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Reginald H. GarrettCharles M. Grisham
Chapter 9Membranes and Membrane
Transport
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Transport
• Energy input drives active transport.• Primary active transport is driven by ATP.• Some transport processes are driven by light
energy.• Secondary active transport is driven by ion
gradients.
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9.8 How Does Energy Input Drive Active Transport Processes?
Energy input drives transport
• In active transport, solutes flow against their thermodynamic potential (against a concentration and/or charge gradient).
• Energy input drives such transport. • Energy source and transport machinery are
"coupled". • Energy source may be ATP, light or a
concentration gradient.
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The Sodium Pump
aka Na+/K+-ATPase
• This is a large protein with 120 kD α-subunits and 35 kD β-subunits. It is an α2β2 tetramer.
• Maintains intracellular Na+ low and K+ high. • Crucial for all organs, but especially for neural
tissue and the brain. • ATP hydrolysis drives Na+ out and K+ in. • Alpha subunit has ten transmembrane helices
with large cytoplasmic domain.
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Na+,K+-ATPase Uses ATP Energy to Drive Sodium and Potassium Transport
Figure 9.48 Schematic (a) and structure (b) of Na+,K+-ATPase.
inside
outside
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Na+,K+-ATPase Uses ATP Energy to Drive Sodium and Potassium Transport
Figure 9.49 A mechanism for Na+,K+-ATPase. The model assumes two principal conformations, E1 and E2. Binding of Na+ ions to E1 is followed by phosphorylation and release of ADP.
• ATP hydrolysis occurs via an E-P intermediate. • Mechanism involves two enzyme conformations
known as E1 and E2.• Cardiac glycosides inhibit by binding to outside.
inside
outside
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Na+,K+-ATPase
ATPATP
E1E1
ATP
E2
2 K+
3 Na+
ATP
ADP
E1
E1
E1
Pi Pi
Pi3 Na+
2 K+Pi
Pi
E2 E2
inside
inside
inside
outside
outside
ADP
outside
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The Sodium Pump
Steps in transport by the Na+/K+-ATPase
• E1 is open to the inside, has a high affinity for Na+ (KM = 0.2 mM) and poor binding of K+.
• So, 2K+ are released and 3 Na+ are bound on the inside. • E1 also has a high affinity for ATP which binds.• Phosphorylation of Asp369 occurs only in presence of Na+
and ATP (needs Mg++).• After phosphorylation the 3 Na+ are tightly bound, ADP
leaves and the E1~P•3 Na+ complex changes conformational to E2~P•3 Na+.
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The Sodium Pump
Steps in transport by the Na+/K+-ATPase
• E2 is open to the outside, has a high affinity for K+ (KM = 0.05 mM) and poor binding of Na+.
• 3 Na+ are released to the outside and E2 ~P binds 2 K+ forming E2 ~P•2 K+.
• Hydrolysis of aspartyl-P occurs only in presence of K+.• Dephosphorylation then occurs giving E2•2 K+ and Pi.• The loss of Pi results in a conformational change back to
E1•2 K+. • 2 K+ is released to the inside and the cycle starts again.
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Free Energy of the Na+,K+-ATPase
The Na+/K+ pump:
3 Na+in <=> 3 Na+
out 2 K+
out <=> 2 K+in
Approx conc.: Na+out = 145 mM
Na+in = 15 mM
K+out = 5
mM K+in = 150
mM = 70 mV
The potential inside = (-) and outside = (+).
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Free Energy of the Na+,K+-ATPase
For Na+ moving in to out at 37oC:
G = RT ln Co/Ci + ZF = 8.314(310) ln 145/15 + 1(96480)(0.070)
Note: Na+ is moving from a region of (-) charge to a region of (+) charge which is energetically unfavorable and this term will contribute to a (+) G so the membrane potential is (+).
G = 5846 + 6754 = 12600 J/mol or 12.6 kJ/mol
G is (+) so this energy must be provided to move 1 mol Na+, and for 3 mol of Na+ = 37.8 kJ.
