Membrane Transport “Pores, Porters and Pumps” CH353 February 26-28, 2008.
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Transcript of Membrane Transport “Pores, Porters and Pumps” CH353 February 26-28, 2008.
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Membrane Transport“Pores, Porters and Pumps”
CH353 February 26-28, 2008
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Summary
• Thermodynamics and Kinetics of Membrane Transport • Classification of Membrane Transport Proteins
– Channels, Porters, Primary Active Transporters• Primary Active Transporters
– driven by hydrolysis of phosphoanhydride bonds• Porters (secondary active transport & facilitated diffusion)
– driven by electrochemical potential• Systems combining active transporters and porters• Channels (for water and ions)• Regulation of ion channels
– voltage and ligand gating– action potential and synaptic function
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Diffusion Across Membranes
Permeability constant (P) depends on:
• Partition constant (K) of solute
Urea, K = 0.0002; Diethylurea, K = 0.01
• Diffusion constant (D) of membrane– is proportional to viscosity
– viscosity of membrane ~100-1000x greater than that of water
• Thickness of membrane (x) 3–5 nm
• K and D vary with lipid composition and position dx within membrane
K =[Solute] membrane
[Solute] aqueous
P =KDx
Diffusion rate is proportional to permeability of solute
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Diffusion Across Membranes
• Thickness (x) and diffusion constant (D) are similar for most membranes
• Thus diffusion across a membrane is proportional to the partition constant of the solute (K) and the difference in concentration (chemical gradient) or electrical gradient across a membrane (membrane potential, Vm)
• Electrochemical gradient / potential: combination of electrical and chemical differences across a cell membrane
For membrane transport:
• partition constant of solute is irrelevant
• depends on electrochemical gradient
dndt
= AP(C1aq – C2
aq)
KDx
= A (C1aq – C2
aq)
Diffusion rate ( )dndt
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Diffusion Accelerated by Transporters
• Diffusion rate is accelerated by lowering its activation energy, ∆G‡
• Transporters lower ∆G‡, providing another path through a membrane
• Facilitated Diffusion: transport down an electrochemical gradient
• Transporters are like Enzymes:
– Lowers ∆G‡ (faster rate)– Substrate specificity– Saturation kinetics– No effect on ∆G of process
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Kinetics of Transporters
α-D-glucose
α-D-mannose
α-D-galactose
Passive Diffusion(no GLUT1)
Init
ial
Ra
te o
f M
on
os
ac
ch
ari
de
Tra
ns
po
rt,
V0 (m
mo
l/m
in)
VmaxTransport of Monosaccharides by GLUT1
0
250
500
0 10 20 30 40 50
External [Monosaccharide] (mM)K0.5
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Kinetics of Transporters
Sout + T T•S complex T + Sin
k1
k-1
k2
k-2
for initial reaction conditions (Sout >> Sin):
assume k-2 = 0 and [T•S] is constant
V0 = k2[T•S] =k2[Tt][S]out
Kt + [S]out
Vmax[S]out
Kt + [S]out
=Vmax
1 + Kt / [S]out
=
Kt is similar to Km; the terms K½ or K0.5 are more commonly used
Ktransport (Kt) = k2 + k-1
k1
Kt is [S] at ½Vmax
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Thermodynamics of Transport
R = gas constant = 8.315 J / mol • K (1.987 cal / mol • K)
T = absolute temperature (K)
Z = charge of solute • number of moles (mol) [electrogenic transport]
F = Faraday constant = 96,480 J / V • mol (23,060 cal / mol • K)
∆ = 2 - 1 = membrane potential
∆G′º + RT ln (P/S) = 0, except for Primary Active Transport
∆G = 0 at equilibrium [resting potential]
∆G of membrane potential1 → 2
∆G of concentration
gradientC1 → C2
∆G of chemical reactions
S → P
∆G = ∆G′º + RT ln ( ) + RT ln ( ) + ZF∆PS
C2
C1
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Resting or Equilibrium Potential
Problem:• The plasma membrane of a neuron is selectively permeable to K+.
• If [K+]in = 140 mM and [K+]out = 4 mM, what membrane potential is needed to balance the transport of K+ out of the cell?
