Water, membranes and ions Flashcards
What is polarity?
The ends/sides of two things are different
What bonds does water have?
- covalent hydrogen bonds
- oxygen is partially negative
- hydrogen is partially positive
What do hydrogen bonds enable a water molecule to interact with?
Four other water molecules
Why is water unique and why is this useful?
- it’s density of ice is less than when it is a liquid
- If water was densest when it was a solid then ice would sink and the water would freeze from the bottom upwards and this wouldn’t work for life on earth
What keeps water on earth in a liquid form?
The hydrogen bonds in water
What is an ion?
Any atom or molecule that has gained or lost one or more electrons
Why are ions important?
- Carry signals in the body
Action potentials - Act as an energy store
Secondary active transport - Interact biochemically with proteins and other molecules
Ca2+/ troponin C in muscle contraction
Mg2+/ ATP
what are ions that are physiologically useful and give examples
- charge carriers or exert osmotic pressure
- Na+
- K+
- Cl-
What are ions that are biochemically useful?
Trace metals e.g. Mg2+, Fe3+, Zn2+
Usually involved in enzymatic reactions or form parts of proteins
Which ion is both physiologically and biochemically useful and how?
- Ca2+
Biochemically useful – signally in muscle
Major charge carrier in heart – use calcium to bring about action potential – physiologically useful
What happens to ions in aqueous solution?
- If you put a positively charged ion in water it will attract the negatively charged oxygen. This forms a shell around the ion
The first shell around the ion is the primary hydration shell - Negatively charged ions attract the positively charged hydrogens to create their hydration shell
What determines the ionic size?
- the hydration shell
- smaller ions will have a bigger hydration shell because they have a higher charge density
Why is the hydration shell important?
- Proteins only interact when they have a hydration shell
- Hydration shell affects mobility in solution
- Hydration shell is the effective ‘size’ of ion
- Hydration shell affects interactions with proteins
What is the lipid bilayer formed from?
- formed from amphipathic lipid
- Hydrophilic polar head (interacts with water)
- Hydrophobic tail (associates with other lipids)
- Amphipathic nature drives formation of bilayers
What does the lipid bilayer enable the cell to maintain?
gradients
what is active transport?
- The movement of a substance in a direction that requires the cell to expend energy
- The substance will be moving against its concentration gradient (or electrochemical gradient in the case of ions)
What is passive transport?
- The movement of a substance in a direction that requires the cell to expend no energy
- The substance will be moving down its concentration or electrochemical gradient
What do membrane proteins do?
Allow cells to establish ion gradients and use them
Whay do pumps do?
- Concentration of ions against gradient needs energy
Cells get this energy from hydrolysis of ATP
Cells use special proteins called pumps
Pumps perform PRIMARY ACTIVE TRANSPORT (because they directly use ATP)
What are the basic features of pumps?
Live in membranes Move ions against gradients Coupled to ATP (usually) Usually fairly slow Nearly always move cations
What does the Calcium pump do?
Hydrolyse ATP and use it to extrude Calcium out of the cell
What does the Sodium potassium ATPase (sodium pump) do?
Takes 3 sodium ions and moves them from the inside of the cell to the outside
Moves 2 potassium ions the opposite direction
Uses energy from hydrolysis of ATP
What are the effects of the Sodium pump?
Generates a Na+ and K+ gradient: Na+ low in cytoplasm, K+ high in cytoplasm
Electrogenic (2+ in, 3+ out – electrical gradient)
How much energy do cells expend keeping the Sodium pump going?
about 25% of ATP
Gradients represent a source of energy. What can they be used to do?
Can be used to transmit information e.g. signalling via ion channels
Can be used to power cellular processes (e.g. transport of other ions via cotransporter – secondary active transport
What do antiporters/exchangers do?
Use concentration gradient or an ion that is low inside and high outside to power the movement of an ion that is low inside and high outside across the membrane
Ions exchanged in opposite directions
What do symporters do?
Use concentration gradient or an ion that is low inside and high outside to power the movement of an ion that is low outside and high inside across the membrane
Ions moved in the same direction
How does the Sodium-Calcium exchanger (antiporter) work?
Low calcium inside the cell and high outside
High sodium outside the cell and low inside
Use sodium gradient moving inside to power the movement of calcium from the inside to the outside
3 sodium’s move inside the cells
1 calcium moved outside the cell
What is an ion channel?
a protein lined hole through the membrane (over-simplification)
How does the ion channel help ions get into cells?
- the channel is lined with amino acids that interact positively with the ion
What do ion channels only work through
diffusion
What are the basic properties of ion channels?
- All ion channels are transmembrane proteins
- All selectively permeable
- Opening and closing are controlled (gating)
- Diverse
How does the selectivity filter in an ion channel work?
- Size: ions have to form the correct interactions
- Charge: ion channels will repel ions of the same charge
What does gating do?