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Free Energy of the Na+,K+-ATPase
For K+ at 37oC:
G = RT ln Ci/Co + ZF = 8.314(310) ln 150/5 + 1(96480)(-0.070)
Note: K+ is moving from a region of (+) charge to a region of (-) charge which is energetically favorable and this term will contribute (-) to G so the membrane potential is (-).
G = 8765 - 6754 = 2011 J/mol or 2.01 kJ/mol
G is (+) so this energy must be provided to move 1 mol K+, and for 2 mol of K+ = 4.02 kJ
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Free Energy of the Na+,K+-ATPase
Total energy required for transport:
G = 37.8 + 4.02 = 41.82 kJ
This occurs concurrent with hydrolysis of 1 mol ATP. At normal physiological concentrations the G for ATP hydrolysis is ~ -51 kJ/mol. Therefore, one concludes that sufficient energy is available to run this pump.
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Calcium Transport Is Accomplished in the Sarcoplasmic Reticulum by Ca2+-ATPase
A process similar to Na+,K+ transport
• Calcium levels in resting muscle cytoplasm are maintained low by Ca2+-ATPase (a Ca2+ pump).
• Calcium is pumped into the sarcoplasmic reticulum (SR) by a 110 kD protein that is very similar to the alpha subunit of Na,K-ATPase.
• Aspartyl phosphate E-P intermediate is at Asp351 and Ca2+-pump also fits the E1-E2 model.
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Calcium Transport Is Accomplished in the Sarcoplasmic Reticulum by Ca2+-ATPase
Figure 9.51 The transport cycle of the sarcoplasmic reticulum Ca2+-ATPase involves at least five different conformations of the protein, represented by the blue-shaded boxes here.
Sarcoplasmic reticulum
Cytoplasm
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The Gastric H+,K+-ATPase
The enzyme that keeps the stomach at pH 0.8
• The parietal cells of the gastric mucosa (lining of the stomach) have an internal pH of 7.4.
• H+,K+-ATPase pumps protons from these cells into the stomach (using energy of ATP) to maintain a pH difference across a single plasma membrane of 6.6 !
• This is the largest known transmembrane gradient in eukaryotic cells.
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The Gastric H+,K+ -ATPase Maintains the Low pH of the Stomach
Figure 9.52 The H+,K+-ATPase and a K+/Cl- cotransport system work together to achieve net transport of H+ and Cl-.
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The Gastric H+,K+-ATPase
• H+,K+-ATPase is similar in many respects to Na+,K+-ATPase and Ca2+-ATPase .
• All three enzymes form covalent E-P intermediates (P-type pumps).
• All three have similar sequences for the large (α) subunit.
• All three are involved in active transport.
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ABC Transporters Use ATP to Drive Import and Export Functions and Multidrug Resistance
• Cells “clean house” with membrane transporters known as multidrug resistance (MDR) pumps.
• MDR pumps are designed to recognize foreign organic molecules in cells and pump them out.
• Among these are the ABC transporters, some export therapeutic drugs from cancer cells, others import nutrients.
• In bacteria, these pumps are used to import nutrients into the cell.
• ABC transporters use the hydrolytic energy of ATP do not phosphorylate the enzyme.
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ABC Transporters Use ATP to Drive Import and Export Functions and Multidrug Resistance
• All ABC transporters consist of two transmembrane domains (TMDs) which form the pore and two cytosolic nucleotide-binding domains (NBDs) that bind and hydrolyze ATP.
• ABC transporters contain p-loops in the NBDs that interact with the phosphates of ATP.
• Bacterial ABC transporters are multimeric (importers tend to be tetrameric and exporters dimeric).
• Eukaryotic ABC pumps are monomeric.
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ABC Transporters Use ATP to Drive Import and Export Functions and Multidrug Resistance
Figure 9.54 Influx pumps in the inner membrane of Gram-negative bacteria bring nutrients into the cell; efflux pumps export cellular waste products.
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ABC Transporters Use ATP to Drive Import and Export Functions and Multidrug Resistance
Figure 9.56 Several ABC transporters are shown in different stages of their transport cycles.MBP = multidrug binding protein.
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9.9 How Are Certain Transport Processes Driven by Light Energy?