Solution:At equilibrium: ∆G concentration gradient = ∆G membrane potential
RT ln = ZF∆[K+]out
[K+]in
∆ = ln (Nernst Equation)[K+]out
[K+]in
RT
ZF
∆ = ln = -93.5 mV(8.315 J/mol•ºK)(310 ºK)
(+1)(98,060 J/mol•V)
4 mM
140 mM
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Thermodynamics of K+ Transport
Group Problem• The resting potential of a neuron is actually -70 mV on the inside• What is the ∆G for transport of K+? • Which direction is K+ spontaneously transported?
Assume: [K+]in = 140 mM; [K+]out = 4 mM; T = 37ºC
R = 8.315 J / mol • K; F = 96,480 J / V • mol
∆G = ∆G′º + RT ln ( ) + RT ln ( ) + ZF∆PS
C2
C1
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Types of Membrane Transport
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Transporter Classification System (http://www.tcdb.org/)
Classes
1. Channels/Pores
2. Electrochemical Potential-Driven Transporters
3. Primary Active Transporters
4. Group Translocators
5. Transport Electron Carriers
8. Accessory Factors involved in Transport
9. Incompletely Characterized Transport Systems
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Transporter Classification System (http://www.tcdb.org/)
1. Channels/Pores
1.A. α-Type channels
1.B. β-Barrel porins
1.C. Pore-forming toxins (proteins and peptides)
1.D. Non-ribosomally synthesized channels
1.E. Hollins
1.F. Vesicle fusion pores
1.G. Paracellular channels
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Transporter Classification System (http://www.tcdb.org/)
2. Electrochemical Potential-Driven Transporters
2.A. Porters (uniporters, symporters, antiporters)
2.B. Non-ribosomally synthesized porters
2.C. Ion gradient-driven energizers
3. Primary Active Transporters
3.A. P-P-bond-hydrolysis-driven transporters
3.B. Decarboxylase-driven transporters
3.C. Methyltransfer-driven transporters
3.D. Oxidoreduction-driven transporters
3.E. Light-driven transporters
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Membrane Transport Systems
1. Channels/Pores (α-Type) Facilitated Diffusion
– Non-gated– Gated (voltage, ligand, signal)
2. Electrochemical Potential-driven Transporters (Porters)– Uniporter Facilitated Diffusion– Antiporter– Symporter
3. Primary Active Transporters (P-P bond hydrolysis driven) – ABC transporter– P-ATPase– F-ATPase
Co-transport against concentration gradient(Secondary Active Transport)
Transport against concentration gradient
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Types of Transport
• Energy from ATP hydrolysis drives transport against electrochemical gradient
• Transport of a solute against its gradient is powered by transport of another down its gradient
TypicallyX = Na+ or H+
electrogenic transport has a net flow of charge contributing to the membrane potential; electroneutral transport does not
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Primary Active Transporters (Pumps)
• A-type, F-type and V-type ATPases– transport uses a rotary mechanism (multi-subunit complexes)– 3 ATPs hydrolyzed (or synthesized) per rotation– 2 to 4 H+ (or Na+) transported per ATP
• P-type ATPases– transport involves phosphorylated Asp and conformation shifts– multi-domain protein has all transporter activities– 1 ATP hydrolyzed; multiple cations (co)transported per cycle
• ATP-binding cassette (ABC) Transporters– each has 2 ABC and 2 transmembrane domains/subunits– transport by dimerization of ABCs and shifting of TMDs– 1-2 ATP hydrolyzed per molecule transported
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F-Type and V-Type ATPases
• integral (F0, V0) and peripheral (F1, V1) multi-subunit complexes
• homologous hexameric ATPase complexes (α3β3 and A3B3)
• homologous rotor complexes (dec12 and DFdc6)
– 1 H+ carrier (Glu) per subunit; F-type transports ~2x more H+ per ATP
• non-homologous a subunits but conserved mechanism• Reversible in vitro but opposite roles in vivo (opposite rotations)
– F-type is ATP synthase using [H+]; V-type is H+ pump using ATP
Nishi & Forgac 2002, Nat. Rev. Mol. Biol. 3:94
