Why are ions needed to pass a membrane and how do they do it?



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Why are ions needed to pass a membrane and how do they do it?


Intro:

Ions will tend to move down their electrochemical gradients in order to decrease Gibbs free energy and increase entropy. However due to their charges, they are not lipid soluble and so they cannot diffuse passively across the lipid bilayer membrane. Instead, they must use protein transporters, made up usually of various peptide subunits which traverse the membrane and have hydrophilic pores to allow ions to pass through the membrane. These pores often have selectivity filters which mostly only certain ions to pass through. The selectivity can be controlled by the number and orientation of negatively charged residues in the pore which must replace the hydration shells of ions. Protein transporters can also be 'gated', that is whether or not they are open or closed depends upon a certain factor such as voltage or ligand binding. Many carriers can also transport ions against their electrochemical gradients, these are known as active transporters and they can be either primary or secondary. Primary transporters use the hydrolysis of ATP to drive transport, where as secondary active transporters use the passive flow of one ion down it's gradient to drive the transport of another ion against it's own gradient. This essay will discuss the structure and mechanism of these protein transporters and the function of them, why the ions are needed to cross membranes in the first place.



F type ATPase:

  • ATP is the major energy carrier for all biological systems, so synthesising ATP is arguably the most important functions of any organism. It is carried out by the F type of a family of transport protein channels named ATPases. Located on the inner mitochondrial membrane, thylakoid membrane and the plasma membranes of all aerobic bacteria.

  • Diffusion of H+ down chemical gradient to drive ATP synthesis. Gradient is set up by v type or electron transport chain or light activated H+ pump bacteriorhodopsin in Halobacterium.

  • Structure is made up of one integral F0 domain and peripheral F1 domain. Each of these in turn is comprised of many subunits. Structure was elucidated by John Walker who won the nobel prize for it in 1997.

  • Process: H+ bing at c-a subunit interface of F0 domain. Sequential binding and release at weakly acidic residues here changes electrostatic interactions within the domain, resulting in it's rotation. The torque generated by the flow of H+ is transmitted to the F1 domain via rotation of the gamma-epsilon central camshaft. F1 acts as a stator and comprises 3 copies each of alpha and beta subunits. ADP and Pi spontaneously bind to each beta subunit. With each 120 degree rotation of the camshaft, there is a conformational change in the beta subunit which causes ATP release. This is termed rotational catalysis.

  • We have seen how the H+ ions cross certain membranes via the F-type ATPase, now we will consider the importance of the energy carrier ATP, the product of this process.

V type ATPase:

  • V-type ATPases are ubiquitous in eukaryotes, and have been determined to have evolved from F-type. They work in the reverse, translocating H+ against it's electrochemical gradient across endomembranes using the hydrolysis of ATP. Again it is a multi-subunit enzyme comprising an integral V0 and peripheral V1 domain. The translocation of H+ at these membranes is vital for the acidification of the compartments they border such as vacuoles and lysosomes. The acidification of lysosomes means that there can be destructive enzymes present in the lysosomes which only work at a pH lower than that of the cytol (usually around 5) and so can break down different sorts of macromolecules such as proteins or nucleic acids.

Na/k pump (P type ATPase):

  • P-type ATPases are primary active transporters, these are structurally quite different to the previous types as they have no rotational catalysis. However alike to V type, they do require the hydrolysis of ATP in order to translocate ions across membranes against their chemical gradient. Often this is used to create an electrochemical gradient which can be used by secondary transporters. An important example of this is the Na/K ATPase or the sodium-potassium pump. It is comprised of two subunits, (100kDa) and beta (35kDa) and transports three Na+ ions out of a cell and two K+ ions into a cell per each molecule of ATP hydrolysed. Hydrolysis of ATP phosphorylates a highly conserves aspartate residue on the cytolic side which causes the conformational change required to release the three Na+ ions outside of the cell. The binding of 2k+ causes dephosphorylation and so the pump returns to it's original conformation and releases the K+ into the cell. This creates a charge imbalance across the membrane and hence is electrogenic. It produces a net membrane current of about -10mv.

