Paper 2 Flashcards
Describe/explain the figure below.
In order to do this study, had to use X-ray crystallography, nowadays we can use cryo-electron microscopy to do these experiments. Easier to crystallise bacteria channels than human channels, so had to justify that it is similar enough to human channels such that if you publish something about the bacterial channel you can make deductions about human channels. Figure 1 shows that yes, human and bacterial channels are similar enough that if you crystalise the bacterial channel KcsA channel you can apply it to human channels.
The last two amino acids sequences are different for the cyclic nucleotide gated channels and because there is so much conservation, the researchers thought that this is one part of the channel that is very important for conferring this massive selectivity for K+ over Na+.
You have a channel, and this channel conducts K+ not Na+, which are both +1 charge and roughly the same size. Yet you have this channel that somehow lets K+ ions through really fast but doesn’t let Na+ through basically at all, and definitely not really fast. So question is, why is that the case? When they did this study, they used two channels, and neither of them were from humans, so this figure is basically saying that “we’re going to study a bacterial channel, and everything that we’re going to publish is on a bacterial channel, but it’s similar enough to all the other K+ channels that there’s something universal about what we’re going to determine.
There are two cyclic nucleotide gated channels which are not K+ selective, and they don’t have the same five amino acids conserved. So if you take away 2 AA that all the others have and that gets rid of your selectivity for K+, then maybe that’s the place that we should look when we want to figure out why the K+ channels are selective for K+ over Na+.
Describe/explain the figure below.
Experimental density maps show the helix structure of the channels. Purpose, especially A-B, is to show the tetramers and display the density of the electrons along the pore region. The reason that they would map out the electron density is to show the fact that the selectivity filter is negatively charged. There are acidic AAs that line the channel to make it negatively charged and to raise the concentration of the cations.
The selectivity filter is at the very beginning of the channel. The residues in the selectivity filter are negatively charged, so they attract cations, which is why anions don’t go through the channel, only cations do. Including the electron density map because you can predict from the AA sequence which stretch of AA will be a helix, which won’t be a helix, which will be B-barrel or sheet, but have to prove it, which is what they did here.
So here: you have this tetramer, you have helixes pointed at the aqueous cavity, and you have negatively charged AAs that line the selectivity filter.
Describe/explain the figure below.
(A) shows a birds eye view of the channel from the EC side. You see the subunits within the tetramer and the green ball is the K+ ion.
(B) is the same thing from a different POV.
This is a tetramer and this is an integral membrane protein. The majority if the protein s in the membrane. When you crystalise these, you can’t keep them in membranes but you have to prove that it looks like it would exist in the membrane and not in the cytosol. The way that these proteins work is that they stabilise in the membrane, which are mad of fat, by having these aromatic AAs near the top of the membrane where the pore is, and near the bottom of the membrane where the ions come out.
So: spans membrane, it’s a tetramer, and aromatic AAs right above and outside of the selectivity filter stabilise the interface between the outside of the cell and the inside of the cell, both at the top and the bottom.
Properties of aromatic AAs - they are polar and hydrophobic, which means that they are comfortable existing at the interface between the outside and inside of the cell, but they have properties that stabilise the channel exactly where it is. They help keep the channel where it is in the membrane.
Describe/explain the figure below
Mutagenesis studies on the channel. They use a different kind of K+ channel, called Shaker, from a fly. Can be easier to do genetic manipulations on fly-derived plasmids than on bacterial-derived plasmids. Switch to a different model system. Point of this figure is to show that the selectivity filter – if you change the nature of the residues that make up the selectivity filter, it no longer becomes selective. The selectivity filter is at the top of the channel, facing outside, and if you change the residues that make it up, it no longer becomes selective. So there is something about those AA residues that is important for channel selectivity.
The carbonyls stabilise the ion as it sheds its water and goes into the channel.
Describe/explain the figure below
(A) – showing the location of different charge densities and also the location of the hydrophobic parts of the channel. Red is neg charged areas at the entry and exit way. The part of the channel after the selectivity filter is where you get yellow, which is the hydrophobic part, which is to help minimise the interaction between the K+ ion and the channel (long pore segment of the channel).
