4. Membranes and membrane proteins

Question 1

Why water (6)?

Answer 1

1. Can interact with charged particles - electrostatic interactions can occur.
2. Hydrophobic and hydrophilic lipids allow self assembly of proteins in water.
3. Thermal fluctuctuations are provided by Brownian motion - water molecules are a good size
4. Adjusts to differnent pH by disociating
5. DNA is charged in water due to pK of water
6. Salts dissolve in water - useful because salts acan influence rate of reactions since many reactions rely on identifcation of specific surfaces.

Question 2

What is the charge density of DNA

Answer 2

-2e per bp

Question 3

Why are electric forces smaller in water?

Answer 3

Water dipoles orient themselves with the field which acts to reduce it.

Question 4

Bjerrum length =

Answer 4

the separation of two particles at which the electrostatic energy is comparable to the thermal

about 0.7 nm in water

Question 5

Poisson Boltzmann equation

Answer 5

d^2V/dx^2 = 2z^2 e^2 c_infinity/DE_0

Question 6

Debye screening lenght

Answer 6

Question 7

Why are positively charged ions often used for signalling?

Answer 7

Because macromolecules are often negative ie DNA, actin

Question 8

Draw a circuit diagram of the Hodgkin Huxley model

Answer 8

Question 9

Define the membrane voltage

Answer 9

= V_in -V_out = V_2 -V_1

Question 10

Derive the Nernst equation

Answer 10

Question 11

Donnnan equilibrium =

Answer 11

in equilibrium the Nernst potential is the same for all ion species and is equal to the membrane potential

Question 12

Name 3 assumptions of the H-H model

Answer 12

1. Different ion channels are independent
2. Activation and inactivation of different channels is independent.
3. Model cell is iospotential across the membrane

Question 13

Derive the Goldman equation

Answer 13

Question 14

How do cells operate far from the Donnan equilibrium?

Answer 14

By pumping ions across the membrane. This requires energy

Question 15

How can cells be divided into cell types?

Answer 15

By their resting potential

Question 16

How much energy is required to pump an ion across a membrane?

Answer 16

eV_nernst + eV_membrane

Question 17

Explain the process of an action potential

Answer 17

1. Depolarisation - Na+ channels activate in response to a depolarisation, which further increases the depolarisation. Na+ enters the cell.
2. A peak is reached before the Na+ equilibrium potential for two reason
   
   1. Na+ channels begin to inactivate spontaneously
   2. K+ channels begin to activate, alllowing K+ ions out.
3. Depolarisation - K+ channels are open
4. Hyperpolarisation - K+ channels take time to inactivate, causing an overshoot

Question 18

What is the range of voltages of an action potential

Answer 18

Resting ~-70mV

Threshold ~-55mV

Peak ~+40mV

Question 19

What is the differnce between myelinated and unmyelinated sections of axions?

Answer 19

Myelinated sections are not excitable - do not produce action potentials - propagate signals passively.

Unmyelinated use action potentials to boost the signal - this is called saltatory conduction

Question 20

3 steps of x-ray crystallography of membrane protein

Answer 20

1. Express and purify the protein
2. Concentrae and crystalise it
3. Perform crystallography

Question 21

Why x-ray crystallography of ion channels hard (3)?

Answer 21

1. Overexpression can be toxic - cells don’t like having lots of holes
2. Purification can be difficult
3. Membrane proteins are amphiphatic - don’t like being in solution

Question 22

Advantage of cryo em for imaging channels?

Answer 22

Involves imaging and aceraging single molecules - crystalisation not necessary.

Question 23

Find the resistance of an ion channel

Answer 23

Question 24

Explain how current is measured using the patch clamp technique

Answer 24

A pipet is polished the the end and sunction is applied through it, creating a seal. Simple electronics can then be attached to analyse the current through the channel.

Question 25

Current through a single ion cannel =

Answer 25

i = g n^4 (V\_membrane - V\_ion)

Question 26

How does K+ selectivity work?

Answer 26

Using hydration energies - the O on a carbonyl (C=O) group mimics the position of Os in water around the ion, called the hydration shell.

Question 27

Why must the interaction strength of the ions with the binding sites in a channel be carefully balanced?

Answer 27

Strong interaction - good selectivity, reduced flow. Weak interaction - poor selectivity, increased flow

Question 28

Why does a multi-ion pore aid flow?

Answer 28

e-e repulsion

Question 29

What evidence is there for a binding mechanism of selectivity?

Answer 29

Current saturates with concentraiton - must be another rate limiting factor

Question 30

Why is Mg2+ not used for signalling?

Answer 30

Very slow dehydration rate.

Question 31

How do ion channels optimise flow rates(4)?

Answer 31

1. Oppositely charged residues sit near mouth of channel 2. The filter region is short compared to the distance across the membrane 3. Selectivity fileter doesn't rely on too strong an interaction 4. Multiple ions in pore - creates repulsion

Question 32

Positive resideues in K+ gate responsible for gating are... and exist in the ... of the channel

Answer 32

argenine S4 alpha helices

Question 33

How can gating of K+ channels be studied (3)?

Answer 33

1. Crystallography - shows there is a confomational change in the structure of the channel. 2. Cysteine accessability studies - replace an amino acid with a very reactive cystein. Measure rate of reaction outside of cell. 3. As above but couple the custein to an environmentally dependent fluorescent probe which reacts to hydrophobic/hydrophilic 4. Measure gating current using patch clamp

Question 34

Difference between Na+ and K+ inactivation

Answer 34

Na+ - ball and chain on linker - IFM linker K+ - ball and chain All Na+ channels inactivate to produce peak of action potential, some K+ channels inactivate to prevent hyperpolarisation.

Question 35

Evidence of ball and chain mechanism (4)

Answer 35

1. Electophysiological - patch clamp studies of Na+ channels in giant squid axions treated with pronase show that this treatment stops inactivation occuring. Suggests a physical mechanism 2. Molecular - add 20 amino acids that make up the ball and chain to non-inactiating K+ channels. Inactivation then occurs. Shows that these residues are important. 3. Structural - can see chain made of alpha helices and a highly disordered ball. Easier for K+ - ball is residies 1-20 and chain 20-45. 4. Remove balls from some subunits of K+ channels. Only one ball is required for inactivation to occur but inactivation is faster when there are multiple.

Question 36

Evidence for multiple differnt open states?

Answer 36

Hard to resolve peaks on graph of open duration against probabilty of that open duration

Question 37

Onset time

Answer 37

T = 1/(k\_open +k\_close)

Question 38

Evidence for two pore Cl channel

Answer 38

Current time plot of a single channel shows two distinct current states

Question 39

What causes myotonic goats?

Answer 39

Defective Cl channel Usually when an action potential fires the Cl gates is open. However, if it gets trapped in the inactive state the overall resistance will go up. This has the effect of activating other channels causing multiple action potentials to fire at once and the muscles to freeze up.

Question 40

What is long QT syndrome

Answer 40

In long QT syndrome the interval bewteen the Q wave and T wave of a heartbeat pulse is unusually long. This means the heart pacemaker may try and fire another action potenital before the previous one has finished. This causes the heartbeat to become unsynchronised, leading to heart attack. Can also cause reduced ventricular refilling, reducing blood flow to the brain.

Question 41

Explain two causes of long QT syndrome

Answer 41

1. Na+ channels don't inactivate properly due to a mutated voltage sensor. This means they keep opening and closing, preveniting depolarisation occuring when it normally would and increasing QT. 2. Some K+ channels don't work - reduces overall K+ current.

Question 42

pV work is given by d

Answer 42

W = -N kT ln (V1/V2)