Transport across Membranes

Question 1

Calculate the free energy to move 1 mol of Na+ from outside a cell ([Na+] = 150mM) to inside the cell ([Na+]=5.0mM) when the membrane potential is -70mV and the temperature is 37C

Answer 1

-70mV = -0.07V
37C = 310K

-15500J/mol = -15.5kJ/mol = NEG = Spontaneous

Question 2

Write out and use equations to describe the thermodynamics of transport.

Answer 2

deltaGtransport = RTln ([A]destination / [A] origin) + (ZA)FdeltaY

deltaY = Membrane Potential Vm
ZA = Charge of molecule of interest

DY is the membrane potential (Vm) (typically ~50mV (negative inside)

Question 3

Explain the origin of an activation energy barrier to passage of a polar substance through a membrane, and describe how membrane proteins lower that barrier.

Answer 3

The activation energy barrier to passage of a polar substance through a membrane arises because the membrane is composed of a hydrophobic interior, which repels polar molecules.
- Electrochemical Gradient

Membrane proteins
- hydrophilic channels or pores in the protein
- specific binding sites on the protein that can bind to the polar molecule and facilitate its passage across the membrane.
- conformational changes that create a hydrophilic pathway across the membrane

Thermodynamics considered when moving AGAINST electrochemical gradient (Active)

Question 4

Distinguish between channel and carrier transporters

Answer 4

• Ionophores and Channels allow molecules to move depending on concentration gradients
• Carriers will move molecules with rate determined both by the gradients and the transporter kinetics (active and passive Transporters)

Passive Transporters:
- Solute movement is determined by Electrochemical gradient
- ∆G solute < 0 (Spontaneous)

Active Transporters
- Movement against the gradient
- ∆G > 0 (+∆G(add’nal process) = ∆GNet < 0
- Requires energy input (coupled to exergonic process)
- Primary or Secondary

Primary Active Transporters:
- Exergonic chemical reaction provides energy for movement against electrochemical gradient (ATP Hydrolysis)

Secondary Active Transporters
- Exergonic solute (ion) transport drives transport of a 2nd molecule against gradient

Question 5

Distinguish between primary and secondary active transport.

Answer 5

Primary Active Transporters:
- Exergonic chemical reaction provides energy for movement against electrochemical gradient (ATP Hydrolysis)

Secondary Active Transporters
- Exergonic solute (ion) transport drives transport of a 2nd molecule against gradient

Question 6

Define the term ionophore.

Answer 6

Molecules that shuttle ions across membranes, down (along) their concentration gradient (passive)
- Carrier Ionophores
- Channel Ionophores

Many are peptide or peptide-like molecules produced by microorganisms
Will destroy trans-membrane electrochemical gradients, affecting secondary active transport processes

Question 7

Describe the structure of valinomycin and its mechanism of action as an antibiotic.

Answer 7

• Valinomycin is a neutral peptide-derivative carrier ionophore
• Not quite peptide: Has aa + Isovaleric Acid + L-Lactic Acid (modified Ala)
• Coordination bonds and H-bonds
• Alternating Ester and Amide linkages
• Six carbonyl groups will form a stable interaction with K+ ions.
• Move ions to low concentration (with gradient)
• 6 H2O in octahedral fashion
• Valinomycin replaces coordination bonds when K+ is in env’t lacking water
• Hydrophobic Exterior
• Lipid-soluble (both bound and unbound) and can move across the membrane.
• Potentially poisonous to any cell.

Question 8

Describe the structure of gramicidin and its mechanism of action as an antibiotic.

Answer 8

Gramicidin
-Peptide-based channel ionophores.
-Monovalent cation ionophore (K+>Na+).
-Gramicidins A, B and C are linear peptide
structures that form a “beta helix” structure.
- Alternating L and D amino acids allow for unusual secondary structure.
- Dimer creates membrane-spanning channel.
- Series of Trp on both sides associate with polar head groups = integral membrane prtn

Alternating L/D means side chains of ind AA in beta-sheet are alternating above and below
Alternating chirality allows new secondary structure

High specificity for K+
Impacts secondary active transport by destroying K+ concentration gradients (membrane potential)

Question 9

List features associated with porins in terms of specificity, secondary, tertiary and quaternary structures, using the E. coli proteins maltoporin and OmpF as examples.

Answer 9

Porins: β-barrel containing transmembrane proteins (not to be confused with aquaporins)
- May be non-selective (except for size) or selective
- Typically trimer of β-barrel each with a pore
- Full of water (continuous with aqueous env’t)

OmpF:
- Antiparallel β-barrel
- selective for molecules < 600Da (110Da/aa = 5aa long fit)

Maltoporin
- Specific to small maltodextrins
- Same β-barrel aspect; opening twisted preferable for Maltose transport
- Homotrimer (C3 symmetry 3 fold)
- Subunits have an 18-stranded β-barrel (antiparallel)
- Opening through each subunit has a left-handed curvature w/ non-polar/aromatic and polar residuces arranged
- Greasy Slide = Individual short disac/trisac structures can fit
- Allows for (α1→4) linked linear chains to pass
- LH helix matches amylose left handed helix

Question 10

Outline how maltoporin creates specificity for specific carbohydrate structures.

