passive and active transport - unifr.ch · 3) active transport transport against a concentration...
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Passive and Active Transport
1. Thermodynamics of transport
2. Passive-mediated transport
3. Active transport
neuron, membrane potential, ion transport
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Membranes
• Provide barrier function – Extracellular – Organelles
• Barrier can be overcome by „transport proteins“ – To mediate transmembrane movements of ions, Na+, K+
– Nutrients, glucose, amino acids etc. – Water (aquaporins)
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1) Thermodynamics of Transport • Aout <-> Ain (ressembles a chemical equilibration) • GA - Go‘
A = RT ln [A] • ∆GA = GA(in) - GA(out) = RT ln ([A]in/[A]out)
• GA: chemical potential of A • Go‘
A: chemical potential of standard state of A
• If membrane has a potential, i.e., plasma membrane: -100mV (inside negative) then GA is termed the electrochemical potential of A
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Two types of transport across a membrane:
o Nonmediated transport occurs by passive diffusion, i.e., O2, CO2 driven by chemical potential gradient, i.e. cannot occur against a concentration gradient
o Mediated transport occurs by dedicated transport proteins 1. Passive-mediated transport/facilitated diffusion: [high] -> [low] 2. Active transport: [low] -> [high] May require energy in form of ATP or in form of a membrane potential
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2) Passive-mediated transport
Substances that are too large or too polar to diffuse across the bilayer must be transported by proteins: carriers, permeases, channels and transporters
A) Ionophores B) Porins C) Ion Channels D) Aquaporins E) Transport Proteins
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A) Ionophores Organic molecules of divers types, often of bacterial origin => Increase the permeability of a target membrane for ions, frequently antibiotic, result in collapse of target membrane potential by ion equilibration
1. Carrier Ionophore, make ion soluble in membrane, i.e. valinomycin, 104 K+/sec
2. Cannel-forming ionophores, form transmembrane channels, gramicidin A, 107 K+/sec
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Valinomycin o One of the best characterized ionophores,
binds K+ ions o Cyclic peptide with D- and L-Aa o Discrimination between Na+, K+, Li+ ?
K+ (r=1.33Å), Na+ (r=0.95Å)
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Gramicidin A o 15 Aa linear peptide o Alternating D- and L-Aa, all hydrophobic o Dimerizes head-to-head to form channel
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B) Porins o Membrane spanning proteins
with β-barrel structure, with central aqueous channel, diameter ~ 7x11Å ~600 D, little substrate selectivity, E. coli OmpF
o Maltoporin, substrate selectivity for maltodextrins, α(1->4)-linked glucose oligossaccharide degradation products of starch, greasy slide
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C) Ion Channels o All organisms have channels for Na+, K+, and Cl-
o Membrane transport of these ions is important for: o Osmotic balance o Signal transduction o Membrane potential
o Mammalian cells: extracellular: 150mM Na+, 4mM K+
intracellular: 12mM Na+, 140mM K+
passive diffusion of K+ ions through opening of K+-channels from cytosol to extracellular space K+-channels have high selectivity of K+ over Na+
selectivity 104
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The K+-channel, KcsA o Streptomyces,Functions as a homotetramer, 158
Aa, 108 ions/sec, Roderick MacKinnon o Selectivity filter allows passage of K+ but not Na+
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The selectivity filter o The ion needs to be
dehydrated to pass through the most narrow opening of the channel
o In the dehydration, water is replaced by hydroxyl groups from the channels amino acids
o These hydroxyls will stabilize K+ but not Na+, because Na+ is much smaller than K+
o Cavity in the middle of the channel = middle of the membrane !!! Contains water
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Ion channels are gated o Channels can be closed and opened upon signal:
o Mechanosensitive channels open in response to membrane deformation: touch, sound, osmotic pressure
o Ligand-gated channels open in response to extracellular chemical stimulus: neurotransmission
o Signal-gated channel, open for example on intracellular binding of Ca2+
o Voltage-gated channel: open in response to membrane potential change, transmission of nerve impulses
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Nerve impulses are propagated by action potentials
o Stimulus of neuron results in opening of Na+ channels -> local depolarization, induces nearby voltage-gated K+ to open as well (repolarization)
o Spontaneous closure of channels before N+ / K+ equilibrium is reached
o Wave of directional transmission to nearby channels -> propagation of signal along the axon = action potential (~10m/sec), no reduction in amplitude (≠electrical wire), can be repetitive (ms)
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Time course of an action potential
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Voltage gated KV channels o Tetramer, S5,S6 ~KcsA, T1 domain in cytosol o Gating by the motion of a protein paddle o S4 helix contains 5 positive charges, spaced by 3
Aa = acts as voltage sensor
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Model for gating of KV channels o Gating by the motion of a protein paddle, S4
helix o Increase in membrane potential, inside
becomes less negative -> baddle moves -> pore opens
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Ion channels have two gates o One to open and one to
close
o T1 domain contains inactivation peptide that blocks pore entrance a few ms after V-dependent pore opening
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Cl- channel differ from cation channels
o Present in all cell types. Permit transmembrane movement of chloride ions along concentration gradient: [Cl-] extracellular: 120mM; intracellular 4mM
o Homodimer with each 18TMDs
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D) Aquaporins o Permit rapid rates of water transport across the
membrane (kidney, Hg2+), (3 109/sec), Peter Agre (1992); 11 genes in human
o AQP1, homotetrameric glycoprotein, 6TMDs, elongated hourglass with central pore
o Constriction region, 2.8Å narrow
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Aquaporins (2) • Only dehydrated water can pass
• Only 1 at the time, reorientation of dipol
• Selectivity filter similar to KscA
• No proton conducting wire allowed
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E) Transport proteins o Erythrocyte glucose
transporter, GLUT1 o Has glucose binding sites on
both sites of membrane (propyl on C1 prevents bdg to outer surface, propyl on C6 prevents bdg on inner site)
o Assymetric bdg sites o 12 TMDs forms tetramer
o Transport proteins alternate between two conformations
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Transport proteins (2)
i.e. GLUT1
i.e. oxalate transporter oxalate in, formate out, (Na+-K+)-ATPase
i.e. Na+ glucose symporter Lactose permease
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Gap Junctions o Form Cell-Cell connections, Connexins form gap junctions o Cells within a organ are in metabolic and physical contact with
neighboring cells through gap junctions, 16-20Å pore, 1000 D o Two apposed plasma membrane complexes o Closed by Ca2+
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Differentiation between mediated and nonmediated transport
mediated
Non-mediated
1. Speed and specificity: Mannitol <-> glucose, structurally similar but uptake of glucose is much faster => must be mediated
