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Cell 1001 Cell Signalling Notes Signalling: the cascade of processes which an extracellular stimulus (typically a neurotransmitter or hormone) effects a change in cell function L1: Signalling through ion channels Ions channels are selective for one type of ion each usually. is a common signalling mo...

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Cell 1001 Cell Signalling Notes Signalling: the cascade of processes by which an extracellular stimulus (typically a neurotransmitter or hormone) effects a change in cell function L1: Signalling through ion channels Ions channels are selective for one type of ion each usually. Ca2+ is a common signalling molecule whose rapid influx can cause very fast (microseconds) exocytosis, muscle contraction etc. Membranes are normally impenetrable to ions due to the hydrophobic lipid layer repelling +/- charges. Given enough time, however, virtually any molecule will diffuse across a lipid bilayer. The rate at which it diffuses varies depending on the size of the molecule and its solubility properties. In general, the smaller the molecule and the more hydrophobic, or nonpolar, it is, the more rapidly it will diffuse across the bilayer. Transmembrane proteins developed as a way of allowing passage for ions and other, larger material e.g. proteins. They exist in two classes: transporters and channels. Transporters A transporter undergoes a series of conformational changes to transfer small water-soluble molecules across the lipid bilayer.

Channels A channel forms a hydrophilic pore across the bilayer through which specific inorganic ions or other small molecules can diffuse. Channels transfer molecules at a much greater rate than transporters. Ion channels can exist in either an open or a closed conformation, and they transport only in the open conformation, which is shown here. Channel opening and closing is usually controlled by an external stimulus or by conditions within the cell.

Channels discriminate mainly on the basis of size and electric charge: if a channel is open, an ion or a molecule that is small enough and carries the appropriate charge can slip through. A transporter allows passage only to those molecules or ions that fit

Cell 1001 Cell Signalling Notes into a binding site on the protein; it then transfers these molecules across the membrane one at a time by changing its own conformation. Ion transport can be passive or active

Passive:  Determined by electrochemical gradient  Moves from high to low along the gradient  Needs to energy input  All channel proteins use passive transport  Cells are ~ negatively charged so many cations can easily diffuse in through channels Active    

Goes against the concentration gradient Requires ATP/energy source Often called “pumps” Coupled transporters: couple the uphill transport of one solute across the membrane to the downhill transport of another  ATP driven pumps: couple uphill transport to the hydrolysis of ATP  Light-driven pumps: found mainly in bacterial cells, couple uphill transport to an input of energy from light

These keep the ion concentrations within a cell at normal levels:  

Na is high outside of the cell K is high inside the cell

Cell 1001 Cell Signalling Notes 

Very little Ca2+ is habitually inside the cell, allowing fine tune reaction to even a small influx

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learn the values in the table above ^ The high concentration of Na+ outside the cell is balanced chiefly by extracellular Cl–. The high concentration of K+ inside is balanced by a variety of negatively charged intracellular ions (anions)

Examples of chemical/electrical gradients within eukaryotes:   

[Ca2+] in cytosol is 100nM, in the ER it’s 0.5mM (A LOT MORE) because the endoplasmic reticulum is the calcium storage space within the cell lysosomes have a much lower pH (4.5) than the cytosol due to large [H+] mitochondria have a slightly higher pH (7.8 vs 7.2 of the cytosol) as they pump out H+ to create a proton gradient

The Na+-K+ pump generates Na+ and K+ gradients across the plasma membrane      

hydrolyses ATP to ADP removes 3 Na+ ions from the cell moves 2 K+ ions into the cell net loss of positivity as there is a high [Na+] outside of the cell it constantly leaks in pump is designed to keep cell at slightly negative polarity

Cell 1001 Cell Signalling Notes

The binding of cytosolic Na+ (1) and the subsequent phosphorylation by ATP of the cytosolic face of the pump (2) induce the protein to undergo a conformational change that transfers the Na+ across the membrane and releases it on the outside (3). The high-energy linkage of the phosphate to the protein provides the energy to drive the conformational change. The binding of K+ on the extracellular surface (4) and the subsequent dephosphorylation (5) return the protein to its original conformation, which transfers the K+ across the membrane and releases it into the cytosol (6).