  • One vital role of this ion movement is apparent in the small intestine of the gut in Na+ coupled glucose transport. The Na/K pump is located at the basolateral membrane of the small intestine epithelial cell, this maintains a low Na+ concentration within the cell, creating a chemical gradient from the gut lumen into the epithelial cells. This causes Na+ to passively diffuse in through a Na+ driven glucose symport, simultaneously transporting glucose against it's concentration graident into the cell from the gut lumen. Glucose can then diffuse across the epithelial cell and out through glucose permease and into the interstital fluid and blood. Without the maintenance of this Na+ gradient by the Na+/k+ ATPase, organisms would not be able to absorb glucose from food into their blood stream and so not receive it's potential energy.

Resting pot:

  • Another use of the Na/k pump is for creating the asymmetries of ion concentrations needed for the set up of the resting potential. The electorgenic nature of the pump also contributes slightly to the resting potential by about -10mv.

  • The resting potential is mainly created by the two pore domain K channels, the name arises due to the fact they are made up from dimers which traverse the membrane creating two pores. K+ permeability at the membrane is high as these channels are usually open and allow K+ ions to 'leak' back out of the axon. Initially, K+ flows down it's chemical gradient out of the cell, however the inside of the axon already had a net negative charge compared with the outside and the leakage of K+ increases the concentration of positive charge outside the cell. This counteracts the outwards flow until a potential difference is reached where the chemical dirve is balenced by the electrical drive leading to an eqilibrium potential which can be calculated using the nernst equation. The equilibrium potential of potassium is around -90mv, the disparity between this and the average resting potential (-65mv) is accounted for by the inwards leakage of Na+ ions down their chemical gradient back into the axon, despite Na+ having a much smaller permeability through the membrane.

Action pot:

  • Some cells are excitable, meaning that stimulation of sensory receptors can cause sensory transducton of the stimulus into a receptor potential. This is a graded response which can cause depolarisation or hyperpolarisation depending on the cell and the stimulus. If the depolarisation reaches threshold, the voltage gated Na channels will be probabistically opened. This causes Na+ to move into the cell down it's electrochemical gradient, the postive ions repel each other and so spread out through the axon. This causes depolarisation at the next Na channel, which then opens and so the Hodgkin cycle continues in this postitive feedback manner. Na channels were first isolated in electric eels and were found to consist of a single large 260kDa alpha subunit, in the mammilian brain this is accompanied by two small subunits beta one and beta two. The alpha subunit is variable but is often divided into four similar domains (I-IV) and each of these split into transmembrane segments s1-s6. S4 has highly conserved positively charges lysines or arginines at every third residue, making the voltage sensor. Movement of this sensor with a particular change in electric field causes opening of the pore loop between S5 and S6.

Neuromuscular junction

  • When an action potential reaches the pre-synaptic membrane, the depolarisation causes the opening of voltage-gated calcium channels which are similar in structure to the voltage-gates Na channels but have variable accessory subunits which control gating, kinetics and selectivities. The influx of Ca2+ ions causes the calcium sensor protein synaptotagmin, found on the membrane of the secretory vesicles containing acetylcholine, to promote exocytosis of acetylcholine into the neuromuscular junction.

  • The post synaptic membrane contains a high density of nicotonic acetylcholine receptors, a type of ligand gated ion channel. It is assembled from 5 subunits, 2 alphas which make the acetylcholine binding site, a beta, delta and either gamma (fetal) or epsilon (adult) subunits. These are arranged circularly through the membrane and each subunit is made up from four segments M1-M4. Hydrophilic residues on M2 make the ion pore. Binding of Ach causes roatation and outward movement of helical rods in the extracellular domain, turning the M2 segments out of the way in the pore and allowing inwards flow of Na+ into the post synaptic cleft and outwards flow of K+. This causes a net depolarisation of the membrane, allowing the action potential to continue through the axon.

Conclu:

  • It is clear that ions are needed to pass membranes on every level of function of organisms. From synthesisng the essential energy carrier molecule ATP, using ATP's potential energy to carry out cellular functions and right up to every nervous system signal, the transport of specific ions across various membranes is vital. It is no wonder then that there has long been a desire to create artificial ion transporters which could be used for our advantages. However, despite the extensive knowledge of ion channel structure and function we have collected via molecular cloning, mutagenesis, x-ray defraction and various other methods, the design and synthesis of artificial carriers has been extremely difficult. However some artificial ion transporters have been created using self assembling protein nanotubes with cylindrical, beta sheet formations. Rates of transport in these have been comparable to their biological counterparts and could prove to be a major method of drug delivery systems in the future.


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