Reginald H. Garrett Charles M. Grisham Chapter 9 Membranes and Membrane Transport.

Reginald H. GarrettCharles M. Grisham

Chapter 9Membranes and Membrane

Transport

Transport

• Energy input drives active transport.• Primary active transport is driven by ATP.• Some transport processes are driven by light

energy.• Secondary active transport is driven by ion

gradients.

9.8 How Does Energy Input Drive Active Transport Processes?

Energy input drives transport

• In active transport, solutes flow against their thermodynamic potential (against a concentration and/or charge gradient).

• Energy input drives such transport. • Energy source and transport machinery are

"coupled". • Energy source may be ATP, light or a

concentration gradient.

The Sodium Pump

aka Na+/K+-ATPase

• This is a large protein with 120 kD α-subunits and 35 kD β-subunits. It is an α2β2 tetramer.

• Maintains intracellular Na+ low and K+ high. • Crucial for all organs, but especially for neural

tissue and the brain. • ATP hydrolysis drives Na+ out and K+ in. • Alpha subunit has ten transmembrane helices

with large cytoplasmic domain.

Na+,K+-ATPase Uses ATP Energy to Drive Sodium and Potassium Transport

Figure 9.48 Schematic (a) and structure (b) of Na+,K+-ATPase.

inside

outside

Na+,K+-ATPase Uses ATP Energy to Drive Sodium and Potassium Transport

Figure 9.49 A mechanism for Na+,K+-ATPase. The model assumes two principal conformations, E1 and E2. Binding of Na+ ions to E1 is followed by phosphorylation and release of ADP.

• ATP hydrolysis occurs via an E-P intermediate. • Mechanism involves two enzyme conformations

known as E1 and E2.• Cardiac glycosides inhibit by binding to outside.

inside

outside

Na+,K+-ATPase

ATPATP

E1E1

ATP

E2

2 K+

3 Na+

ATP

ADP

E1

E1

E1

Pi Pi

Pi3 Na+

2 K+Pi

Pi

E2 E2

inside

inside

inside

outside

outside

ADP

outside

The Sodium Pump

Steps in transport by the Na+/K+-ATPase

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

• So, 2K+ are released and 3 Na+ are bound on the inside. • E1 also has a high affinity for ATP which binds.• Phosphorylation of Asp369 occurs only in presence of Na+

and ATP (needs Mg++).• After phosphorylation the 3 Na+ are tightly bound, ADP

leaves and the E1~P•3 Na+ complex changes conformational to E2~P•3 Na+.

The Sodium Pump

Steps in transport by the Na+/K+-ATPase

• 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 and E2 ~P binds 2 K+ forming E2 ~P•2 K+.

• Hydrolysis of aspartyl-P occurs only in presence of K+.• Dephosphorylation then occurs giving E2•2 K+ and Pi.• The loss of Pi results in a conformational change back to

E1•2 K+. • 2 K+ is released to the inside and the cycle starts again.

Free Energy of the Na+,K+-ATPase

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 mol ATP. At normal physiological concentrations the G for ATP hydrolysis is ~ -51 kJ/mol. Therefore, one concludes that sufficient energy is available to run this pump.

Calcium Transport Is Accomplished in the Sarcoplasmic Reticulum by Ca2+-ATPase

A process similar to Na+,K+ transport

• Calcium levels in resting muscle cytoplasm are maintained low by Ca2+-ATPase (a Ca2+ pump).

• Calcium is pumped into the sarcoplasmic reticulum (SR) by a 110 kD protein that is very similar to the alpha subunit of Na,K-ATPase.

• Aspartyl phosphate E-P intermediate is at Asp351 and Ca2+-pump also fits the E1-E2 model.

Calcium Transport Is Accomplished in the Sarcoplasmic Reticulum by Ca2+-ATPase

Figure 9.51 The transport cycle of the sarcoplasmic reticulum Ca2+-ATPase involves at least five different conformations of the protein, represented by the blue-shaded boxes here.

Sarcoplasmic reticulum

Cytoplasm

The Gastric H+,K+-ATPase

The enzyme that keeps the stomach at pH 0.8

• The parietal cells of the gastric mucosa (lining of the stomach) have an internal pH of 7.4.

• H+,K+-ATPase pumps protons from these cells into the stomach (using energy of ATP) to maintain a pH difference across a single plasma membrane of 6.6 !

• This is the largest known transmembrane gradient in eukaryotic cells.

The Gastric H+,K+ -ATPase Maintains the Low pH of the Stomach

Figure 9.52 The H+,K+-ATPase and a K+/Cl- cotransport system work together to achieve net transport of H+ and Cl-.

