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KcsA is a potassium channel found in the plasma membrane of Streptomyces lividans. Monomers come together to create a heterotetramer, forming a pore down the centre, that is fashioned like an inverted tepee. Each monomer contains two transmembrane alpha helices with a pore loop (P-loop) of approximately 30 amino acid residues connecting them. The transmembrane (TM) domains which are closer to the C-terminal of the protein (M2 helices) from each monomer form the lining of the pore. The P-loop also faces the pore and is responsible for creating the selectivity of the channel to potassium ions. The channel contains a selectivity filter and a large central cavity (10Å diameter).
2.1 KcsA Activation
A decrease in pH causes the channel of KcsA to open. The mechanism by which this works is unknown as no X-ray crystal structures have been produced that have the last 30 residues of the C-terminal domain and the first 22 residues of the N-terminal domain attached. However, Cortes et al have created a model of the full length KcsA using electron paramagnetic resonance (EPR) and site-directed spin labelling (SDSL). This model has shown that the C-terminus extends approximately 50Å into the cytoplasm and contains three alpha helices. The C-terminus contains a group of charged residues (R117, E118, E120, R122 and H124) that are thought to be responsible for this pH sensor activity but it does not form part of the activation gate. Histidine-25 is located at the N-terminus end of M1 yet is in close proximity to E118 (3.95Å) and was first identified as the sole pH sensor. Thompson et al have carried out studies of KcsA mutants to determine the mechanisms of pH sensing. The double mutant of E118A and E120A will open up to a pH of 7 showing that these residues are partially but not fully responsible for the pH-sensor activity. Glutamate 118 and 120 interact with R122 in the closed state forming a network of both intersubunit and intrasubunit salt bridges and hydrogen bonds. The protonation of glutamate at acidic pH causes the salt bridges to break and an increase in the net charge of the M2 helices. A mutant of H25R in addition to the E118A/E120A enables KcsA to open up to pH 9 indicating that these are the main components of the pH sensor. Histidine 25 becomes positively charged when the charge of the M2 helices increase due to its intersubunit interaction with E118. Destabilization of the crossing bundle occurs either through electrostatic repulsion of H25 with R117, R121 and R122 or the mainly hydrophobic bundle increasing in hydrophilicity. As the C-terminus is located intracellularly, protons are unlikely to be the natural activation ligand as cells usually undergo homeostasis to keep the pH of the cytoplasm fairly neutral. The C-terminus is therefore more likely to form a receptor for a ligand which is as yet unknown.
Activation of KcsA involves the movement of the helices so that the channel pore becomes large enough for potassium ions to flux through. The M1 helices move as if rigid whereas the movement of the M2 helices is more complex. The M2 helices rotate outwards and tilt ~8o both along the x-y plane away from the permeation path and along the z-axis towards the membrane normal. From the extracellular side the M2 helices twist anticlockwise by ~30o. Residues 107-108 appear to act as a pivot point as the intersubunit distance in proximity of these changes minimally. The increase in diameter around these residues is ~2Å whereas in the inner vestibule there is an increase of ~10Å. This movement of the M2 helices is congruent with two distinct mechanisms where either the movement is scissor-like and fully rigid, or the movement is semi-rigid as the helix undergoes some kinking.
2.2 Potassium Selectivity of KcsA
The selectivity filter created by the P-loop is 12Å length and contains the amino acid sequence TVGYG. Rather than the side chains of these residues facing the channel, the backbone does instead. The oxygen atoms from the backbone carbonyl groups are able to coordinate with a potassium ion such that they mimic the hydration shell of potassium ions found in the cytoplasm. This is because the eight oxygen atoms within the selectivity filter are configured in a square antiprism around the potassium ion like four water molecules. This creates specificity for potassium ions over sodium ions as Na+ ions have a smaller diameter than potassium (XÅ compared to 1.33Å) meaning the carbonyl groups are therefore at a greater distance from the ion, making coordination energetically unfavourable. However, Sansom et al have shown that there is a degree of flexibility to the selectivity filter, which could allow the slight flux of Na+ through the channel. The energy required for this accommodation could still makes the flux of sodium ions unfavourable. KcsA shows a selectivity of K+≈Rb+>Cs+> Na+. KcsA is termed a 'long pore channel' due to the transport of two K+ ions through the selectivity filter at one time in single file.
There are four coordination sites for potassium ions in the selectivity filter termed S1 to S4. In addition to these there is a S0 site which forms an intermediate stage of hydration. The potassium is hydrated by 4 oxygen atoms from water and 4 oxygen atoms provided by the carbonyl group of the four glycine 79 residues. Another coordination site termed SEXT has also been witnessed but only at 100oK. This site is located extracellularly and the K+ ion has a hydration shell formed only by water molecules. The large central cavity of the pore contains one potassium ion which is also fully hydrated by water molecules.
2.3 KcsA Inactivation
Inactivation of KcsA produces a conformation that is in the open state but does not allow the flux of ions. KcsA undergoes activation-coupled inactivation as inactivation occurs in the same pH range with which activation increases. The mechanism by which inactivation occurs is unlike other potassium channels with similar structure because the N-terminal type structure associated with the ball and chain theory of inactivation is lacking. The inactivation mechanism is more similar to that of voltage-gated potassium channels, which contain 6 TM helices in each monomer, which have a C-terminal type inactivation. The C-type inactivation state is in a non-conductive conformation but it remains sensitive to the external K+ concentration.