Help to open an ion channel
What are the different type sof gating?
Mechanical Second messenger inhibitory/activating Phosphorylation Leak (open most of the time) Ligand-gated Voltage-gated Proton-gated G-protein-gated Temperature-gated
How are ion channels usually classified and how are ligand-gated channels classified?
Gating
Ion selectivity
E.g. voltage-gated potassium channel
But for ligand-gated channels, named after natural ligand (activating molecule), e.g. GABAA receptor – chloride channel whose opening and closing is controlled by Gabba-aminobutyric acid – a neurotransmitter. When two molecules of GABA bind to the receptor the chloride channel opens
When are ligand-gated channels opened?
When an activating ligand (agonist) binds to them
What are ligand gated ion channels split into?
Cys-loop receptors
Ionotropic glutamate receptors
Give examples of Cys loop receptors
- Nicotinic AChR – cation channel gated by acetylcholine
- GABAa
- 5HT3 receptor
- Inhibitory glycine receptor
Give features of ionotropic glutamate receptors
- Mainly located in CNS
- All cation channels
- Structurally different to Cys loop receptors but operate in similar way
- Respond to glutamate
What do all ligand gated channels have?
- Pore – lets ions through
- Ligand binding site – tells channel to open in response to ligand binding
- Coupling mechanism – couples channel opening to ligand binding
- Desensitization mechanisms – close channel if ligand binds for too long
What is the operation of a typical ligand-gated ion channel?
- Normally in closed state
- When ligand binds channel opens
- After ligand has been bound for a while the receptor enters desensitized state – ligand is still bound but channel closes
What is the structure of the nicotinic acetylcholine receptor (it is an example of a ligand-gated channel)
- Pentamer of five similar subunits
- About 50% of the protein is outside the cell – portion of the protein where the acetylcholine binding sites are found
- Portion in the membrane – alpha helices found here
- Half way through the membrane you find the gate – determines whether the pore is opened or closed
What led to the characterisation of nAChR?
- Taiwanese snake
Taiwanese banded Krait produces toxin which is an almost irreversible ligand at acetylcholine receptor – it is an agonist and blocks the receptors
Paralyses the snakes prey
The toxin is useful in purifying the nAChR – if you want to characterise a protein you need a good ligand that binds to it - Electric fish:
Electric fish – torpedo electric ray. Uses electric shocks to paralyse it’s prey
Electric organ is similar to muscle
Stores it’s energy within the electrochemical gradients in the organ – gradient set up by sodium pump
Energy release triggered by ‘molecular switch’ (nAChR)
What causes a voltage gated ion channel to open?
Membrane potential changes (usually depolarisation) and the channel opens
Give examples of voltage gated ion channels
- Calcium channels (Cav)
- Sodium channels (Nav)
- Potassium channels (Kv)
Essentially what type of proteins are voltage gated ion channels?
tetrameric proteins
What is the structure of the Potassium voltage gated channel?
- Tetramer of four equivalent subunits
- Crosses the membrane 6 times fully – 6 transmembrane domains (TM domain) in each subunit
- Membrane dipping domain between 5th and 6th domain (dips but doesn’t go all the way through). It forms the lining of the channel
- The fourth transmembrane domain is the voltage sensor
How many Kv channels does the human genome have?
50 Kv channel generally subdivided into 12 families
Where in evolution does the Kv channel appear?
Early - present in prokaryotes
What does the TPC family look like and why?
looks like two Kv subunits joined together – thought to have evolved by gene duplication – potassium channel like structures strung together
What is the structure of the Cav and Nav channels?
- The pore forming subunit of Cav and Nav is called the alpha subunit – consists of four copies of a voltage gated potassium like structure strung together in a single peptide
- The alpha subunit has four pseudo-subunit domains, each of which contains 6 transmembrane domains, for a total of 24
- Each of the four segments in the alpha subunit called a pseudo-subunit
- Instead of having four separate subunits that come together the alpha subunit folds so that the four pseudo-subunits form the channel
- Main difference between Cav and Nav is that instead of having four separate subunits they have four subunits joined together to make one long peptide
What are the different Calcium channel subunits and what could the native channel possibly be?
- A Cav 1.1-1.4
- a Cav 2.1-2.3
- a Cav 3.1-3.3
- 4B 4a2(sigma) 8gamma subunits
- Native channel possibly 1a: 1B: 1a2(sigma) – 3 subunits
What are the different Sodium channel subunits and what could the native channel possibly be?
- a Nav 1.1-1.9
- 4B subunits
- Native channel possibly 1a: 1B
What are voltage-gated ion channels all selective for?
Cations
What is the knock on mechanism?
- Selectivity filter strips off the hydration shell from our ion
- Our ion moves into the channel and knocks forward ions already in the channel
- In the Kv channel the potassium ion interacts with the oxygen molecules in the channel – this means that the hydration shell is striped from the ion
- If the ion is too small it won’t be able to interact with the oxygen and will keep it’s hydration shell
- If the ion is too big it won’t be able to fit through the channel
How does the sodium channel open?