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Vacuolar (V-type) ATPases
• Structure and Activity:– ATP-hydrolyzing peripheral complex (V1) (640 kDa)
– H+-translocating integral assembly (V0) (260 kDa)
– 6 c subunits in rotor: maximum 2 H+ transported per ATP (higher pH gradients than for F-type ATPases)
– Electrogenic transport: requires transport of anion (e.g. Cl-)
• Functions:– pH regulation in organelles (lysosomes, endosomes, vacuoles)– In specialized cells (on plasma membrane) : renal acidification,
bone resorption, sperm maturation, cytoplasmic pH regulation– Multiple isoforms for specialized functions
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V-type ATPase H+ Transport Mechanism
• Subunit A hydrolyzes ATP changing its conformation
• This causes 120º rotation of rotor (subunits DFdc6)
• Proteolipid ring of c subunits moves past subunit a, having an essential Arg (R735), and 2 hemichannels open to either cytoplasm or lumen
• The Arg removes H+ from a Glu (E) on each c subunit; H+ exits to lumen
• H+ from cytoplasm neutralizes the charged Glu on c subunit, allowing it to rotate into the lipid bilayer
H+
H+
EE
ATP ADP + Pi
Adapted from Forgac 2007, Nat. Rev. Mol. Biol. 8:917
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V-type ATPase H+ Transport Mechanism
H+ from cytoplasm enters hemichannel in subunit a
H+ neutralizes charge on Glu of subunit c in the proteolipid ring
Essential Arg of subunit a removes H+ from Glu on subunit c
H+ dissociates from Arg and exits through hemichannel to lumen
Cycle for 60º Rotation
Forgac 2007, Nat. Rev. Mol. Biol. 8:917
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V-ATPase H+ Transport Reaction
H+in EH RH+ H+
out
R E-E- R
H+in
EH E– EH
RH+ R
H+out
H+in ↔ H+
out (~360º cycle)
H+ exchange on c subunit (~60º cycle)
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Regulation of V-type ATPases
Reversible Dissociation• V1 and V0 dissociate under low
glucose conditions (yeast, insects)• Aldolase may be glucose sensor• RAVE complex required for
reassembly of V-ATPase• PI3K dependence in kidney cells
Plasma Membrane Localization• transport of HCO3- to cytoplasm• adenylate kinase activation• cAMP synthesis• endocytosis of V-ATPase
Forgac 2007, Nat. Rev. Mol. Biol. 8:917
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P-type ATPases
Superfamily of active transporters (ATPases) including:– Na+K+ ATPase: maintains intracellular high [K+] and low [Na+]– Ca2+ ATPase (plasma membrane): Ca2+ homeostasis (< 0.2 μM)
– Ca2+ ATPase (SERCA): concentrates [Ca2+] in SR (~10 mM)
– H+K+ ATPase: gastric acidification (pH ~1)
– H+ ATPase: maintains membrane potential in plants and fungi
Characterized by: – Reversible phosphorylation of ATPase during transport cycle– Sensitivity to phosphate analogs, e.g. vanadate– Structural homologies (sequence and 3D structure)
SERCA is prototype for structure of P-type ATPases
Structures for Na+K+ ATPase and H+ ATPase (Dec 2007)
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P-type ATPases
• 3 cytoplasmic domains:
N – Nucleotide (ATP) binding
P – Phosphorylation (Asp)
A – Actuator (TGES motif)• multiple transmembrane helices
(10) having ion binding sites and transient channels to cytoplasm and to outside of cell (or lumen)
• phosphorylation and binding of nucleotides and ions result in conformational shifts causing:– opening/closing channels– changes to ion-binding affinity
Kuhlbrandt 2004, Nat. Rev. Mol. Biol. 5:282
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Ca2+ ATPases
Sarco-endoplasmic reticulum Ca2+ ATPase (SERCA)• Pumps Ca2+ from cytoplasm to sarcoplasmic reticulum
(SR) in skeletal muscle cells (induces relaxation)• [Ca2+] = 0.1 μM in resting cell; 1 μM in contracting cell;
and 2 mM in SR• ~80% of integral protein in SR
Plasma membrane Ca2+ ATPase• pumps Ca2+ from cytoplasm out of cell (ubiquitous)• allosterically activated by Ca2+-calmodulin • accelerates pump when [Ca 2+] is high
