chapter 13: membrane channels and pumps copyright © 2007 by w. h. freeman and company berg tymoczko...

Chapter 13:Membrane Channels and Pumps

Copyright © 2007 by W. H. Freeman and Company

Berg • Tymoczko • Stryer

BiochemistrySixth Edition

Types of Transport

Simple Diffusion

Simple diffusion moves molecules across a membrane with a concentration gradient. The rate of diffusion varies linearly with concentration and the diffusion constant of the given molecule.

Diffusion Constants

Water 5 x 10-3 Urea 3 x 10-6

Glycerol 3 x 10-6

Glucose 5 x 10-8

Cl- 7 x 10-10 K+ 5 x 10-12 Na+ 1 x 10-12

Passive Mediated

This method also transports with a gradient but requires a protein to mediate transport across the membrane and differs from simple diffusion by:

rate of diffusion and specificityexhibits saturation behaviorsusceptable to inhibitionsusceptable to inactivation

Although ionophores are different they behave in this manner also.

Active Transport

This method moves molecules against a gradient and requires a protein to mediate transport across the membrane as well as an energy source which may be ATP.

The protein may effect movement in one of three ways:

Uniport moves one molecule in either direction. Symport moves two molecules in the same direction. Antiport moves two molecules in opposite direction .

Types of Transport Proteins

These are gated systems for active transport

Ionophores

Carriers or channel formers that transport ion.

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.

Valinomycin

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 end to end to span the membrane and K+ ions flow through the core of the helix.

Transports K+ at a rate of 107 ions/sec.

Gramicydin

The dimer has adjacent N-terminal residues. H-bonds are like parallel beta sheet.

K+ ions flow through the hollow core.

Membrane Transport

Site Requires sat’d Gradient energy

(1) Simple diffusion No with No

(2) Channels and pores Less with No

(3) Passive transporters Yes with No

(4) Active transporters Yes against Yes

(2) requires an integral peptide or carrier, is limited by rate of diffusion and molecular size. (3) & (4) involve integral proteins.

Free Energy of Transport

Gtransport = Gchem. + Gelect.

G for movement from inside a membrane to outside due to a concentration gradient (chem.):

[Cout] Gtransport = 2.3 RT log ------

[Cin]

G for movement from inside a membrane to outside due to a potential gradient (charge):

Gtransport = zF

The potential inside = (-) and outside = (+).

Thermodynamics, cont.

• When both a concentration gradient and a charge gradient are involved the equation is:

[Cout] Gtransport = 2.3 RT log ------- + zF

[Cin]

, the membrane potential, is the charge difference across the membrane in volts, z is the charge on the species being moved and F is Faraday’s constant, 96500 joules/volt.

G Calculation

Concentration gradient only: Assume that “A” is 0.05 mM extracellular and that the intracellular concentration is maintained at 15 mM at 37oC. For movement outside to inside:

G = RT ln Ci /Co = 8.314(310) ln 15 x 10-3/0.05 x 10-3

= 2577 (ln 300) = 2577 (5.7) = 14700 J/mol or 14.7 kJ/mol

(G is (+) so this energy must be provided in order to move 1 mol of “A”)

G Calculation

Assume that ATP is available to provide energy for this transport. Go' = -30.5 kJ/mol

Available concentrations: ATP = 2.5 mM; ADP = 1.5 mM and Pi = 0.5 mM

G = Go' + RT ln ([ADP][Pi]/[ATP]) G = -30500 + 2577ln ([1.5x10-3][0.5x10-3]/[2.5x10-3 ]) G = -30500 + 2577 ln (3 x 10-4)

G = -30500 + 2577 (-8.111) = -30500 - 20900 G = -51.4 kJ/mol

There is sufficient energy from 1 mol of ATP hydrolysis to move 3 mols of “A”.

G Calculation

Membrane potential:

What membrane potential would be needed to move 1 mol of “A” ?

G = zF 14700 = 1(96480) = 14700/96480 = 0.152 V = 152 mV

G Calculation

Normal membrane potentials range from ~ 60 mV to 100 mV. Assume a membrane potential of 60 mV.

What intracellular concentration of “A” could be reached if driven by this potential ?

zF = RT ln Ci /Co

1(96480)(0.06) = 2577 (ln Ci /0.05 x 10-3) 5789 / 2577 = 2.246 = ln Ci /0.05 x 10-3 9.454 = Ci /0.05 x 10-3 and

Ci = 4.72 x 10-4 Therefore, a potential difference of 60 mV could not maintain the desired 15 mM conc.

Gtransport vs C2/C1

Gtransport vs (21)

Na+/K+ Pump G Calculation

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 = (+).


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.


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


Total energy required for transport:

G = 37.8 + 4.02 = 41.82 kJ

This occurs concurrent with hydrolysis of 1 ATP. At normal physiological concentrations the G for ATP hydrolysis is ~ -51 kJ/mol, so sufficient energy is available.

Typical Pumping Scheme

Types of Pumps

• Primary Pumps (direct energy source, e.g. ATP):

P-type ATPases (use energy from ATP and involve phosphoryation of the transport protein): Na+/K+ ATPase, Ca++ ATPase, H+/K+ ATPase

ABC ATPases (use energy from ATP but no phosphorylation): Small molecule pump

• Secondary pumps (no ATP, e.g. ion gradient): H+/lactose - lactose permease in E.coli Na+/glucose – plasma membrane ATP/ADP transporter - mitochondria

Ca++ ATPase

The Ca++ pump in the sarcoplasmic reticulum (SR) is a primary transport (75%).