Answer 10

Maltoporin
- Specific to small maltodextrins
- Same β-barrel aspect; opening twisted preferable for Maltose transport
- Homotrimer (C3 symmetry 3 fold)
- Subunits have an 18-stranded β-barrel (antiparallel)
- Opening through each subunit has a left-handed curvature w/ non-polar/aromatic and polar residuces arranged
- Greasy Slide = Individual short disac/trisac structures can fit
- Allows for (α1→4) linked linear chains to pass
- LH helix matches amylose left handed helix

Question 11

Define a specificity/selectivity filter.

Answer 11

Gives channel selectivity
- Channel functional groups are arranged to interact with very specific molecules
- Other molecules either can’t interact or are repelled

Ion Selective Channels
- Allow for rapid ion movement across a membrane.
- Motion is down concentration gradient
- Rates approach free-diffusion limits.
- Highly selective (Fit, size, coordination bond)
- May be gated (opened/closed)

Question 12

Describe the structure of the K+ channel from S. lividans.

Answer 12

K+ Channel Structure (Integral Mbrn Channel Prtn):
4 identical subunits (C4 Symmetry) Homotetramer
- Core channel portion of larger structure
- 2 trans-membrane helices
- Additional a-helix found in core

K+ channel is formed between subunits
- A single channel passes from one side to the other of the bilayer
- A series of K+ binding sites exist along the interface

Question 13

Outline the structural features of the K+ channel from S. lividans that allows for the specific transport of K+ ions.

Answer 13

• Openings on either end are Negatively Charged (C-terminal (NEG) end of alpha-helix)
• Third helix in each subunit is oriented with its negative dipole towards the channel opening

• The K+ channel presents a series of 4 K+ binding sites.
• As a new ion enters, the previously bound ions move further down the channel in alternating binding sites.
• Na+ ions are too small to interact with the channel (10,000 times lower rate).
• Channel does not change shape as potassium binds

Question 14

Describe the structural features of the Cl- transporter and compare/contast it with the K+ transporter from S. lividans

Answer 14

Anion Channel (trickier than Cations)
- Channel through centre of each subunit (not at interface (K+))
- Homodimer
- 18 transmembrane alpha-helices that are tilted relative to membrane
- Selectivity filter created by alpha-helices (N-terminal dipoles) and Hydroxyl-containing amino acid (Ser/Tyr) (not positive charges, partial pos - selective for anions without getting “stuck”)

Both Polytopic Mbrn Proteins

Question 15

Describe the general features of bovine erythrocyte aquaporin and explain how it prevents proton movement across membranes.

Answer 15

Aquaporin:
- Allow for cross-membrane movement of water, excluding other solutes and H+ (H3O+) = move water without protons
- Homotetramer with multiple transmembrane a-helices.
- Water-channel formed at centre of each subunit

Proton Movement via Proton jumping is prevented by hydrogen-bonding interactions in the channel
- Bond Asn to prevent proton movement

Question 16

Describe the function, specificity, and proposed structure of the erythrocyte glucose transporter GLUT1.

Answer 16

GLUT1:
- Multipass (polytopic) membrane protein. Member of the MFS superfamily
- 12 transmembrane helices in single polypeptide, many contain polar amino acids.
- Passive carrier for D-glucose.
- 50,000 times faster movement of D-glucose across the erythrocyte membrane than simple, unassisted diffusion

As a carrier, GLUT1 does not have a continuous passage from one side of the membrane to the other.
Binding of glucose combined with a conformational shift leads to the binding site being opened to the interior
Transport is dependent on the rate of the steps in this process.

Question 17

Classify GLUT1 in terms of energy use in transport, number of solutes, and direction of solute transport.

Answer 17

GLUT1:
- uniporter (single glucose)
- facilitated diffusion process = passive carrier/transporter
- bidirectional; direction determined by [glucose] gradient

As a carrier, GLUT1 does not have a continuous passage from one side of the membrane to the other.
Binding of glucose combined with a conformational shift leads to the binding site being opened to the interior
Transport is dependent on the rate of the steps in this process.

Question 18

Describe features of an amphipathic helix and their role in membrane transport.

Answer 18

Arrangement of amino acids around the helix places polar and non-polar residues on opposite faces.

Groups of helices associating in the membrane can create a polar area near the center.

Non-polar regions will be oriented towards the lipid core.

Question 19

Sketch graphs to illustrate the kinetics of glucose transport into erythrocytes and explain the interactions between solute and transporter that determine the shape of each graph.

