Membrane Protein Channels Potassium ions queuing up in the potassium channel Pumps: 1000 s-1 Channels: 1000000 s-1

Pumps & Channels The lipid bilayer of biological membranes is intrinsically impermeable to ions and polar molecules. Permeability is conferred by two classes of membrane proteins, pumps and channels. Pumps use an energy source (ATP or light) to drive the thermodynamically uphill transport of ions or molecules. Channels, in contrast, enable downhill or passive transport (facilitated diffusion).

Pumps and channels

Facilitated diffusion Facilitated diffusion requires specific membrane transport proteins. Since the number of such membrane transport proteins is limited, they can be saturated if the concentration of molecules to be transported is high. Thus, facilitated diffusion has the characteristic property of saturation at high "substrate" concentration. Passive diffusion does not rely upon such transport proteins, and therefore, does not display such saturation at high substrate concentrations ("substrate" referring to the molecule being transported)

Propagation of nerve impulses We shall examine three channels important in the propagation of nerve impulses: the ligand gated channel (for which the acetylcholine receptor is the model and which communicates the nerve impulse between certain neurons); and the voltage gated Na+ and K+ channels, which conduct the nerve impulse down the axon of a neuron.

Nerve communication across synapses Ligand gated channel Acetylcholine is a cholinergic neurotransmitter 50 nm synaptic cleft Synaptic vesicles have 10000 acetylcholine molecules

Nerve communication across synapses Synchronous export of 300 vesicles in response to a nerve impulse. Acetylcholine concentration increases from 10nM to 0.5mM in a ms. The binding of acetylcholine to the postsynaptic membrane changes its ionic permeabilities. The conductance of Na+ and K+ increases in 0.1 ms, leading to a large inward current of Na+ and a smaller outward flux of K+.

Nerve communication across synapses The inward Na+ current depolarises the plasma membrane and triggers an action potential. Acetylcholine opens a single type of cation channel, which is almost equally permeable to Na+ and K+. This change in permeability is mediated by the acetyl-choline receptor. Ligand-gated

Acetylcholine receptor 2a, b, g, d Pseudo five fold symmetry The acetyl choline receptor is a pentamer of four kinds of membrane spanning subunits. 2 alpha a beta, gamma and delta subunit. They are clearly very similar and probably arose by duplication and divergence from a common ancestral gene They are arranged in a ring and forma pore in the centre of the complex.which runs through the membrane. The extracelular domain is primarily beta strands it has four transmembrane hydrophobic alpha helices and a single alpha helix inside the cell. You can see the arrangement looking from above which each subunit differently colored.

Acetyl choline receptor - a ligand gated ion channel Here we see a comparison of the structures in the closed and open forms. These are electron microscope reconstructions of the receptor. Hole in the centre of the open channel is larger than the pore in the closed channel. M1-4 indicate the membrane spanning alpha hekices of one subunit. M-2 lines the pore of the membrane and due to the similarity of these alpha helices provides 5-fold symmetry within the pore

Importance of amino acids lining the pore Amino acid sequences of these helices suggest that there are alternating ridges of small polar or netral amino acids (ser, thr, gly In the closed form, it appears that the alpha chain has kink in its structure whweras it is linear when oopen. If we look at the alpha helix lining the channel we can see at a critical position there are two bulky amino acids valine and leucine which are bulky and hydrophobic and so when in position they close the cahnnel. But upon binding of acetylcholine, it appears that the chain rotates and moves these bulky chains away from the pore rendering it permeable to ions.

Importance of amino acids lining the pore Closed Open Red bulky non polar therefore hydrohobic replaced with small polar amino acids allowing passage of ions Na and K

(M2) (M2) Flexible loops

Voltage gated channels (K+ & Na+ channels) The nerve impulse is an electrical signal produced by the flow of ions across the plasma membrane of a neuron. The interior of the neuron has a high concentration of K+ and a low concentration of Na+. These gradients are produced by a Na+-K+ ATPase.