Despite the lack of a voltage-sensor TM helix, initiation of inactivation does show some voltage dependence but termination does not. It has been demonstrated that between +150mV and -150mV there is an increase in the normal probability of opening by a factor of 60-100 when comparing steady-state activity. Aspartate 80 and glutamate 71 have destabilising interactions, as do aspartate 80 and tryptophan 67, that cause the conductive conformation of the selectivity filter to become unstable. An E71C mutant of KcsA eliminates this voltage dependence thus this residue is responsible for the gating charge of the selectivity filter. Glutamate 71 must be subjected to some ionization to possess the voltage-sensor properties and it is probable that the proton is donated by the carboxylate group of aspartate 80 as together they form a carboxyl-carboxylate interaction. The position of the side chain of E71 changes position with a change in direction of the transmembrane voltage field. KcsA with an E71A mutation has a higher Po (0.95-0.99) compared to wild type (0.05-0.15) meaning the channel is effectively constitutively open. This mutation increases the side chain flexibility of aspartate 80, allowing reorientation thus weakening its hydrogen bonding with tryptophan 67.
Interaction of KcsA with Membrane Lipids
The plasma membrane is formed from a bilayer of lipid molecules. These lipid molecules are able to interact with the proteins that span the bilayer, defining their topology, insertion into the membrane and structural integrity. The structural integrity of KcsA is manipulated by lipid membrane through the lateral pressure it exerts on the protein. The lipids can be annular, nonannular or integral lipids. Annular lipids surround the protein, binding weakly, creating a casing between the protein and the bilayer. They are probably important for positioning the protein vertically across the membrane. Nonannular lipids bind within clefts, such as those between subunits of a protein and bind with a higher affinity than annular lipids. These provide the protein with stability and aid integration into the membrane. They may also be necessary for the association of subunits. Integral lipids are lipids that bind to the protein in unusual positions, contributing to protein folding and assembly. They may bind in a manner such that the plane of the membrane is above the headgroup or the lipid is non-perpendicular to the membrane. KcsA has been shown to interact with both annular and nonannular lipids. The nonannular lipid binding sites are located at the interface between the subunits of the tetramer meaning there are a possible four nonannular lipids bound at any one time to the channel. The crystal structure of KcsA has been obtained that has lipid molecules present at the nonannular site. The structure of this lipid can not be resolved as there is no constraint of the lipid head group within the site so Fig. X shows the occupied nonannular sites of KcsA as diacylglycerol (DAG) but it is thought likely that the lipid is phosphatidyl glycerol (PG).
The selectivity of the nonannular lipid binding sites for different anionic lipids has been studied using fluorescence quenching of tryptophan residues 67 and 68. These are constituents of the pore helix and are in close proximity to the sn-1 chain of the lipid when bound at the nonannular site. The binding of brominated anionic phospholipids therefore causes quenching of the tryptophan residues. The nonannular sites show selectivity for anionic lipids with cardiolipin being the most favourable with a binding constant of 7.65 mole fraction-1. This is thought to be due to this being one of the main components of the native membrane. The composition of the membrane of Streptomyces lividans is not fully known but this prediction is based on the composition of other prokaryotic membranes. PG also has high affinity for this site but has weaker binding than the other anionic lipids tested with a binding constant of 1.46 mole fraction-1. However, as KcsA is expressed in E.coli, the lipid that has been crystallised with the protein is likely to be PG because it constitutes the majority of anionic phospholipids within the membrane at around 20%. Overall, the binding of the anionic lipids to the nonannular site is with moderate affinity that only varies slightly with the different phospholipid head groups. This is because the binding occurs through close contact of the negatively charged lipid head groups with the positively charged residues in KcsA; meaning as long as the lipid is anionic it should bind in the cleft.
Electrospray ionization mass spectrometry experiments have reinforced that there is preferential binding of anionic lipids to KcsA as PG and phosphatidic acid (PA) bind with higher affinity than phophatidylethanolamine (PE) which in turn has a higher binding affinity than phosphatidylcholine (PC). However, only the monomer of KcsA was detected indicating that the lipids probably bind at the annular sites. Marius et al have also demonstrated that PG and PA bind an annular site with a two to three times greater affinity than PC. These annular sites are located intracellularly whereas the extracellular annular sites are non-specific; correlating with the lack of charged residues clusters at this face. Positively charged residue arginine 27 is located on the intracellular side and probably forms a favourable interaction with the anionic phospholipids while in contrast the extracellular side contains the negatively charged residue glutamate 120 that causes the unfavourable interaction with PS.
Marius et al have demonstrated that in the absence of POPG KcsA doesn't open but as the anionic lipid content is increased the probability of opening (Po) increases. KcsA was thought to have an intrinsically low Po as a membrane containing 25% POPG would give a Po of ~2.5% but altering the content to 100% has shown that this is due to the POPG content of the membrane as the Po increases to ~65%. The effect that POPG has on the Po is consistent with cooperativity. With an increase in the number of sites that require occupation, there is an increase in cooperativity. It has been proposed that for the potassium channel to open, at least three of the four nonannular sites must be occupied.
Gel electrophoresis of KcsA has shown that the addition of PG to a PC bilayer mildly stabilizes the tetramer because of the electrostatic interactions that form between the two entities. Addition of PA to a PC bilayer has a greater stabilizing effect because the phosphomonoester head group forms hydrogen bonds with basic arginine side chains. These effects are established by R64, R89 and R27.
This investigation will study the effect a mutation of arginine 64 to leucine will have on the ability of PG to bind in the nonannular site. This will be studied by solid-sate nuclear magnetic resonance (NMR). It is hypothesised that this mutation will prevent binding as it will remove one of the positive charges thought necessary for the anionic lipid to bind.