Answer 19

Rates of transport in carriers is analogous to Michaelis-Menten enzyme kinetics, and has similar equation (under similar assumptions):
- Kt is equivalent to Km (concentration of solute where Vo = ½ Vmax).
- Kt for GLUT1 is lower than blood glucose levels.
- Initial [S] on one side is 0 (no reverse transport)

Vo=Vmax[S]out / Kt+[S]out

Question 20

Describe the defining characteristics of P-type ATPases.

Answer 20

P-Type ATPases (P=Phosphorylation involved)
- Primary active transporters
- Hydrolysis of ATP provides energy to move solutes up concentration gradients.
- Reversible conformational change associated with phosphorylation/dephosphorylation.

Examples:
Na+K+ATPase; SERCA; Flippases; proton pumps

Question 21

Outline the structural features of the sarcoplasmic and endoplasmic ATPase (SERCA, Ca2+ ATPase) pumps.

Answer 21

SERCA pump Structure:
* Mr 111,000 -> 1000aa long (110Da/aa)
* single polypeptide
* Multipass/Helical bundle

4 domains
(1) Transmembrane (M) domain
- 10 transmembrane alpha helices
- Contains 2 Ca++ binding sites near centre
- involved in Ca++ translocation

(2) Phosphorylation (P) domain
- cytosolic
- Asp sidechain phosphorylated by ATP induces a conformational change in the M domain

(3) Nucleotide-binding (N) domain
- Binds ATP
- cytosolic

(4) Actuator (A) domain
nnects N & P to M domain; require change in TMD to allow transport
- Connects conformational changes in N and P domains to M domain
- Change in TMD required for transport

Sarcoplasmic and Endoplasmic Reticulum CAlcium pumps. (Ca2+ ATPase)
- P-type ATPases.
- Responsible for the transport of calcium out of the cytoplasm and into sarcoplasmic reticulum during muscle relaxation.

Question 22

Describe the sequence of events occurring in one cycle of action of the SERCA pump.

Answer 22

Two conformations for the transporter (E1 and E2).
E1: high-affinity Ca++ binding site exposed to cytosol.
E2: low-affinity Ca++ binding site exposed to lumen.

Phosphorylation of P-domain = switch to E2 = Ca++ dissociation
Hydrolysis of P-domain = dephosphorylation = Switch to E1
Phosphorylation site is distant from Ca++ binding site (~45Å)

Question 23

Explain the essential physiological roles of the SERCA pump.

Answer 23

Responsible for the transport of calcium out of the cytoplasm and into sarcoplasmic reticulum during muscle relaxation.

Question 24

List the defining characteristics of MFS transporters, as exemplified by the E. coli lactose permease transporter.

Answer 24

MFS Transporters:
* Single polypeptides (Multipass, helical bundles)
* 12 or 14 transmembrane helices arranged in two semi-symmetrical pairs of 6 or 7 helices.
* Includes lactose transporter and GLUT1.

Lactose Transporter: (LacY) Structure
Single polypeptide
* 417 amino acids long
* 12 transmembrane helices
* 2 domains

1 lactose binding site
* Between domains
* Near center of membrane

Multiple families of transporters with relatively low sequence similarity but similar predicted topologies.
Found in bacteria, archaea and eukarya.

• Lactose import is via the active symport lactose transporter (secondary active).
• Proton concentration outside the cell is relatively high (lower pH)
• The proton gradient is maintained by proton pumps
• Proton import is down the electrochemical gradient, driving lactose import up its concentration gradient.

E2: High Lactose Affinity (open to periplasm)
E1: Low lactose affinity (open to cytoplasmic face)

Question 25

Describe the sequence of events occurring in one cycle of action of the E. coli lactose permease transporter.

Answer 25

Lactose Transporter: (LacY)
* Lactose import is via the active symport lactose transporter (secondary active).
* Proton concentration outside the cell is relatively high (lower pH)
* The proton gradient is maintained by proton pumps
* Proton import is down the electrochemical gradient, driving lactose import up its concentration gradient.

E2: High Lactose Affinity (open to periplasm)
E1: Low lactose affinity (open to cytoplasmic face)

• Binding of lactose and protonation of specific residues in the transporter drives a conformational shift.
• Two domains shift (“rock”) relative to each other, opening the binding site to the interior where both lactose and a proton dissociate.

Multiple families of transporters with relatively low sequence similarity but similar predicted topologies.
Found in bacteria, archaea and eukarya.

Question 26

Outline the effect of either inhibition of proton pumping or mutations on the transport of lactose by the lactose permease

Answer 26

• Inhibition of the proton pump (by CN-) leads to lactose export.
• Mutation of Glu or Arg residues involved in transport uncouples lactose import from the proton gradient.

Both conditions lead to lactose transport down its concentration gradient.
- Lactose transporter acts as a passive carrier
- Will equilibrate at lower concentration (once reaches [lactose]media)