1. Membrane Transport “Pores, Porters and Pumps”
CH353 February 26-28, 2008

2. 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

3. Diffusion Across Membranes
Diffusion rate is proportional to permeability of solute
Permeability constant (P) depends on:
Partition constant (K) of solute
Urea, K = ; Diethylurea, K = 0.01
Diffusion constant (D) of membrane
is proportional to viscosity
viscosity of membrane ~ x 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 = KD x

4. 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
Diffusion rate ( ) dn dt dn dt
= AP(C1aq – C2aq) KD x
= A (C1aq – C2aq)

5. 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

6. Kinetics of Transporters
Vmax
α-D-glucose
α-D-mannose
Initial Rate of Monosaccharide Transport, V0 (mmol/min)
α-D-galactose
Passive Diffusion
(no GLUT1)
K0.5

7. 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 =
Vmax
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

8. Thermodynamics of Transport
∆G of membrane potential
y1 → y2
∆G of concentration gradient
C1 → C2
∆G of chemical reactions
S → P
∆G = ∆G′º + RT ln ( ) + RT ln ( ) + ZF∆y P S C2 C1
R = gas constant = 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]

9. 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∆y
[K+]out
[K+]in RT ZF
[K+]out
[K+]in
∆y = ln (Nernst Equation)
∆y = ln = mV
(8.315 J/mol•ºK)(310 ºK)
(+1)(98,060 J/mol•V)
4 mM
140 mM

10. 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 = J / mol • K; F = 96,480 J / V • mol
∆G = ∆G′º + RT ln ( ) + RT ln ( ) + ZF∆y P S C2 C1

11. Types of Membrane Transport

12. Transporter Classification System (http://www.tcdb.org/)
Classes
Channels/Pores
Electrochemical Potential-Driven Transporters
Primary Active Transporters
Group Translocators
Transport Electron Carriers
Accessory Factors involved in Transport
Incompletely Characterized Transport Systems

13. Transporter Classification System (http://www.tcdb.org/)
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

14. Transporter Classification System (http://www.tcdb.org/)
Electrochemical Potential-Driven Transporters
2.A. Porters (uniporters, symporters, antiporters)
2.B. Non-ribosomally synthesized porters
2.C. Ion gradient-driven energizers
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

15. Membrane Transport Systems
Channels/Pores (α-Type) Facilitated Diffusion
Non-gated
Gated (voltage, ligand, signal)
Electrochemical Potential-driven Transporters (Porters)
Uniporter Facilitated Diffusion
Antiporter
Symporter
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

16. 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

17. 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

18. 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

19. 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

20. V-type ATPase H+ Transport Mechanism
ADP + Pi
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+ E E H+
Adapted from Forgac 2007, Nat. Rev. Mol. Biol. 8:917

21. 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

22. V-ATPase H+ Transport Reaction
H+in ↔ H+out (~360º cycle)
H+in EH RH H+out R E- R E-
H+ exchange on c subunit (~60º cycle)
H+in EH E–
RH+ R
H+out

23. 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

24. 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)

25. 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

26. 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

27. Transport Cycle for SERCA
Overall Reaction:
2 Ca2+in H+out + ATP → 2 Na2+out H+in + ADP + Pi
E1-ATP
2 Ca2+
E1~P
2 Ca2+
E2-P
2 Ca2+
2-3 H+
ADP
2-3 H+
inside
outside
ATP Pi
2 Ca2+
3 Ca2+
E1-ATP
2-3 H+ E2
2-3 H+
E2-P
2-3 H+
[Inside] K½ K½ [Outside]
Ca+: 1 μM 0.1 μM high 2 mM
E1 has high affinity for Ca2+
E2 has low affinity for Ca2+

28. 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

29. Mechanism of P-Type ATPases
Kuhlbrandt 2004, Nat. Rev. Mol. Biol. 5:282

30. 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)

31. 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+
inside
outside
ATP Pi
3 Na+
3 Na+
E1-ATP
2 K+ E2
2 K+
E2-P
2 K+
[Inside] K½ K½ [Outside]
Na+: mM 0.6 mM high 145 mM
K+: mM high mM mM

32. 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

33. 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

34. 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

35. 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

36. ATP Induced Conformational Changes
ModBC-A without ATP
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
Sav1866 with ATP analog
Hollenstein et al. 2007, Curr. Opin. Struct. Biol. 17: 412

37. 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

38. Electrochemical Potential-driven Transporters (Porters)

39. 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]

40. 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
Sin 1 2 3 4
Kinetic
Model

41. Properties of Glucose Transporters
GLUT1
GLUT4
GLUT2
Initial Rate / Maximum Rate, V0 / Vmax
Physiological Range of Blood [Glucose]

42. Insulin Regulation of GLUT4-Mediated Glucose Transport in Muscle Cells
Insulin increases rate of glucose transport ~15 x

43. Structures of MFS Porters from E. coli
Alternating Access Model – “Rocker Switch” Mechanism
Locher et al. 2003, Science 301: 603

44. 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

45. 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

46. Structure of Lactose Permease and Proposed Transport Mechanism
3D structure of lactose permease with bound substrate (red) and essential Glu325 and Arg302 (green)
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

47. 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

48. 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