2. Saturation 3. Competition, with similar substrates 4. Inactivation, heat, protease...
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3) Active Transport Transport against a concentration gradient,
endergonic process, frequently requires ATP or other energy sources (i.e., trans-membrane potential = secondary active transport !)
Example: Glucose concentration in blood is around 5mM Glut1 cannot increase the intracellular glucose concentration in the erythrocyte above this 5mM this would require an energy consuming transporter
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Classes of ATPases All consume intracellular ATP for the transport
process:
1. P-type ATPases undergo internal phosporhylation during transport cycle, i.e. Na+, K+, Ca2+
2. F-type ATPase, proton transportes in mitochondria, synthesize ATP
3. V-type ATPase, acidify a cellular compartment: vacuole, lysosome
4. A-type ATPase transport anions across membranes
5. ABC transporters, ATP-binding cassette, transport wide variety os substances for example ions, small metabolites, lipids, drugs
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A) (Na+-K +)-ATPase o Plasma membrane, (αβ)2 tetramer o Antiporter that generates charge seperation
across membrane -> osmoregulation and nerve excitability 3Na+(in) + 2K+(out) + ATP + H2O -> 3Na+(out) + 2K+(in) + ADP + Pi
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(Na+-K +)-ATPase (2) o ATP transiently phosphorylates Asp to form a
high energy intermediate = All P-type ATPases o While every single reaction step is reversible,
the entire transport cycle is not
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(Na+-K +)-ATPase (3)
Step 5 is inhibited by cardiac glycosides -> increase in Na+
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Cardiac Glycosides o Increase intensity of heart muscle contraction
Digitalis contains digitoxin Oubain (wabane), arrow poison (East African Ouabio tree)
o Belong to the steroids, inhibit (Na+-K+)-ATPase Block step 5 -> increase intracellular [Na+]
-> stimulate (Na+-Ca2+) antiporter -> increases intracellular Ca2+ and ER stores -> reinforces muscle contraction (higher Ca2+ peaks)
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B) Ca2+-ATPase o Transient [Ca2+] increase triggers many processes,
such as: o Muscle contraction o Neurotransmitter release o Glycogen breakdown
o Cytosolic [Ca2+] ~0.1 µM, extracellular 1500µM
o Concentration gradient (1000x) is maintained by active transport across the plasma membarne and the ER by Ca2+-ATPases, antiport of protons
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Ca2+-ATPase (2)
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C) ABC transporters are responsible for drug resistance
o If anti-cancer drugs do not show any positive effect, this is frequently due to overexpression of the P-glycoprotein, a member of the ABC transporter superfamily or multidrug resistance (MDR) transporters
o Built from 4 modules: 2x cytoplasmic nucleotide binding sites, 2x TMDs with 6 helices each,
o In bacteria these 4 domains can be coded by 2 or 4 single proteins; in eukaryotes all 4 domains on a single protein
o In bacteria, ABC transporters can act as importers or exporters, in eukaryotes only as exporters (?)
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Structure of an ABC transporter
o Sav1866 from Staphylococcus
o Homodimer, intertwined subunits
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CFT is an ABC transporter
o CFTR, cystic fibrosis transmembrane conducting channel, is the only ion transporting of the ca 100 known ABC transporters (see Box 3-1)
o Allows Cl- ions to flow out of the cell, by ATP hydrolysis
o More than 1000 mutations known, most frequent is Phe508 deletion, protein is functional, but improperly folded and degradet in the ER before transport to PM
o Homozygous have lung problems, thick mucus in the airways -> suffer from chronic lung infections, early death
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D) Ion Gradient-Driven Active Transport
o Free energy of the electrochemical gradient can be utilized to power active transport for example uptake of glucose by symport with Na+ (which is transported down the gradient)
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Lactose permease requires a proton gradient
o E. coli lactose permease (galactoside permease), utilizes proton gradient to symport lactose and H+
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The lactose permease o High affinity Bdg site extracellular o Low affinity intracellular o Binds sugar only in combination with proton