Bacteriorhodopsin is a light-driven proton pumpProtein opsin and retinal chromophore
• Retinal is bound to opsin via a Schiff base linkage. • The Schiff base (at Lys216) can be protonated, and
this site is one of the sites that participate in H+ transport.
• The carboxyl groups of Asp85 and Asp96 also serve as proton binding sites during transport.
• These Asp residues lie in hydrophobic environments.
• Their carboxyl pKa values are near 11.
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9.9 How Are Certain Transport Processes Driven by Light Energy?
Figure 9.57 The Schiff base linkage between the retinal chromophore and Lys216.
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9.9 How Are Certain Transport Processes Driven by Light Energy?
• Lys216 is buried in the middle of the 7-TMS structure of bR, and retinal lies mostly parallel to the membrane and between the helices.
• Light absorption converts retinal from all-trans to 13-cis configuration, triggering conformation changes that induce pKa changes.
• This facilitates proton transfers from Asp96 to the Lys Schiff base to Asp85 and net proton transport across the membrane.
• The transmembrane proton hopping causes cis-retinal to convert back to the trans form.
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9.10 How Is Secondary Active Transport Driven by Ion Gradients?
• The gradients of H+, Na+ and other cations and anions established by ATPases can be used for secondary active transport of various substrates.
• Many amino acids and sugars are accumulated by cells in transport processes driven by Na+ and H+ gradients.
• Many of these are symports, with the ion and the transported amino acid or sugar moving in the same direction.
• In antiport processes, the ion and the transported species move in opposite directions.
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AcrB is a Secondary Transport System
• AcrB is the major MDR transporter in E. coli.• It is responsible for pumping a variety of
molecules.• AcrB is part of a tripartite complex that bridges
the E. coli inner and outer membranes and spans the entire periplasmic space.
• AcrB works with AcrA and TolC to transport drugs and other toxins from the cytoplasm across the entire cell envelope and into the extracellular medium using energy from a proton gradient.
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AcrB is a Secondary Transport System
Figure 9.59 A tripartite complex of proteins comprises the large structure in E. coli that exports waste and toxin molecules. The transport pump is AcrB, embedded in the inner membrane.
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AcrB is a Secondary Transport System
• AcrB is a secondary active transport system and a H+-drug antiporter.
• As protons flow spontaneously inward through AcrB in the E. coli inner membrane, drug molecules are driven outward.
• Remarkably, the three identical subunits of AcrB adopt slightly different conformations, denoted loose (L), tight (T), and open (O).
• These three conformations are three consecutive states of a transport cycle.
• As each monomer cycles through L, T, and O states, drugs enter tunnel, are bound and then exported.
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AcrB is a Secondary Transport System
Figure 9.60 In the AcrB trimer, the three identical subunits adopt three different subunits. Possible transport paths of drugs through the tunnels are shown in green.
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AcrB is a Secondary Transport System
Figure 9.61 A model for drug transport by AcrB involves three different conformations.
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Ionophores
Ionophores are carriers or channel formers that transport ions.
Carrier: Valinomycin – a cyclic depsipeptide.
It has 12 residues and the bonding arrangement alternates -ester-peptide-ester-peptide-
(-L-Val-D-hydroxyisoVal-D-Val-L-Lactate-)3
Carries K+ in the center of the cyclic structure.
Transports K+ at a rate of 104 ions/sec.
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Valinomycin
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Ionophores
Channel former: Gramicydin – a helical peptide.
It has 15 residues that alternate in stereochemistry except for one Gly.
Formyl-V-G-A-L-A-V-V-V-W-L-W-L-W-L-W-ethanolamine
L L D L D L D L D L D L D L
Has mostly non-polar sidechains.
It dimerizes N-term to N-term to span the membrane and K+ ions flow through the core of the helix.
Transports K+ at a rate of 107 ions/sec.
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Gramicydin
The dimer has adjacent N-terminal residues.
Not an α-helix. It is more like a cylinder of parallel beta sheet. H-bonds are like those in parallel beta sheet.
K+ ions flow through the hollow core.
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End Chapter 9Membranes and Membrane
Transport