Lecture 15 - Membrane transport

Question 1

What are lipid bilayers freely permeable to?

Answer 1

Freely permeable to non-polar and low molecular weight molecules.

Question 2

Permeability coefficients decrease with increasing what and what? (2 things)

Answer 2

Increasing polarity and molecular weight

Question 3

Why is almost all transport mediated by proteins?

2 reasons

Answer 3

Unassisted diffusion is too slow for biological activity and proteins shield the solute from unfavourable reactions with the bilayer

Question 4

Difference between channel and carrier proteins?

Answer 4

Channel proteins create pores in the bilayer - diffusion from one side to another

Carrier proteins - binding sites that cycle between two sides of the membrane

Question 5

What are the 4 consequences of proteins being held responsible for membrane transport?

Entrusting membrane transport to proteins…

Answer 5

1. Acceleration (carriers: up to several thousand ions per second. Channels: 10^3 times faster - close to the free diffusion rate).
2. Selectivity (specificity of interaction between protein and transported solute)
3. Regulation (gated channels - channel proteins: opening and closing in response to a binding event (ligand gating) or to changes in membrane potential - voltage gating.
4. Coupling (co transport of solutes and inputs of energy)

Question 6

What are the two factors that determine the direction of spontaneous movement across a membrane?

Answer 6

1 Concentration (solutes move from region of high to low conc)

2 Membrane potential

Question 7

Please describe how membrane potential affects movement across a membrane

Answer 7

A negative membrane potential favours the uptake of cations and the release of anions (negative)

Question 8

What are the two gradients that combine to give the electrochemical gradient?

Answer 8

Concentration gradient and electrical gradient combine to give the net driving force

Question 9

When does passive transport occur

Give an example

Answer 9

Occurs when channels and carriers facilitate movement down an electrochemical gradient

e.g. Na+ diffuses into nerve cell when action potential opens a voltage gated Na+ channel

Question 10

Active transport occurs when:

Answer 10

An input of energy allows a carrier to transport a solute against its electrochemical gradient

Question 11

Where can energy needed for active transport come from?

Answer 11

Metabolism, light, electrochemical gradients

Question 12

Examples of carriers that use ATP as an energy source (act as pumps)

Answer 12

Na+ K+ ATPase
Ca2+ ATPase
H+ ATPase
H+ pyrophosphates in the tonoplast of plant cell vacuoles

Question 13

What does reversible phosphorylation do?

Answer 13

Causes a cyclic sequence of conformational changes that move the binding site for the transported ion from one face of the membrane to the other, and back again

Question 14

Bacteriorhodopsin is an example of light energy in active transport. How?

Answer 14

The absorption of light can be used to drive energy in active transport. It is a light driven proton pump in Halobacterium salinarium - light is absorbed the retinal chromophore.

Each bacteriorhodopsin is folded into seven closely packed transmembrane alpha helices and contains a single light absorbing group (chromophore) which gives the protein a purple colour. Retinal is covalently linked to a lysine side chain of the bacteriorhodopsin protein. When activated by a single photon of light, the excited chromophore changes its shapes and causes a series of small conformational changes in the protein that leads t the transfer of one H+ from the inside to the outside of the cell. In bright light, each molecule can pump several hundred protons across per second. The light-drive proton transfer establishes a proton gradient across the plasma membrane which in turn drives production of ATP by a second protein in the cell’s plasma membrane. Energy stored in the proton gradient also drives other energy-requiring processes in the cell.

Question 15

Outline the mechanism of Na|, K+ ATPase

Answer 15

1. Binding of 3 intracellular Na+
2. Phosphorylation of an Asp residue by ATP –> induces a conformational change
3. Release of Na+ to extracellular space
4. Binding of 2 extracellular K+
5. Dephosphorylation reverse conformational change
6. Release of K+ into cytosol

Question 16

Outline the mechanism of Ca2+ ATPase

It is a P type pump that pumps Ca2+ into the sarcoplasmic reticulum

The purpose of the pump is to move the Ca2+ from the cytosol back into the SR (after these Ca2+ ions have bee released into the cytosol from the SR through calcium release channels - stimulating the muscle to contract).

Answer 16

The Ca2+ pump is located in the sarcoplasmic reticulum membrane.

In the pump, amino acid side chains protruding from the transmembrane helices form two centrally positioned binding sites for Ca2+ .

In the pump’s ATP bound nonphosphorylated state, the binding sites are only accessible from the cytosolic side of the SR membrane. Ca2+ binding triggers conformational changes that close the passageway to the cytosol and activates phosphotransfer reaction in which the terminal phosphate of ATP is transferred to an aspartate that is highly conserved . The ADP then dissociates and is replaced with a fresh ATP, causing another conformational change that opens a passageway to the SR lumen through which the two Ca2+ ions exit. They are replaced by two protons and a water molecule that stabilise the empty Ca2+ binding sites and close the passageway to the SR lumen.

Transient self-phosphorylation of the pump is an essential characteristic of all P-type pumps.

