Membrane Transport
“Pores, Porters and Pumps”
CH353 February 26-28, 2008
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
Diffusion Across Membranes
Diffusion rate is proportional
to permeability of solute
Permeability constant (P) depends on:
• Partition constant (K) of solute
[Solute] membrane
K=
[Solute] aqueous
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
P=
KD
x
• K and D vary with lipid composition
and position dx within membrane
Diffusion Across Membranes
•
•
•
dn
Diffusion rate ( dt )
dn
= AP(C1aq – C2aq)
dt
=A
KD
(C1aq – C2aq)
x
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
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
Kinetics of Transporters
Transport of Monosaccharides by GLUT1
Vmax
Initial Rate of Monosaccharide Transport,
V0 (mmol/min)
500
α-D-glucose
α-D-mannose
250
α-D-galactose
0
0
K0.5
10
20
30
External [Monosaccharide] (mM)
40
50
Passive Diffusion
(no GLUT1)
Kinetics of Transporters
k1
Sout + T
k2
T•S complex
k-1
k-2
T + Sin
for initial reaction conditions (Sout >> Sin):
assume k-2 = 0 and [T•S] is constant
k2[Tt][S]out
Vmax[S]out
Vmax
V0 = k2[T•S] =
=
=
Kt + [S]out
Kt + [S]out
1 + Kt / [S]out
Ktransport (Kt) =
k2 + k-1
k1
Kt is [S] at ½Vmax
Kt is similar to Km; the terms K½ or K0.5 are more commonly used
Thermodynamics of Transport
∆G = ∆G′º + RT ln (
∆G of
chemical
reactions
S→P
P
C2
+ RT ln C + ZF∆y
S
1
)
( )
∆G of
concentration
gradient
C1 → C2
∆G of
membrane
potential
y1 → y2
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)
∆y = y 2 - y 1 = membrane potential
∆G′º + RT ln (P/S) = 0, except for Primary Active Transport
∆G = 0 at equilibrium
[resting potential]
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
[K+]out
RT ln + = ZF∆y
[K ]in
∆y =
[K+]out
RT
∆y =
ln [K+]
ZF
in
(Nernst Equation)
(8.315 J/mol•ºK)(310 ºK)
4 mM
ln
= -93.5 mV
(+1)(98,060 J/mol•V)
140 mM
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
P
C2
∆G = ∆G′º + RT ln ( S ) + RT ln ( C ) + ZF∆y
1
Types of Membrane Transport
Transporter Classification System
(http://www.tcdb.org/)
Classes
1.
2.
3.
4.
5.
Channels/Pores
Electrochemical Potential-Driven Transporters
Primary Active Transporters
Group Translocators
Transport Electron Carriers
8. Accessory Factors involved in Transport
9. Incompletely Characterized Transport Systems
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
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
Membrane Transport Systems
1.
Channels/Pores (α-Type)
– Non-gated
– Gated (voltage, ligand, signal)
Facilitated Diffusion
2.
Electrochemical Potential-driven Transporters (Porters)
– Uniporter
Facilitated Diffusion
– Antiporter
Co-transport against concentration gradient
(Secondary Active Transport)
– Symporter
3.
Primary Active Transporters (P-P bond hydrolysis driven)
– ABC transporter
– P-ATPase
Transport against concentration gradient
– F-ATPase
Types of Transport
Typically
X = Na+ or H+
•
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
electrogenic transport has a net flow of charge contributing
to the membrane potential; electroneutral transport does not
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
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
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
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
ATP
ADP + Pi
H+
E
E
H+
Adapted from Forgac 2007, Nat. Rev. Mol. Biol. 8:917
V-type ATPase H+ Transport Mechanism
Cycle for 60º Rotation
H+ from cytoplasm
enters hemichannel
in subunit a
H+ neutralizes charge
on Glu of subunit c in
the proteolipid ring
H+ dissociates from
Arg and exits through
hemichannel to lumen
Essential Arg of subunit
a removes H+ from Glu
on subunit c
Forgac 2007, Nat. Rev. Mol. Biol. 8:917
V-ATPase H+ Transport Reaction
H+in ↔ H+out (~360º cycle)
H+in
RH+
EH
E-
E-
R
R
H+ exchange on c subunit (~60º cycle)
H+in
EH
E–
RH+
H+out
EH
R
H+out
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
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)
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
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
Transport Cycle for SERCA
Overall Reaction:
2 Ca2+in + 2-3 H+out + ATP → 2 Na2+out + 2-3 H+in + ADP + Pi
E1-ATP
2 Ca2+
E1~P
2 Ca2+
E2-P
2 Ca2+
2-3 H+
ADP
2-3 H+
outside
inside
E1-ATP
2-3 H+
[Inside]
Ca+: 1 μM
Pi
ATP
2 Ca2+
K½
0.1 μM
E1 has high affinity for Ca2+
E2
2-3 H+
3 Ca2+
E2-P
2-3 H+
K½
high
[Outside]
2 mM
E2 has low affinity for Ca2+
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
Mechanism of P-Type ATPases
Kuhlbrandt 2004, Nat. Rev. Mol. Biol. 5:282
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)
Transport Cycle for Na+K+ ATPase
Overall Reaction:
3 Na+in + 2 K+out + ATP → 3 Na+out + 2 K+in + ADP + Pi
E1-ATP
3 Na+
E1~P
3 Na+
E2-P
3 Na+
2 K+
ADP
2 K+
outside
inside
E1-ATP
2 K+
[Inside]
Na+: 12 mM
K+: 140 mM
Pi
ATP
3 Na+
K½
0.6 mM
high
E2
2 K+
3 Na+
E2-P
2 K+
K½
high
0.2 mM
[Outside]
145 mM
4 mM
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
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
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
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
ATP Induced Conformational Changes
• Coupling helices of ABC
transporters with ATP are
closer than those without
ModBC-A without ATP
Sav1866 with ATP analog
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
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
Electrochemical Potential-driven
Transporters (Porters)
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]
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
S • T1
Kinetic
Model
Sout
2
3
1
T1
S • T2
4
T2
Sin
Properties of Glucose Transporters
Kinetics of Glucose Transporters
Initial Rate / Maximum Rate, V0 / Vmax
1.0
GLUT1
GLUT4
GLUT2
0.5
0.0
0
10
20
External [Glucose] (mM)
Physiological Range of Blood [Glucose]
30
40
Insulin Regulation of GLUT4-Mediated
Glucose Transport in Muscle Cells
• Insulin increases
rate of glucose
transport ~15 x
Structures of MFS Porters from E. coli
Alternating Access Model – “Rocker Switch” Mechanism
Locher et al. 2003, Science 301: 603
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
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
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
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
Glycerol-3-Phosphate : Phosphate Antiport
by Rocker Switch Mechanism
Huang et al. 2003, Science 301: 616;
Law et al. 2007, Biochem 46: 12190
• Transporter alternates between
conformations facing outward
(Co) and inward (Ci)
• Binding phosphate or glycerol-3phosphate draws 2 arginines
together facilitating the Co ↔ Ci
conformation switch
• Conformation changes are rate
limiting and temperature
dependent
• Binding phosphate or glycerol-3phosphate is temperature
independent