Intracellular Ca2+ Concentrations Are Kept Low by Ca2+ Pumps The lower the background concentration of free Ca2+ in the cytosol, the more sensitive the cell is to an increase in cytosolic Ca2+. Eukaryotic cells maintain very low concentrations of free Ca2+ in their cytosol (about 10–4 mM) in the face of very much higher extracellular Ca2+ concentrations (typically 1–2 mM). This huge concentration difference is achieved mainly by means of ATP-driven Ca2+ pumps in both the plasma membrane and the endoplasmic reticulum membrane, which actively pump Ca2+ out of the cytosol. Like the Na+-K+ pump, the Ca2+ pump is an ATPase that is phosphorylated and dephosphorylated during its pumping cycle.

Cell 1001 Cell Signalling Notes 2 Ca2+ are returned to the sarcoplasmic reticulum from the cytosol in a muscle cell via this pump. The Pump has four main domains: Ca2+ binding region, activator domain, ATP binding region and the phosphorylation domain (contains aspartic acid). The polypeptide chain of the protein crosses the membrane as 10 a helices.

ATP binding and the consequent phosphorylation of an aspartic acid in the transporter trigger conformational changes that bring the nucleotidebinding and activator domains into close proximity. This movement in turn leads to a rearrangement of the transmembrane helices, which eliminates the Ca2+-binding sites and releases Ca2+ ions into the lumen of the sarcoplasmic reticulum. Note that the pathway Ca2+ ions take through the protein allows the ions to avoid contact with the lipid bilayer.

Ion gradients can be used to drive secondary (coupled) transport Downhill movement of one solute is coupled to the uphill movement of another. Any mix of inorganic ions and organic molecules can occur in tandem. Symports move both molecules in the same direction, one with its concentration gradient and one against its concentration gradient. Atiports move the molecules in opposite directions, one with and one against its concentration gradient. In animal cells, an especially important role is played by symports that use the inward flow of Na+ down its steep electrochemical gradient to drive the import of other solutes into the cell. The epithelial cells that line the gut, for example, transfer glucose from the gut across the gut epithelium and also possess a glucose–Na+ symport, which they can use to take up glucose from the gut lumen by active transport, even when the concentration of glucose is higher inside the cell than in the gut. Because the electrochemical gradient for Na+ is steep, when Na+ moves into

Cell 1001 Cell Signalling Notes the cell down its gradient, the sugar is, in a sense, “dragged” into the cell with it (because the binding of Na+ and glucose is cooperative— the binding of one enhances the binding of the other—both molecules must be present for coupled transport to occur.) (textbook example, not examined.) The Na gradient is used to drive Ca2+ efflux via the Na+/Ca2+ exchanger (antiporter) Na is moved down its concentration gradient, which is coupled with the extraction of Ca against its concentration gradient. Both cations have a greater concentration outside of the cell. Often called the NCX for short.

ION CHANNELS Have ion selectivity (dependant on shape & diameter of pore and distribution of charged amino acid groups lining the channel) and the ability to close (unlike other channel proteins.) Each ion in aqueous solution is surrounded by a small shell of water molecules, and the ions have to shed most of their associated water molecules in order to pass, in single file, through the selectivity filter in the narrowest part of the channel. There, ions make important but very transient contacts with atoms in the amino acids that line the walls of the selectivity filter. Precisely positioned atoms allow the channel to discriminate between ions that differ only minutely in size, and also to limit the maximum rate of diffusion.

Cell 1001 Cell Signalling Notes

Selectivity Filter in K+ channels K+ ions in the vestibule are still cloaked in their associated water molecules. The narrow selectivity filter, which links the vestibule with the outside of the cell, is lined with carbonyl oxygen atoms (red) that bear a partial negative charge and form transient binding sites for the K+ ions that have shed their watery shell. Voltage Gated Ion Channels Voltage gated ion channels have specialized charged protein domains called voltage sensors that are extremely sensitive to changes in the membrane potential: changes above a certain threshold value exert sufficient electrical force on these domains to encourage the channel to switch from its closed to its open conformation, or vice versa. The voltage-sensing domain works via regularly spaced lysine and arginine residues in the S4 region which are capable of sensing changes in the potential. A conformational change occurs during polarisation that moves the S4 unit downwards.