Muscle resting state: Ca++

SR = 0.1 μM

Muscle contraction, Ca++ released to cytosol Ca++

cyt = 15 mM

Ca++ levels must be reduced to relax muscle

Reaction:

2 Ca++cytosol + ATP <=> 2 Ca++

SR + ADP + Pi

Ca++ ATPaseThe transporter is a monomeric 110 kD protein and

binds 2 Ca++. It cycles between two major conformations, E1 and E2, has 10 transmembrane helices and 4 domains.

Transmembrane domain binds 2 Ca++ N domain binds ATP (nucleotide) P domain accepts P at Asp351 A domain actuator

The pump is regulated by calmodulin which activates the pump at high cytosolic Ca++ levels. KM of the ATPase goes from 20 μM to 0.5 μM.

Calmodulin binds 4 Ca++, two in each domain.

Ca++ ATPase, events

E1 is open to the cytosol.

2 Ca++ are bound from the cytosol. E12 Ca++ then ATP binds which traps the Ca++.

Phosphorylation of Asp351 occurs using ATP (needs Mg++) and E1~P2 Ca++ADP is formed.

ADP is then released triggering a conformational change to E2 ~P2 Ca++.

Ca++ ATPase, events

E2 is open to the SR.

E2 ~P2 Ca++ does not bind Ca++ well and the 2 Ca++ are released to the SR giving E2 ~P.

Dephosphorylation of E2 ~P occurs giving E2 and Pi.

This results in a conformational change back to E1 and the cycle starts again.

Ca++ Pump 0.1 M in cytoplasm1.5 mM in sarcoplasmic ret.

Sarcoplasmic reticulum Ca++ ATPase

Noncovalentbinding of Ca++

(green)

(SERCA)

Na+/K+ ATPase, events

The Na+/K+ pump in the plasma membrane is an antiporter and is a primary transport system:

3 Na+in + 2 K+

out + ATP <=> 3 Na+out + 2 K+

in + ADP + Pi

The transporter has 2 types of subunits (α = ~120 kD and β = ~35 kD), is an α2 β2 tetramer and cycles between two major conformations, E1 and E2. It has 10 transmembrane helices.

The mechanism is reported to be ordered sequential.


E1 is open to the inside, has a high affinity for Na+ (KM = 0.2 mM) and poor binding of K+.

So, 3 Na+ are bound from inside the cell. E1 also has a high affinity for ATP and it binds.

Phosphorylation of Asp369 occurs only in presence of Na+ and ATP (needs Mg++).

After phosphorylation the 3 Na+ are tightly bound and ADP leaves.

The E1~P3 Na+ complex then causes a conformational change to produce E2 ~P3 Na+.


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 the cell and E2 ~P binds 2 K+ from outside forming E2 ~P2 K+.

Hydrolysis of Asp-P occurs only in presence of K+.

Dephosphorylation occurs giving E22 K+ and Pi

A conformational change resuts giving to E12 K+

2 K+ is then released to the inside and the cycle starts again.

ABC ATPaseATP binding casette (ABC) transporters move

small molecules across a membrane and vary in specificity for substrate.

They bind ATP and use the energy of ATP hydrolysis for transport but do not phosphorylate the transporter.

Most are found in the plasma membrane and are pumps but some have been shown to be ion channels.

They are members of a superfamily of transporters that represents the oldest line of transporters known.

ABC ATPaseThe ABC transporters typically contain two

membrane spanning domains and two ATP binding casettes (nucleotide binding domains - NBDs) where binding and hydrolysis of ATP occurs. Some are single polypeptide chains, others are dimers each containing a membrane spanning domain of about six transmembrane helices and an NBD.

These proteins contain p-loops as do the NMP kinases. The amino acid residues in the P-loop interact with the phosphate groups of the bound nucleotide.

ABC ATPase

E1 is open to the inside of the cell and binds substrate E1S. E1S changes conformation, retains substrate but allows 2 ATPs to bind: E1S2 ATP. Binding of ATP induces a conformational change to E2S2 ATP.

E2 is open to the outside of the cell and S exits outside, leaving E22 ATP. Hydrolysis of ATP then occurs and both ADP and Pi leave.

E2 reverts to E1 and the cycle begins again.

ABC ATPase

H+/Lactose Transporter

Lactose permease in E. Coli is a symporter and is a secondary transport system.

It is another transporter that exhibits two states. E1 has high lactose affinity and is open to the outside of the cell. E2 has low lactose affinity and is open to the inside of the cell.

Lactose permease has two membrane spanning domains each containing six transmembrane helices.

There is some evidence to indicate that this is a random multisubstrate process.

H+/Lactose Transporter

E1 is open to the outside and binds a proton (Glu269 ?) giving E1~H. Then lactose binds from the outside to form E1~HLactose and the E1 sites are full. A conformational change occurs to give E2~HLactose.

E2 is open to the inside, so, E2~HLactose loses lactose giving E2~H.

A proton is then lost to the inside and the E2 sites are empty. A conformational change occurs back to E1 and the cycle starts again.

Lactose Permease

chapter 13: membrane channels and pumps copyright © 2007 by w. h. freeman and company berg tymoczko...

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