- Open rapidly but inactivated after 1ms
- Sodium channel exists in:
Resting state
Open state
Inactivated state - Transitions between the states are dependent on voltage
- Changes between states are more probable as the membrane depolarises
- Voltage sensor has charges in it – if you put a charged substance in an electrical field and then change the electrical field the charged particle will move – this happens with the voltage sensor – as we depolarise the membrane the voltage sensor moves and opens the gate of the channel
- Sodium ions can now cross the membrane – as they cross it will increase the membrane potential even more – as it gets even more positive the inactivation gate will swing up and block the channel
How does the voltage sensor work?
Voltage sensor has charges in it – if you put a charged substance in an electrical field and then change the electrical field the charged particle will move – this happens with the voltage sensor – as we depolarise the membrane the voltage sensor moves and opens the gate of the channel
Does the potassium channel open more slowly or quickly than the sodium channel?
More slowly
What are structure function studies?
- when you clone subunits and put them into a plasmid expression vector and mutate single amino acids or even whole sections of subunits. You then put the plasmid into a mammalian cell line and compare the properties of the mutant receptor to the wild type using techniques such as patch clamp, calcium imaging or even simple radioligand binding assays.
What is the problem with structure function studies?
It is a relatively slow way to go about things – nAChRs are big proteins with over 2500 amino acids in total, which is a lot of ground to cover using mutagenesis. It is also an indirect method – if we mutate amino acid 190 which is a tyrosine to glycine then acetylcholine affinity dramatically drops – does this mean tyrosine 190 is involved in binding acetylcholine or does it mean that changing this amino acid is nowhere near the acetylcholine binding site but is important for the overall structure of the receptor that when the mutation is made, the receptor no-longer folds properly
What did Dutch researchers clone and obtain an x ray crystal structure of?
a snail (Lymnae Stagnalis) protein that they called the ‘acetylcholine binding protein; (AChBP). It was a water soluble protein that seemed to be the N terminal extracellular domain of an nAChR • It is thought that the AChBP evolved from a nicotinic receptor subunit that had its transmembrane domains deleted. It has a reasonably high sequence homology with the N terminal extracellular domains of mammalian nicotinic receptor subunits
What is the synaptic role of AChBP?
- Acetylcholine is removed from mammalian synapses by the action of an enzyme, acetylcholinesterase
- In the snail an extra mechanism is present. When acetylcholine (ACh) is released into synapses in the snail’s nervous system, it binds not only to nicotinic acetylcholine receptors (nAChRs) on the post synaptic neurone, but also on nearby glial cells. In response, the glial cells release AChBP into the synapse. AChBP acts as a molecular sponge for acetylcholine, lowering its levels in the synapse
What can AChBP act as a template for?
A process called homology modelling
What happens in homology modelling?
a protein whose structure has been worked out by an experimental method such as X ray crystallography is used as a template for another protein. This method will only work if the two proteins have a similar amino acid sequence and are thought to have similar overall structures
What is the process of homology modelling?
- First the amino acid sequences are aligned to give the best match between identical and similar amino acids in the two sequences. The alignment process may also require gaps to be inserted in order to take account of differences in the sequences
- Next, the alignment is used to map the unknown protein onto the 3D structure of the known protein. This process gives the researcher a rough structure for the unknown protein that can be refined using computer molecular modelling techniques.
Using homology modelling techniques what have researchers been able to do?
use the AChBP as a template for nicotinic acetylcholine receptors and other members of the ligand-gated ion channel family such as the GABA(A) receptor, 5HT3 receptor and inhibitory glycine receptor
What have searches of prokaryotic genomes revealed?
ligand-gated channel-like sequences in several eubacteria and even an archaebacterial genus
What proteins were crystallised from eubacteria
- ELIC, from the gram-negative bacterium Erwinia chrysanthemi is a cation selective channel that can be activated by amines such as GABA
- GLIC from the cyanobacterium Gloeobacter violaceus was crystalised. GLIC is also a cation channel but is gated by protons.
What is the structure of ELIC and GLIC and what are they similar to?
- they are similar in their overall structure to eukaryotic channels such as the nicotinic receptors
- they are homopentamers
- each of their subunits has four transmembrane domains
Why are cys-loop receptors called this?
because the ‘cys loop’ is a structural feature found in these channels; a protein loop in the extracellular domain by a bond between two cysteine residues (S-S bond)
What is absent in the bacterial relatives of the nicotine receptor, so what are they called instead?
In the bacterial relatives of the nicotine receptor the cys-loop is absent. Members of this receptor family are now known as pLGIC: pentameric ligand-gated ion channels. The pLGIC family includes both the eukaryotic and prokaryotic proteins