Cell 1001 Cell Signalling Notes Summary Slides:           

Ions exist in gradients across both the PM and organelles. Ion transport is affected by transporters and ion channels. Transport can be passive or active. Ion gradients are generated by active transport (via P-type ATPases) and coupled secondary transport (exchangers). Na+ and K+ gradients are established by the Na+-K+ pump. Ca2+ gradient across the PM is generated by PMCA (plasma membrane Ca2+ ATPase) and NCX (Na/Ca exchanger). Ca2+ gradient across the ER is generated by SERCA (sarco/endoplasmic reticulum Ca2+-ATPase). Voltage-gated Na+, K+ and Ca2+ channels share a similar tetrameric structure. These channels comprise voltage sensing and pore forming domains. Key residues in these domains allow selective ion transport in response to voltage changes

L2: Electrical signalling Membrane Potential: difference in voltage between the interior and exterior of the cell, typically -70 mV at rest (but can vary) Charge on the inside of the cell is more positive than on the outside if all the ions were to be added up, so to depolarise the cell “fixed anions” are kept in the cell (these are negatively charged proteins and PO4- groups.) Electrical and chemical gradients are often opposing forces for the ions in the cell: the chemical gradient supports K+ leaving the cell as these is a high concentration inside, but the electrical gradient supports K+ remaining in the cell as it is negative. The equilibrium potential for K+ comes form this push-pull dynamic between the two opposing forces. The actual value of the resting membrane potential in animal cells is chiefly a reflection of the K+ concentration gradient across the plasma membrane, because, at rest, this membrane is chiefly permeable to K+, and K+ is the main positive ion inside the cell. The Nernst equation can be used to quantify the equilibrium potential of an ion.

Cell 1001 Cell Signalling Notes Thus we find the equilibrium potential for K+ to be about -90mV (using 5mMfor the extracellular concentration and 140mM for the intracellular concentration.) For Na+ the equilibrium potential is +60mV as there is more outside than inside.

The membrane potential comprises charge imbalance of all permeable ions. The resting membrane potential arises primarily due to K+ imbalance. Most cells are more permeable to K+ than other ions (due to “leak channels” letting K+ in at random.) Other ions do not leak as much and so their potential is close to zero. Resting membrane potential is thus close to the equilibrium potential for K+ (-90 mV.) The Goldman-Hodgkin-Katz equation allows calculation of the membrane potential by accounting for all ions and their permeability (don’t need to memorise equation.) It shows that K+ permeability is much larger than Cl-, which in turn is greater than Na+. Cl- and Na+ are so small they are effectively negligible, which is why membrane potential is based on K+. The Nernst equation is a simplified version of the GHK equation. Neurotransmission Action Potentials (Na+ & K+) A typical neuron has a cell body, a single axon, and multiple dendrites. The axon conducts signals away from the cell body toward its target cells, while the multiple dendrites receive signals from the axons of other neurons. Although a local change in membrane potential will spread passively along an axon or a dendrite to adjacent regions of the plasma membrane, it rapidly becomes weaker with increasing distance from the source. Over short distances, this weakening is unimportant. But for long-distance communication, such passive spread is inadequate. Neurons solve this long-distance communication problem by employing an active signaling mechanism: a local electrical stimulus of sufficient strength triggers an explosion of electrical activity in the plasma membrane that is propagated rapidly along the membrane of the axon and sustained by automatic renewal all along the way. This traveling wave of electrical excitation, known as an action potential, or a nerve impulse, can carry a message, without the signal weakening, from one end of a neuron to the other at speeds of up to 100 meters per second.

