exerted by the
macromolecules (colloids)
ΠPlasma ~ 25 Hgmm
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Transmembrane transport utilizing transport molecules (proteins)
Channels and pores (passive transport)
Carrier proteins:
transporters (passive – facilitated diffusion)
pumps (active)
Driving forces:
Passive trp.: uncharged particles (glucose, urea) – concentration difference
charged particles (ions)– electro-chemical potential difference
it is determined by the Nernst equation (see later)
Active trp.: consumption of metabolic energy (ATP hydrolysis)
Specificity (selective permeability or binding)
Transport rate/saturation (Tmax): number of the carriers, transport kinetics
(analogy: enzyme kinetics -Michaelis-Menten equation)
Temperature dependency
Activation/regulation: „Gating“ (conformation change)
covalent/non-covalent modification
protein expression/protein trafficking (translocation)
Selective inhibition (competitive/non competitive blockers, pore blockers etc.)
Electroneutral or electrogenic: net flux of charged particles (ions)
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 binding
stretch (e.g.: skin 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 permeability
of the membranes (e.g.: kidney collecting duct epithelium - effect of ADH)
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Functional model of the ion channels – interaction between the
ions and the surface of the pore region
Spontaneous oscillation (conformation
change) of the channel molecule allows
the guided transition of the ion
– selectivity filter!
IC
potential energy
IC
EC
conformation
Filter mechanism of a calcium channel:
Carboxy residues of 4 glutamate
molecules
EC
Example I: ligand-gated ion channelnicotinic acetylcholine receptor
(motor endplate, autonomic nervous system)
Ionotropic receptor: the transmitter receptor is an
ion channel
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Voltage gated ion channels
Example II.: voltage gated Na+-channel
(Axons – node of Ranvier, muscle cells
myocardium)
-Gating: voltage sensor (charged residues)
-other regulatory elements (inactivation gate)
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)
G-protein regulated
K+ channel
G-protein
Ach-receptor
(7 TM protein)
Ach receptor – G-protein – G-protein-gated K+ channel
Schmidt/Thews: Physiologie des Menschen 27. Auflage 1997
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2016.09.07.
Collection of human ion channels (and membrane receptors) as drug targets:
IUPHAR-database: www.iuphar-db.org
IUPHAR: International Union of Basic and Clinical Pharmacology
2013 August: 2272
2015 August: ~5500!!
VGIC: Voltage-Gated Ion Channel; LGIC: Ligand-Gated Ion Channel
Carrier-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 concentration
difference or electro-chemical gradient of the transported substrate
SLC (Solute Carrier) – superfamily (more than 50 subclasses)
Active (primary, secondary, tertiary): uphill transport against the concentration difference/
electro-chemical gradient of transported substrate
ABC (ATP-binding Casette)- transporters and ATPases superfamilies
Primary active trp.: pumps, ATPase-es
Secondary/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+ in
Transport 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
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2016.09.07.
Transport kinetic: analogy with the enzyme kinetic of biochemical reactions
(see Michaelis-Menten equation)
Transport rate
saturation
Diffusion through
the membrane or channel
Transport by a carrier
Vmax is proportional to the
number of active carriers
It is also dependent on the velocity
of the elementary cycle
(e.g.: temperature, regulators)
Concentration (gradient) of the substrate
Schmidt/Thews: Physiologie des Menschen 27. Auflage 1997
Example I.: facilitated diffusion – glucose transporter molecule
(single duty-cycle of the uniporter – reversibility)
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2016.09.07.
Example II.: primary active transport – Na+-K+ ATPase (pump)
electrogenic antiport
Jens C Skou – Nobel price for Chemistry 1997
ECF
ICF
Selective blockers:
Heart glycosides
(Digoxin, Ouabain)
Digitalis lanata- foxglove
Effect of the inhibition of
ATP synthesis
(lack of O2 or intoxication:
DNP - Dinitrophenol)
Schmidt/Thews: Physiologie des Menschen 27. Auflage 1997
Example III.: symport and antiport mechanisms in the secondary
active transport processes
NCX: Na-Ca Exchanger
SGLT: Na-Glucose Linked (Luminal)
Transporter (Robert K. CRANE, 1960)
Schmidt/Thews: Physiologie des Menschen 27. Auflage 1997
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GLUT
Lumen of the tubulus
SGLT: Sodium-Glucose
Luminal Transporter
Lumen of the tubulus
example IV.: secondary and tertiary active transports in the proximal tubuli
of the kidney (transepithelial transport)
Amino
acids
Schmidt/Thews: Physiologie des Menschen 27. Auflage 1997
Transport of macromolecules and corpuscular objects:
endo- and exocytosis
Exocytosis
ECF
Specialized form: release of
neurotransmitters in the
chemical synapses
cytoplasm
Receptor-mediated endocytosis:
ECF
Endocytosis
Transferrin complex (iron transport)
-transferrin receptor
Lipoproteins – lipoprotein receptor
Opsonisation – phagocytosis of
antigene-antibody complexes
cytoplasm
Schmidt/Thews: Physiologie des Menschen 27. Auflage 1997
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2016.09.07.
Regulation of glucose uptake of the cells: insulin-induced translocation
of GLUT-4 molecules
Rejection Coefficient
•• Solutes retained by the membrane
•– Lower solubility in water or
•– Diffuse more slowly through the membrane
•• Rejection coefficient
Cpi= conc. of solute i in permeate
Cri= conc. of solute i in retentate
•
•
•
•
•
•
Rejection Coefficient
• Ranges:
– 1–0
• When ri=0
– the membrane is completely permeable
• When ri= 0
– the membrane is completely impermeable
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Pore formation of the hydrophilic AA residues of transmembrane domains
3.6 AA / turns
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Voltage-current function of the ion flux through a specific
Ion channel (population) - rectification
Im
Outward rectifying
Ohmic current*
(no rectification)
Inward rectifying
Example: outward rectifying channel
(menthol (TRPM8) receptor)
Em
* The slope of the IV-curve is proportianal to the resistance of the membrane
Development of the polarisation of epithelial cells (kidney tubuli)
Mechanism for lumen formation in epithelia. Top. Confocal sections of MDCK cells forming cysts. Images were taken at the indicated
number of hours after putting individual cells into a 3D collagen matrix. Cysts are stained for an apical marker (gp135, red); adherent
junction marker (beta-catenin, green) and nuclei (blue). Scale bar 5 µm. Bottom. General mechanism for generation of lumens,
including apical membrane biogenesis, vectorial transport of apical vesicles to the plasma membrane, secretion, and regulated
expansion.
Curr Opin Cell Biol. 2008 Apr;20(2):227-34. Epub 2008 Feb 20.
Regulation of cell polarity during epithelial morphogenesis.
Martin-Belmonte F, Mostov K.
Madin-Darby Canine Kidney Epithelial Cells (MDCK Line)
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