The Gastric H+,K+-ATPase

• H+,K+-ATPase is similar in many respects to Na+,K+-ATPase and Ca2+-ATPase .

• All three enzymes form covalent E-P intermediates (P-type pumps).

• All three have similar sequences for the large (α) subunit.

• All three are involved in active transport.

ABC Transporters Use ATP to Drive Import and Export Functions and Multidrug Resistance

• Cells “clean house” with membrane transporters known as multidrug resistance (MDR) pumps.

• MDR pumps are designed to recognize foreign organic molecules in cells and pump them out.

• Among these are the ABC transporters, some export therapeutic drugs from cancer cells, others import nutrients.

• In bacteria, these pumps are used to import nutrients into the cell.

• ABC transporters use the hydrolytic energy of ATP do not phosphorylate the enzyme.


• All ABC transporters consist of two transmembrane domains (TMDs) which form the pore and two cytosolic nucleotide-binding domains (NBDs) that bind and hydrolyze ATP.

• ABC transporters contain p-loops in the NBDs that interact with the phosphates of ATP.

• Bacterial ABC transporters are multimeric (importers tend to be tetrameric and exporters dimeric).

• Eukaryotic ABC pumps are monomeric.


Figure 9.54 Influx pumps in the inner membrane of Gram-negative bacteria bring nutrients into the cell; efflux pumps export cellular waste products.


Figure 9.56 Several ABC transporters are shown in different stages of their transport cycles.MBP = multidrug binding protein.

9.9 How Are Certain Transport Processes Driven by Light Energy?

Bacteriorhodopsin is a light-driven proton pumpProtein opsin and retinal chromophore

• Retinal is bound to opsin via a Schiff base linkage. • The Schiff base (at Lys216) can be protonated, and

this site is one of the sites that participate in H+ transport.

• The carboxyl groups of Asp85 and Asp96 also serve as proton binding sites during transport.

• These Asp residues lie in hydrophobic environments.

• Their carboxyl pKa values are near 11.


Figure 9.57 The Schiff base linkage between the retinal chromophore and Lys216.


• Lys216 is buried in the middle of the 7-TMS structure of bR, and retinal lies mostly parallel to the membrane and between the helices.

• Light absorption converts retinal from all-trans to 13-cis configuration, triggering conformation changes that induce pKa changes.

• This facilitates proton transfers from Asp96 to the Lys Schiff base to Asp85 and net proton transport across the membrane.

• The transmembrane proton hopping causes cis-retinal to convert back to the trans form.

9.10 How Is Secondary Active Transport Driven by Ion Gradients?

• The gradients of H+, Na+ and other cations and anions established by ATPases can be used for secondary active transport of various substrates.

• Many amino acids and sugars are accumulated by cells in transport processes driven by Na+ and H+ gradients.

• Many of these are symports, with the ion and the transported amino acid or sugar moving in the same direction.

• In antiport processes, the ion and the transported species move in opposite directions.

AcrB is a Secondary Transport System

• AcrB is the major MDR transporter in E. coli.• It is responsible for pumping a variety of

molecules.• AcrB is part of a tripartite complex that bridges

the E. coli inner and outer membranes and spans the entire periplasmic space.

• AcrB works with AcrA and TolC to transport drugs and other toxins from the cytoplasm across the entire cell envelope and into the extracellular medium using energy from a proton gradient.


Figure 9.59 A tripartite complex of proteins comprises the large structure in E. coli that exports waste and toxin molecules. The transport pump is AcrB, embedded in the inner membrane.


• AcrB is a secondary active transport system and a H+-drug antiporter.

• As protons flow spontaneously inward through AcrB in the E. coli inner membrane, drug molecules are driven outward.

• Remarkably, the three identical subunits of AcrB adopt slightly different conformations, denoted loose (L), tight (T), and open (O).

• These three conformations are three consecutive states of a transport cycle.

• As each monomer cycles through L, T, and O states, drugs enter tunnel, are bound and then exported.


Figure 9.60 In the AcrB trimer, the three identical subunits adopt three different subunits. Possible transport paths of drugs through the tunnels are shown in green.


Figure 9.61 A model for drug transport by AcrB involves three different conformations.

Ionophores

Ionophores are carriers or channel formers that transport ions.

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 N-term to N-term 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.

Not an α-helix. It is more like a cylinder of parallel beta sheet. H-bonds are like those in parallel beta sheet.

K+ ions flow through the hollow core.

End Chapter 9Membranes and Membrane

Transport

Reginald H. Garrett Charles M. Grisham Chapter 9 Membranes and Membrane Transport.

Documents

Transcript of Reginald H. Garrett Charles M. Grisham Chapter 9 Membranes and Membrane Transport.