Question 17

What are the properties of ion channels

Answer 17

1. High selectivity - the bacterial K+ channel transports K+ rather than Na+ because the energetic cost of removing water of hydration is compensated for K+ by favourable interactions with precisely positioned residues within the channel
2. Open and closed states
3. Regulation - channels can respond to changes in membrane potential, ligand binding, or mechanical stress

Ligands may be extracellular e.g. neurotransmitters or intracellular e.g. ions or nucleotides

4. Transient opening

Question 18

What is lactose permease

Answer 18

A symporter that uses H+ electrochemical gradient to drive uptake of lactose across the e.coli membrane
It is a carrier as no hydrolysis of ATP occurs

Question 19

How does lactose permease work? Describe the cycle

Answer 19

1. Opening to the binding pocket faces outside the cell - an extracellular proton binds to a residue in the permease
2. When protonated the permease binds lactose from outside the cell
3. The structure changes and releases lactose to inside of the cell and a proton.
4. The permease everts to complete the cycle

A cyclic series of conformational changes transfers H+ and lactose from one side of the membrane to the other

Question 20

Mechanism of Na+ K+ ATPase

Answer 20

1. Binding of 3 intracellular Na+
2. Phosphorylation of an Asp residue by ATP inducing a conformational change
3. Release of Na+ to extracellular space
4. Binding of 2 extracellular K+
5. Dephosphorylation reversing the previous conformational change
6. Release of K+ into the cytosol

Question 21

What is the concentration of K+ and Na+ inside and outside of cells?

Answer 21

Conc. of K+ is higher inside cells and reverse is true for Na+ and the pump maintains these concentration differences

Question 22

Why is the pump said to be electrogenic?

Answer 22

It pumps out three positive ions for every two it pumps in - it drives a net electric current across the membrane creating an electric potential (the cell’s inside being relatively negative to the outside)

Question 23

Describe the structure of the bacterial K+ channel

Answer 23

Four identical subunits - each with 2 transmembrane alpha helices - forming a cone shaped channel that narrows towards the external surface of the membrane - wide end facing outside of the cell where the K+ ions exit from the channel

Question 24

Describe the selectivity filter and why it is important

Answer 24

The polypeptide chain that connects two transmembrane helices forms a short alpha helix ( the pore helix) and a crucial loop that protrudes into the wide section of the cone to form the selectivity.

The selectivity filter loops from the four subunits to form a short, narrow core that is lined by the carbonyl oxygen atoms of their polypeptide backbones

Highly conserved amino acid sequence Thr-Val-Gly-Tyr-Gly. The selectivity filter explains the selectivity of the ion channel - it contains 4 binding sites for K+ –> remember, only 2 non-adjacent sites can be occupied at the same time due to electrostatic repulsion between the cations

Question 25

Please outline the mechanism of the bacterial K+ channel

Answer 25

1. Hydrated K+ ions enters the pore from the cytosol and remains hydrated until reaching the selectivity filter 2. The water of hydration is removed (the K+ ion must lose all of its bound water molecules) and dehydrated K+ binds sequentially to the sites in the selectivity filter (interacts with the carbonyl oxygens lining the filter) 3. The electrostatic repulsion arising from the binding of a second K+ pushes the first K+ into the extracellular space 4. Voltage gating of the channel is caused by a rearrangement of the transmembrane helices, opening and closing the cytosolic entrance to the channel

Question 26

Describe co-transporters and give 2 examples

Answer 26

Co-transporters use the spontaneous movement of one solute down its electrochemical gradient to drive the active transport of a second solute Na+-glucose co transport in the membrane of animal cells H+-nitrate co-transport across the plasma membrane of plant cells It works because secondary active transport depends on primary active transport to establish the electrochemical gradients for the Na+ and H+ ions.

Question 27

What is key to the mechanism of Na+ K+ ATPase?

Answer 27

Na+ dependent phosphorylation of the carrier and the K+ dependent dephosphorylation is key

Question 28

Textbook mechanism of Na+ K+ ATPase

Answer 28

The concentration of K+ is normally much higher inside than outside cells and vice versa for Na+. The pump is found in literally all plasma membranes of animal cells. and it maintains these differences. Like the Ca2+ pump it actively pumps Na+ out of the cell against a steep electrochemical gradient and pumps K+ in. Loads of energy is consumed in order to do this. The Na+K+ pump drives 3 positively charged ions out of the cell for every two it pumps in. Drives a net electric current across the membrane, tending to create an electric potential, with the cell's inside being negative relative to the outside.

Question 29

Further information/description of the bacterial K+ channel

Answer 29

The structure of the selectivity filter explains the ion selectivity of the channel. A K+ ion must lose almost all of its bound water molecules to enter the filter - where it interacts with the carbonyl oxygens lining the filter. The oxygen are rigidly spaced at the exact distance to accommodate a K+ ion . Therefore, an Na+ ion cannot enter the filter because carbonyl oxygen atoms are too far away from the smaller Na+ ion to compensate the energy expense associated with the loss of water molecules required for entry.