Unit 12: Membrane Transport

Membrane transport can be classified into…

Membrane transport can be classified into…

passive and active transport

What is passive transport?

• The transported molecules/ions travel down their concentration gradient

• Can be non-mediated (AKA simple diffusion) or protein-mediated (AKA facilitated diffusion)

What is active transport?

The transported molecules/ions migrate against the concentration gradient → requires some input of energy to pump them across the membrane

The rate of simple diffusion depends on…

• The magnitude of the substance concentration gradient and its solubility in the membranes nonpolar core

• The hydrophobicity, charge and size of the molecule

What kind of molecules will diffuse more readily across the membrane’s lipid bilayer?

Non-polar

What type of passive transport requires special proteins or passive transporters?

Facilitated (mediated) diffusion

In facilitated (mediated) diffusion, what do passive transporters do and not do?

They do not shift the equilibrium of the transport process, but simply facilitate the rate of diffusion by creating a favorable passage

Illustrate the difference between mediated and simple diffusion with the example of glucose transport.

• Glucose can passively diffuse at a slow rate across the membrane unless phosphorylated (via glycolysis)

• The rate of diffusion can be accelerated if glucose crosses the membrane through a special transporter, such as GLUT1.

• GLUT1 facilitates glucose transport across the plasma membrane of mammalian cells.

1. Glucose transporter opening appears on the extracellular side of the cell

2. Glucose binds to GLUT1 outside of the cell → changes conformation of the transporter

3. Glucose transporter opening appears on the intracellular side

4. The bound glucose molecule is released inside the cell.

What special channel proteins facilitate water transport?

Aquaporins

There are 13 different aquaporins in mammals. What is their overall function?

They mediate transmembrane movement of water in the kidney, salivary glands, and lacrimal glands

Aquaporins not only accelerate diffusion of water across cell membranes, but they also…

Enable high selectivity (up to 3 × 10^9 molecules/sec) - they allow passage of water molecules but do not allow hydronium ions to go through

What is an example of an aquaporin and its structure?

AQP1

• Homotetramer - each monomer acts as a water channel

• The transmembrane domain of each monomer is composed of 6 α-helices

• There is an elongated central pore with the narrowest diameter of 0.28 nm → corresponds to the size of a water molecule

Explain the mechanism for high selectivity of aquaporins.

• Two Asn molecules in the central opening can form 2 hydrogen bonds with water molecules

• Hydronium ions cannot pass through because they can only form 1 hydrogen bond

What are porins?

Transmembrane proteins that form pores through which biological molecules can go through to cross biological membranes via facilitated diffusion

What is the general structure of porins?

They often have a ß-barrel structural motif for their transmembrane domains

Where are porins found and what is their function?

• In the outer membrane of gram-negative bacteria

• In the mitochondrial membrane of eukaryotes

• They allow flow of nutrients into cells, while metabolic waste and toxins are translocated out of the cells

Explain OmpF porin from Escherichia coli and its structure.

• The length of the channel is ~5.5 nm (same as the thickness of the lipid bilayer)

• The pore diameter is ~0.7 nm → only molecules smaller than 600 Da can go through

Explain maltoporin from E. coli and the presence of greasy slide.

• Facilitates diffusion of maltodextrin (products of starch degradation)

• Consists of a ß-barrel motif composed from 18 antiparallel ß-strands

Greasy slide

• 6 contiguous aromatic amino acid residues with their side chains are arranged in a left-handed helix

• Continuous exchange of hydrogen bonds as maltodextrin moves through the channel

• The presence of a Tyr residue on the opposite side of the channel from the greasy slide enable selective entering of the ligands from the required shape of the substrate

What are ionophores?

Passive transporters that allow passage of ions across biological membranes

• Can be subdivided into carrier ionophores and channel-forming ionophores

What are carrier ionophores?

They include lipid-soluble ions → shielding their charge and increasing permeability of the membrane of the membrane for ions

What are channel-forming ionophores?

Proteins that span the membrane and form pores through which ions can diffuse

What is an example of carrier ionophores?

Valinomycin

• A macrocyclic molecule consisting of alternating L-Val, D-hydroxyisovaleric acid, D-Val, and L-lactic acid residues connected to each other via peptide and ester binds

• Specifically binds potassium ions over sodium ions

Why does valinomycin favor potassium ions over sodium ions?

The K+ radius minimizes steric strain and potential for degrees of rotation

What is an example of a channel-forming ionophore?

Potassium channels

• Passive channel-forming protein transporters that specifically facilitate diffusion of K+ through the membrane

• Homotetramer → each subunit is composed of two α-helices connected via a short loop

• Inner helices of the four subunits form a channel that tapers in width from the intracellular side (~0.6 nm) to the extracellular side (~0.3 nm)

• The middle contains a 1-nm wide cavity that is lined with hydrophobic amino acid residues

What is found at the entrance of the potassium channel and why is this significant?

• Negatively charged amino acids to attract the positively charged K+ but repel anions

What enables the selectivity of the potassium channel towards K+?