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Transport Cycle for SERCA
Overall Reaction:
2 Ca2+in + 2-3 H+
out + ATP → 2 Na2+out + 2-3 H+
in + ADP + Pi
E2-P2-3 H+
E2-P2 Ca2+
E1-ATP2-3 H+
E1-ATP2 Ca2+
E22-3 H+
E1~P2 Ca2+
2-3 H+
2 Ca2+
inside
2-3 H+
3 Ca2+
outside
[Inside] K½ K½ [Outside]
Ca+: 1 μM 0.1 μM high 2 mM
ATP Pi
ADP
E1 has high affinity for Ca2+ E2 has low affinity for Ca2+
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Mechanism of SERCA
• A-domain is connected to 3 transmembrane helices
• ATP binding to N-domain causes it to tip toward the P-domain, displacing the A-domain
• This opens a channel from the cytoplasm for Ca2+ entry
• Phosphorylation of P-domain causes N-domain to move back, allowing A-domain to return
• This occludes the bound Ca2+
• ADP is released allowing A-domain to turn into ADP binding site and bind to P- and N-domains
• This opens the channel to the lumen for to Ca2+exit
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Mechanism of P-Type ATPases
Kuhlbrandt 2004, Nat. Rev. Mol. Biol. 5:282
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Na+K+ ATPase
• maintains [K+] and [Na+] in cell; pumps 3 Na+ out and 2 K+ in
• electrogenic transport accounts for some of membrane potential
• tetramer of α2β2 subunits with tissue specific subunits/isoforms
• sensitive to ouabain, digoxin and palytoxin
• α subunit has similar 3D structure and mechanism as SERCA
• 3D structure shows that Na+ and K+ may have same binding sites (Olesen et al 2007, Nature 450:1036)
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Transport Cycle for Na+K+ ATPase
Overall Reaction:
3 Na+in + 2 K+
out + ATP → 3 Na+out + 2 K+
in + ADP + Pi
E2-P2 K+
E2-P3 Na+
E1-ATP2 K+
E1-ATP3 Na+
E22 K+
E1~P3 Na+
2 K+
3 Na+
inside
2 K+
3 Na+
outside
[Inside] K½ K½ [Outside]
Na+: 12 mM 0.6 mM high 145 mM
K+: 140 mM high 0.2 mM 4 mM
ATP Pi
ADP
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ATP-Binding Cassette (ABC) Transporters
• Superfamily of active transporters for both import and export of diverse molecules across membranes
• ABC importers found only in bacteria; require additional binding protein
• Each transporter has 2 transmembrane domains (TMDs) and 2 nucleotide-binding domains (NDBs)
• The NDBs are conserved, interchangable structures the TMDs vary with the molecule transported
• Dimerization of NBDs changes conformation of TMDs directing alternate access to either side of membrane
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Structures of ABC Transporters
• ABC importers: separate subunits for NBDs and TMDs• ABC exporters: single multidomain polypeptide
Hollenstein et al. 2007, Curr. Opin. Struct. Biol. 17: 412
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Structure of the B12 Transporter
• ABC importer for vitamin B12 is tetramer of NDBs and TMDs (BtuC2D2)
• requires periplasmic B12 binding protein (BtuF)
• ABC exporters do not need a binding protein
Locher 2004, Curr. Opin. Struct. Biol. 14: 426
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NBDs of ABC Transporters
• Cooperative binding and hydrolysis of ATP
• 2 NBDs form head-to-tail dimers with 2 ATPs sandwiched between
• ATP binding site (P) of one domain next to the hydrolysis site (P) of the other domain
• NBDs have binding sites for conserved coupling helices from TMDs
Hollenstein et al. 2007, Curr. Opin. Struct. Biol. 17: 412
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ATP Induced Conformational Changes
• Coupling helices of ABC transporters with ATP are closer than those without
ATP Switch Model:• 2 conformations: open dimer
(- ATP), closed dimer (+ ATP)• Binding of solute to TMDs
activate NBDs• ATP binding provides power
for transport (closed NBDs)• ATP hydrolysis restores
transporter (open NBDs)Higgins & Linton 2004
Nat. Struct. Mol. Biol. 11: 918
Hollenstein et al. 2007, Curr. Opin. Struct. Biol. 17: 412
ModBC-A without ATP
Sav1866 with ATP analog
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Human ABC Proteins
12 Sub-family A (ABC1) – lipid transport