(B) – a different perspective on the channel. The long narrow selectivity filter by the wide cavity at the entrance of the channel. Go down into another channel leading into the cytosol. Shows us how the channel is structured in terms of charge density and atomic structure.
You have negatively charged amino acids (red), which allow this + charged ion to coast through the channel. But, you have these non polar AAs that keep the pore open to the right diameter. If you don’t have them then the pore closes, which makes it non-selective and possibly even non-conductive.
Have some AAs that keep the pore open, others that attract ions into the pore, and others that allow cations to go through the pore easily.
Pore is 34 Angstroms long, the selectivity filter is only 12 Angstroms long. So the cations don’t have to go through the selectivity filter for very long, and after can get conducted easily through the aqueous pore.
OVERALL: Have this negatively charged tunnel that the cations get conducted through.
Describe/explain the figure below
A and B are the maps showing the positions of ion Rb+ for a, Cs+ for b, and these ions were used in place of K+ as a model, because they are more electron dense. And the map is showing the positions of the ions in the selectivity filter, showing that two ions can be in the selectivity filter at once, in two different positions (inner ion position, outer ion position).
C – the diffuse electron density in the cavity centre likely reflects a hydrated cation cloud rather than an ion binding site (the lowering of the electrostatic barrier facing a cation crossing a lipid bilayer).
They used these more e- dense substitute ions, do X-ray crystallography with the channel they were already working with and this type of ion added in instead of K+. They found out that there are two ions in the selectivity filter at a time, and there’s this density of a single cation in this aqueous pore that is being stabilized by the dipole moment formed by the carbonyls on the AAs. So basically ions don’t like going through lipid bilayers, because they are made up of fat, so if the ion was going through, and there is a massive energy barrier in the middle, the ion would want to go back out from where it came rather than keep on going. But if you have this dipole moment facing directly into the middle, then you will have a negative charge (shown in F7 as the red part of the helix), the cation will be electrostatically stabilised in the middle. This allows the ion to get over the energy barrier, so it keeps on conducting through the pore. Chemical gradient and electrical gradient wants K+ to come into the cell, and this gives it the opportunity to do that.
Describe/explain the figure below
Demonstrates the mechanisms that help overcome the electrostatic issue. You have this cation stabilised in the middle of the pore (and of the lipid bilayer), where it doesn’t want to be. You have these helixes pointed directly towards the place that the cation doesn’t want to be. The helixes form a dipole moment which puts this negative charge around the aqueous pore. This negative charge stabilises the positive charge of the cation enough so that it will continue going through the cell.
The selectivity filter occludes water, is not big enough for the K+ to enter while it’s surrounded by water. K+ sheds the cage of water in order to go thru the selectivity filter. Na+, which is smaller, is not able to be stabilised in the selectivity filter, so if it sheds its water, it’s too small, doesn’t get stabilised, and isn’t able to go into the aqueous pore and doesn’t go through (as much).
Stabilised: need the staying forces to be greater than the leaving forces. Electrostatic interactions can cause two things to stay together, or two things to push apart. So if the electrostatic interactions were so great that your + ion did not leave, that would be bad, but the concern is that the ion doesn’t want to be in the middle. So the electrostatic forces between the dipole moment and the ion are large enough to overcome the ion’s desire to not be in the middle of a fatty bilayer, for long enough that it will exit the channel from the other direction.
Describe/explain the figure below
Have an idea of where the ions are on the density map.
Aromatic AAs are right above and outside the selectivity filter, and are essential in making the selectivity filter the selectivity filter.
Summarise the paper
They used X-ray crystallography to do this.
They used 2 diff channels, KcsA (bacterial channel) and Shaker (fly channel).
Critical question is how can this channel conduct K+ ions really fast but somehow doesn’t allow Na+ into the channel.
The K+ channel is a tetramer, so 4 identical subunits that form an inverted cone. There are Aromatic AAs around the selectivity filter that keep the filter open at the right distance and they also stabilize the channel inside of the bilayer.