Cell 1001 Cell Signalling Notes A stimulus that causes a sufficiently large depolarization to pass a certain threshold value, promptly causes voltage-gated Na+ channels to open temporarily at that site, allowing a small amount of Na+ to enter the cell down its electrochemical gradient. The influx of positive charge depolarizes the membrane further (that is, it makes the membrane potential even less negative), thereby opening more voltage-gated Na+ channels, which admit more Na+ ions and cause still further depolarization. This process continues in a self-amplifying fashion until, within about a millisecond, the membrane potential in the local region of membrane has shifted from its resting value of about –60 mV to about +40 mV. This voltage is close to the membrane potential at which the electrochemical driving force for movement of Na+ across the membrane is zero—that is, at which the effects of the membrane potential and the concentration gradient for Na+ are equal and opposite and Na+ has no further tendency to enter or leave the cell. The Na+ channels have an automatic inactivating mechanism, which causes them to rapidly adopt (within a millisecond or so) a special inactive conformation, where the channel is unable to open again. Even though the membrane is still depolarized, the Na+ channels will remain in this inactivated state until a few milliseconds after the membrane potential returns to its initial negative value. The membrane is further helped to return to its resting value by the opening of voltagegated K+ channels. These also open in response to depolarization of the membrane, but not as promptly as the Na+ channels, and they then stay open as long as the membrane remains depolarized. As the action potential reaches its peak, K+ ions (carrying positive charge) therefore start to flow out of the cell through these K+ channels down their electrochemical gradient. The rapid outflow of K+ through the voltage-gated K+ channels brings the membrane back to its resting state more quickly than could be achieved by K+ outflow through the K+ leak channels alone. Returning to the Goldman-Hodgkin-Katz equation, we can now work out that Na+ permeability is greater than K+ during the active phase as the voltage gated ion channels are open. Now the other ion equilibrium potentials are irrelevant and the cell’s membrane potential becomes that of Na+, +60mV. Once the voltage gated K+

Cell 1001 Cell Signalling Notes channels open, the membrane potential now rests of the equilibrium potential of K+ as it is now the most permeable ion. Neurotransmitter release (Ca2+) At most synapses, the plasma membranes of the transmitting and receiving cells— the presynaptic and the postsynaptic cells, respectively— are separated from each other by a narrow synaptic cleft (typically 20 nm across), which the electrical signal cannot cross. For the message to be transmitted from one neuron to another, the electrical signal is converted into a chemical signal, in the form of a small signal molecule known as a neurotransmitter. When the action potential reaches the terminal, the neurotransmitters are released from the nerve ending by exocytosis. The depolarisation of the nerveterminal plasma membrane caused by the arrival of the action potential transiently opens voltage-gated Ca2+ channels, which are concentrated in the plasma membrane of the presynaptic nerve terminal. Because the Ca2+ concentration outside the cell is more than 1000 times greater than the free Ca2+ concentration in the cytosol, Ca2+ rushes into the nerve terminal through the open channels. The resulting increase in Ca2+ concentration in the cytosol of the presynaptic cell triggers the fusion of some of the synaptic vesicles with the plasma membrane, releasing the neurotransmitter into the synaptic cleft. Post synaptic ligand-gated ion channels Their function is to convert the chemical signal carried by a neurotransmitter back into an electrical signal.

Ligand-gated ion channels 

Electrical signalling is

Cell 1001 Cell Signalling Notes

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enhanced at excitatory synapses. (Excitatory neurotransmitters stimulate the postsynaptic cell, encouraging it to fire an action potential.) The receptors for excitatory neurotransmitters, mainly acetylcholine and glutamate, are ligand-gated cation channels. Neurotransmitter bind= channels open to allow an influx of cations = depolarisation of the plasma membrane toward the threshold potential required for triggering an action potential 

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Electrical signalling is dampened at inhibitory synapses. Inhibitory

neurotransmitters discourage the postsynaptic cell from creating an action potential. The receptors for inhibitory neurotransmitters, mainly g-aminobutyric acid (GABA) and glycine are ligand-gated Cl– channels. When the neurotransmitter binds, the channels open, but very little Cl– enters the cell at this point because the driving force for movement of Cl– across the membrane is close to zero at the resting membrane potential. Cl- will neutralize the effects of an excitatory neurotransmitter, whereby the depolarisation from the Na+ will be dampened by the Cl- also moving into the cell

Inotropic glutamate receptors   

Glutamate binds to the ligand binding domain in the extracellular region This depolarises the membrane, activating Na+ gated ion channels & Ca2+ channels, causing an action potential Thus receptor is found at excitatory synapses

GABAA receptors    

binds the neurotransmitter GABA GABA contains a disulfide linkage in the binding domain permeable to Clions It can repolarise/hyperpolarise the membrane by bringing in negative charge + “cys-loop receptor” Thus found at inhibitory synapses

Cell 1001 Cell Signalling Notes Nicotinic acetyl choline receptors      

binds acetyl choline allows Na+ to pass through found at excitatory synapses has an extracellular ligand gated channel cys-loop receptor can also depolarise muscle cells which causes contraction as the Ca2+ is released from the sarcoplasmic reticulum

Summary Slides: 1. Membrane potential comprises charge imbalance of all permeable ions but arises primarily due to the K+ gradient. 2. ...