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

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

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

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

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

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

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

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

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

Types of Membrane Transport

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

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) 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

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

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

H+

H+

EE

ATP ADP + Pi

Adapted from Forgac 2007, Nat. Rev. Mol. Biol. 8:917

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

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)

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

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+

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

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

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

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

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

Sout

S • T1 S • T2

T1 T2

Sin1

2

3

4

KineticModel

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]

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

• 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