Outside the cell, you have K+ ions and Na+ ions. When K+ and Na+ are outside the cell, they are surrounded by water; they have to shed their cage of water in order to go through any channel.
The pore of the channel, which is where the ions go through, is surrounded by neg charged amino acids. (Makes sense, conducting a cation).
In the middle of the channel is an aqueous pore. There’s water inside the cell, water in aqueous pore, and water leading from aqueous pore into outside the cell. The selectivity filter which is only 12 Angstroms long does not allow water through, so when the ions come through they have to shed their cage of water.
The s. Filter is exactly the right size such that K+, when it sheds its water cage, fits in, but Na+ is too small to be stabilized to it leaves.
Will have 2 K+ in the selectivity filter at a time. Because they both carry + charge, they will push each other through the filter.
There are 2 helixes pointed towards the aqueous pore. The ions do not want to be in the middle of a membrane because they are charged. So in order to overcome that energy barrier, there must be something that stabilized the ions that are in the aqueous pore. 2 things do that: 1 – unlike selectivity filter the aqeous pore is full of water, so cage reforms around the ion inside the pore. 2 – we have four helixes pointed directly at the aqueous pore, which have a dipole moment that has strong negative charge facing towards the aqueous pore, this stabilizes the ions enough to overcome the energy barrier that would otherwise prevent them from being conducted down through the membrane from the outside of the cell to the inside.
There are carbonyls on the functional groups fo the AAs that face towards the selectivity filter, which is what stabilizes the ions in the selectivity filter and what defines the size of that region. Selectivity filter is able to select for K+ over Na+ because it is the right size to stabilize K+ (w/o water cage) but not Na+ (w/o water cage).
The K+ channel conducts the K+ so fast because 1–it allows 2 ion in the selectivity filter which pushes them through. 2 – there’s a aqueous cavity in the middle which overcomes the electrostatic barrier the ion passing through the lipid bilayer. 3 - there are helixes pointed directly at the aqueous cavity that form a dipole moment that stabilizes the positive charge of the ion with their neg charge. –> conducts fast, and only conducts K+ ions.
KscA Channel
Outside of cell has K+ and Na+ in the outside space, and moving towards the inside of the cell.
4 subunits, identical subunits which coalesce to form a pore in the inside layer, forming an inverted tepee. Pore is made up of 3 thirds: selectivity filter makes up first third, aqueous cavity makes up second third, and third third is where the ion pass through (rest of the pore)
There’s water everywhere, outside cell, inside cell, and in aqueous cavity and last third of the channel. Ions outside the cell surrounded by water, by cage of water/hydration shell.
Electrostatic interactions between ions and oxygens on the water stabilise the ions energetically.
Selectivity filter made up of amino acids with carbonyl groups (partially negatively charged) with oxygens pointed directly towards the selectivity filter. It is the first thing the ions see when passing from outside to inside.
K+ ions are bigger than Na+. In terms of size, the ions are bigger when hydrated, and neither can fit into the selectivity filter. Both ions have some motivation to enter selectivity filter, but in order to do so they must lose their hydration shells, which neither of them want to do. The reason that K+ is willing to enter s.f and shed hydration shell is that it is exactly the right size to fit into the s.f. The O-s are close enough (from carbonyl groups) to the K+ in the s.f that they can take the place of the O-s from H2O cage. Na+ is so small so far away from carbonyl O-s, so not electrostatically stable enough to stay in s.f and get conducted through the pore.
S.f holds 2 ions at a time. When only one is in there, it’s happy, when 2, they repel each other, and the one further down the s.f. get’s pushed through to the aqueous cavity.
When ion enters the aq. Cavity, it is surrounded by water. And each subunit contributes a pore helix, directed towards the aq. Cavity. These have a dipole moments, one neg one positive, and neg ends face directly towards the aq cavity. Neg. charges of the dipoles stabilise the + charge of the ions so ion is stabilised in the middle of the pore, in aq cavity.