Action potential – signals are send along neurons by transient depolarization & repolarization Depolarization beyond a threshold causes Na+ ions to flow in leading to further depolarisation & more Na+ influx Causes –60 to + 30 mV in a ms K+ ions flow out restoring the membrane potential Requires therefore that there are specific channels and that they are opened by change in membrane potential but then closedafter a brief period of time. There must be specific ion channels

Potassium & Sodium Channels Structural similarity Hydrophobic except S4 – positively charged (Arg, Lys) Isolated protein is 260kd. They have four internal repeats with sequence similarity. Each repeat has hydrophobic segments S1, S2, S3, S5, S6. S4 is positively charged segment. K+ channel is homologous to one segment of the sodium channel. In practice the K= channel is made up of 4 subunits coming together to form a channel Hydrophobic segment suggest membrane spanning domains and s4 proposed to act as voltage sensor Protein purified on the basis that it could bind tetrodotoxin (from the puffer fish) binds Na+ channels with Ki ~ 1nM – lethal dose for adult 10ng.

Structure of the potassium ion channel (tetramer) Potassium channel as seen earlier consists of pore forming segemnts S5 and S6

Potassium ion channel Six-transmembrane-helix voltage-gated (Kv)

View of a hypothetical Kv-type K+ channel

Ionised ions in solution have spheres of hydration

Path through the K+ channel Closed channel. K= ion can pass into the channel a distance of 22Angstroms into the pore in its hydrated shell Can penetrate 2/3 of the membrane until the pore becomes too constricted. At this point K must give up the water molecules and interact with amino acids within the protein Membrane reduced from 34 Angstroms to 12 angstroms. Once ion has given up its water it must form polar interactions within the protein. Inside cell

Selectivity filter Thr-Val-Gly-Tyr-Gly (TVGYG) The restricted part of the pore is made from residues contrivbuted by the two transmembrane alpha helices. Act as the selectyivity filter. There are 5 residues that are conserved across the family of K channels with sequence Carbonyl groups are directed into the channel and interact with the positively charged K ion. Thr-Val-Gly-Tyr-Gly (TVGYG)

Selectivity of K+ channel Ions with radius > 1.5Å are too big to fit through the channel of 3.0Å diameter Na+ is not so well solvated by the protein Why doesn’t Na move through channel even though it is smaller than K ion.

Energetic basis of ion selectivity Energetic cost of dehydration is high. Potassium is able to interact favourably with the carbonyl groups in the channel. Favourable interaction with Carbonyl groups

ion is too small and therefore the interactions are less favourable and energetically too low. ie energy gained in interaction with carbonyls is less than the energy required for the channel to desolvate the ions.

Model for potassium channel ion transport Four potassium binding sites. Ion is desolvated and binds to site within the pore and it can bind any of 4 sites within that pore. As each subsequent ion binds its positive charge repels the adjacent ion further up the channel. And pushes any ion in the way further up the channel. As each subsequent ion enters the channel it releases an ion on the other side of the channel. This results in rapid flow through the channel as required.

Voltage gated requires substantial conformational change S1 to S4 form paddles that are involved in the opening and closing of the channel. S4 forma s a helix which is lined with positively charged residuesS1 to S4 lie within the membrane. In closed state the paddle lies horizontal position. Upon depolarisation the paddle moves into vertical position and the base of the channel is allowed to pull apart increasing the access to the selectivity pore. Model for activation - S1 to S4 form the voltage responsive paddles S4 Increased access

A channel can be inactivated within milliseconds of opening

The channel can be inactivated by occlusion of the pore “Ball and chain model” Trypsin cleaved channel (cytoplasmic tail) does not inactivate. Neither does a mutant lacking 42 N-terminal residues Channel is cleaved by trypsin Adding back the peptide 1-20 restores inactivation

Ball and chain model for channel inactivation In closed position ball rotates freely within cytoplasm. Channel opens and ball finds complimentary site in open pore and occludes it. Therefore channel only opens briefly.

Na+ and K+ channels work together to give the action potential Na+ in first Then K+ out

Reorientation of helix 6 opens channel