11 Sub-family B (MDR/TAP) – multi-drug resistance / T-cell antigen processing
13 Sub-family C (CFTR/MRP) – cystic fibrosis transmembrane conductance regulator / multiple resistance pump
4 Sub-family D (ALD) – peroxisomal fatty acyl-CoA
1 Sub-family E (OABP)
3 Sub-family F (GCN20)
8 Sub-family G (WHITE) – eye pigment, cholesterol
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Electrochemical Potential-driven Transporters (Porters)
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Major Facilitator Superfamily
• Largest group of porters (>5000 in all kingdoms, 54 in human)
• Diverse in function (uniporters, symporters, antiporters)
• Most have 12 transmembrane helices (some with 14 and 24)
• Low sequence homology but similar predicted topology
Examples• Sugar Porter Family (2.A.1.1)
– Glucose transporters (human) GLUT1 – GLUT12 [Uniporters]
• Organophosphate:Pi Antiporter Family (2.A.1.4)
– Glycerol- Phosphate transporter (E. coli) GlyT [Antiporter]
• Oligosaccharide:H+ Symporter Family (2.A.1.5)
– Lactose permease (E. coli) lacY [Symporter]
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Model for Glucose Transport by GLUT1
• Transporter has 2 conformations– T1 facing outside; T2 facing inside
• Transport of glucose proceeds by alternate access model (rocker switch)
• Rate limiting step: T1 ↔ T2 (step 4)
– demonstrated using labeled glucose
Sout
S • T1 S • T2
T1 T2
Sin1
2
3
4
KineticModel
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Properties of Glucose Transporters
GLUT1
GLUT2
GLUT4
Init
ial
Ra
te /
Ma
xim
um
Ra
te,
V0
/ V
ma
xKinetics of Glucose Transporters
0.0
0.5
1.0
0 10 20 30 40
External [Glucose] (mM)
Physiological Range of Blood [Glucose]
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Insulin Regulation of GLUT4-Mediated Glucose Transport in Muscle Cells
• Insulin increases rate of glucose transport ~15 x
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Structures of MFS Porters from E. coli
Alternating Access Model – “Rocker Switch” Mechanism
Locher et al. 2003, Science 301: 603
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Lactose Transport in E. coli
• Lactose permease lacY uses electrochemical H+ gradient for symport of lactose (secondary active transport)
• H+ gradient is generated by oxidative respiration (electron transport)
• Import of lactose is sensitive electron transport inhibitors
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Inhibiting Secondary Active Transport of Lactose by lacY Mutants or Cyanide
• Glu325 and Arg302 are both essential for coupling transport of H+ and lactose
• lacY mutants are active in facilitated diffusion but not secondary active transport
• Collapse of H+ electrochemical gradient produces same result
• High intracellular lactose diffuses out when respiration is poisoned
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Structure of Lactose Permease and Proposed Transport Mechanism
a) 3D structure of lactose permease with bound substrate (red) and essential Glu325 and Arg302 (green)
b) protonation of amino acid side chains, e.g. Glu325 and Arg302 may change ionic interactions and switch conformations; with alternate access to cytoplasm or periplasmic space
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Structure of Glycerol-3-Phosphate Transporter of E. coli
3D structure of Glycerol-3-phosphate transporter with substrate binding amino acids Arg45 and Arg269
Huang et al. 2003, Science 301: 616
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Glycerol-3-Phosphate : Phosphate Antiport by Rocker Switch Mechanism
• Transporter alternates between conformations facing outward (Co) and inward (Ci)
• Binding phosphate or glycerol-3-phosphate draws 2 arginines together facilitating the Co ↔ Ci conformation switch
• Conformation changes are rate limiting and temperature dependent
• Binding phosphate or glycerol-3-phosphate is temperature independent
Huang et al. 2003, Science 301: 616;Law et al. 2007, Biochem 46: 12190