The presence of a signature sequence TVGYG in the selectivity filter (top of the pore)

• Provides the C=O groups of the backbone to coordinate “naked” K+ (w/o the hydration shell) )

What is the main function of ion channels?

To discharge ion gradients across membranes, which are established by ion pumps (active transporters)

How do ion channels prevent uncontrolled flux of ions through the channels?

Ion channels normally open and close transiently in response to a specific signal

What are the four types of ion channels?

1. Mechanosensitive channels

2. Ligand-gated channels

3. Signal-gated channels

   1. Voltage-gated channels

What do mechanosensitive channels open in response to?

A physical stimulus causing local membrane deformation

What do ligand-gated channels open in response to?

A ligand bound from the outside of cells (e.g. neurotransmitter)

What do signal-gated channels open in response to?

An intracellular stimulus (e.g. Ca2+)

What do voltage-gated channels open in response to?

Changes in membrane potential (e.g. nerve impulses)

What are gap junctions and what do they form?

• Tubular structure that are response for exchange of small molecules and ions between cells

• They form intercellular channels

The intercellular channels of gap junctions do not allow what to pass through? What are these channels controlled by?

• Biopolymers (e.g. nucleic acids or proteins)

• Controlled by Ca2+ concentration

Gap junctions channels open and close with what concentrations of calcium?

• Open at [Ca2+] less than 100 nM

• Close at increase of [Ca2+] until it reaches ~0.5 nM

Based on the number of substances protein transporters move across membranes, as well as the mutual relation ship in the flow direction of each substance, protein transporters are classified as…

1. Uniports (one substance is translocated)

2. Symports (two substances are simultaneously transported in the same direction)

3. Antiports (two substances are simultaneously transported in the opposite direction)

What are primary active transporters?

They pump molecules or ions against their concentration gradient by relying on hydrolysis of ATP

What are secondary active transporters?

They pump molecules or ions against their concentration gradient by using the energy of ion gradients as a driving force for the transport process

What are the several classes of primary active transporters and what do they transport?

1. P-type ATPases - Na+, K+, and Ca2+

2. F-type ATPases - Protons across the membranes of bacteria and mitochondria

3. V-type ATPases - Function in plant vacuoles and animal lysosomes; they resemble the F-type ATPases

4. A-type ATPases - Anions

5. ATP-binding cassette - transporters

Na+-K+ ATPase, also known as Na+/K+ pump, can be classified as an ___, and why?

• Antiport

• Pumps Na+ out of the cell and K+ into the cell

• The ions are not simultaneously translocated though

How does the sodium-potassium pump work?

It uses the energy of ATP hydrolysis to translocate 3 sodium ions outside and 2 potassium ions inside the cell

Sodium ion concentration is greater ___ the cell, and potassium concentration is greater ___ the cell.

Outside; inside

Explain the conformational changes that take place in the sodium/potassium pumps.

• The proteins undergo conformation changes from E1 to E2 conformational states in response to phosphorylation of its Asp residue to yield aspartyl phosphate

• The phosphate group is donated by bound ATP

• Phosphorylation occurs only when the protein has Na+ bound

• Hydrolysis of the anhydride bond occurs only when K+ is bound inside the protein

How does Ca2+-ATPase work?

Pumps calcium ions out of the cytosol into the lumen of the sarcoplasmic reticulum

How is Ca2+-ATPase needed?

To maintain low concentration of Ca2+ inside the cell

What is the net equation of the transport process by Ca2+-ATPase.

Explain the conformational changes in Ca2+-ATPase

E1

• Has the ion-binding sites exposed to the cytoplasm

• Has high affinity to Ca2+

• Binds ATO

• P-type ATPase → undergoes transient phosphorylation by transferring a phosphate group from bound ATP to its amino acid residue

ABC transporters are responsible for…

• Pumping small molecules including drugs out of either bacterial or eukaryotic cells (exporters, AKA effluxes)

• Pumping nutrients into bacterial cells (importers)

How does lactose permease work?

• Transports lactose into the cell using the energy of proton gradient established as a result of oxidative metabolism

What is the net equation for the translocation process for lactose permease?

Explain the conformational state changes in lactose permease?

E1

• Low affinity to lactose

• Lactose-binding site facing the interior of the cell

E2

• High affinity to lactose

• Lactose-binding site exposed to the periplasm

Conformational changes are triggered by lactose binding and protonation

What is the driving force for glucose translocation?

The Na+ gradient, which is maintained by Na+/K+-ATPase

Free energy for translocating of an uncharged compound across a membrane depends on what?

The difference of the compound’s concentration between the compartments separated by the membrane

By analyzing this equation, we can see that the translocation process inside cells is…

Spontaneous ▲G < 0 if [C]in < [C]out

• IF the compound is moving DOWN its concentration gradient

Free energy charge for ion translocation across biological membranes depends on both…

• The difference between ion concentration across the membrane

• The membrane potential

• The charge of the translocated ion (z)

What equation is used to determine the fractional saturation of the transporter with the ligand?