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C7 > 4. Membranes and membrane proteins > Flashcards

4. Membranes and membrane proteins Flashcards

1
Q

Why water (6)?

A
  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.
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2
Q

What is the charge density of DNA

A

-2e per bp

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3
Q

Why are electric forces smaller in water?

A

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

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4
Q

Bjerrum length =

A

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

about 0.7 nm in water

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5
Q

Poisson Boltzmann equation

A

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

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6
Q

Debye screening lenght

A
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7
Q

Why are positively charged ions often used for signalling?

A

Because macromolecules are often negative ie DNA, actin

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8
Q

Draw a circuit diagram of the Hodgkin Huxley model

A
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9
Q

Define the membrane voltage

A

= V_in -V_out = V_2 -V_1

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10
Q

Derive the Nernst equation

A
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11
Q

Donnnan equilibrium =

A

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

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12
Q

Name 3 assumptions of the H-H model

A
  1. Different ion channels are independent
  2. Activation and inactivation of different channels is independent.
  3. Model cell is iospotential across the membrane
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13
Q

Derive the Goldman equation

A
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14
Q

How do cells operate far from the Donnan equilibrium?

A

By pumping ions across the membrane. This requires energy

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15
Q

How can cells be divided into cell types?

A

By their resting potential

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16
Q

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

A

eV_nernst + eV_membrane

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17
Q

Explain the process of an action potential

A
  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
18
Q

What is the range of voltages of an action potential

A

Resting ~-70mV

Threshold ~-55mV

Peak ~+40mV

19
Q

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

A

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

20
Q

3 steps of x-ray crystallography of membrane protein

A
  1. Express and purify the protein
  2. Concentrae and crystalise it
  3. Perform crystallography
21
Q

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

A
  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
22
Q

Advantage of cryo em for imaging channels?

A

Involves imaging and aceraging single molecules - crystalisation not necessary.

23
Q

Find the resistance of an ion channel

A
24
Q

Explain how current is measured using the patch clamp technique

A

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.

25
Q

Current through a single ion cannel =

A

i = g n^4 (V_membrane - V_ion)

26
Q

How does K+ selectivity work?

A

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.

27
Q

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

A

Strong interaction - good selectivity, reduced flow.

Weak interaction - poor selectivity, increased flow

28
Q

Why does a multi-ion pore aid flow?

A

e-e repulsion

29
Q

What evidence is there for a binding mechanism of selectivity?

A

Current saturates with concentraiton - must be another rate limiting factor

30
Q

Why is Mg2+ not used for signalling?

A

Very slow dehydration rate.

31
Q

How do ion channels optimise flow rates(4)?

A
  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
32
Q

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

A

argenine

S4 alpha helices

33
Q

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

A
  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
34
Q

Difference between Na+ and K+ inactivation

A

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.

35
Q

Evidence of ball and chain mechanism (4)

A
  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.
36
Q

Evidence for multiple differnt open states?

A

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

37
Q

Onset time

A

T = 1/(k_open +k_close)

38
Q

Evidence for two pore Cl channel

A

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

39
Q

What causes myotonic goats?

A

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.

40
Q

What is long QT syndrome

A

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.

41
Q

Explain two causes of long QT syndrome

A
  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.
42
Q

pV work is given by d

A

W = -N kT ln (V1/V2)

C7 (5 decks)

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