MCB2 Midterm 1

d) Well before the myelin can reform after a bout of the autoimmune disease, patients partially recover when newly synthesized voltage-gated Na+ channels distribute along the full length of the axon. Explain this partial recovery:

They'll be able to transmit the AP now but it will be significantly slower than with Myelin

At the resting membrane potential of the cell, the ∆G of Na+ to enter the cell is -3.16Kcal/mole of Na+.The ∆G of K+ is +0.57 kCal/mole K+ to enter the cell The Na+/K+ ATPase pump maintains the different concentrations of low Na+ and high K+ inside the cell.- 1. How much Kcal are required to transport 3 Na+ out and 2 K+? 2. How many molecules of ATP will be used? ATP hydrolysis generates a ∆G of -7.3 kCal/mol

1. > 10.62Kcal/mol 2. at least 2 ATP

a) What allows the unidirectional propagation of the action potential (AP) from the cell body of a neuron down its axon towards the synaptic terminals?

1. Inactivation of Na+ channels due to chain/ball mechanism 2. Hyperpolarization due to open voltage-gated K+ channels and the inactivation of Na+ voltage-gated channels

To increase the speed of propagation of the action potential, neurons can grow larger (squid giant axon), be myelinated, or both. Explain how these two processes increase the speed of conductance

1. Larger size decrease resistance in the axon and allow charges to move to depolarize the membrane faster 2. Myelin allows AP to jump from one node of Ranvier to the next without talking the time to develop an AP in between

3. The first reactions of glycolysis, sometimes referred to as "pump priming" or investment phase, involve the hydrolysis of adenosine triphosphate (ATP). Explain at least two advantages of this energy expenditure.

1. Phosphorylation keeps glycolytic intermediates polar, and thus impermeable to the plasma membrane. 2. Increasing the energy of phosphorylated molecules so they yield more energy.

A patient infected by a bacterium was treated with an antibiotic that kills this bacterium. However, after getting better, the infection relapsed in the patient as the bacteria had become resistant to the antibiotic. This resistance was mapped to the locus of an ABC transporter. The transporter is made of three proteins encoded by three genes that are in the same operon (remember MCBI?) Suggest at least two types of mutations that could affect this locus and why this would cause resistance to antibiotics.

1. The 3 genes are controlled by the same promoter and a mutation that increases expression will allow the ABC transporter to expel the antibiotic more efficiently 2. There could be a local amplification of the gene which causes higher expression and an increase in the number of transporters in the membrane of the bacteria

How do hepatocytes, erythrocytes, and brain neurons manage to have different transporters on their surface, considering the fact that they share the exact same genome?

This is due to differential gene expression whereby the transcription factors that specify the cell type control the expression of one or the other GluT, activating one, repressing the other(s)

b) GABAergic interneurons restrain brain activity by releasing GABA that binds to a GABAgated ionotropic Cl- channel. [Cl-]i is 8mM while [Cl-]o is 120mM. The Nernst equation gives an equilibrium membrane potential of -70mV. Explain why GABA is inhibitory since its membrane equilibrium potential is so close to the resting membrane potential.

Although the resting potential doesn't change, Cl- 'clamps' the neuron at that value and it takes much more Na+ to depolarize.

The brain is a heavy consumer of glucose: It is the last organ that should suffer from food deprivation. If there were a specific GluT gene expressed in brain neurons, imagine what would be its biochemical properties

Because the brain is a heavy consumer of glucose, there must be a large number of GluT (Brain) transporters within the membrane in order to ensure efficient glucose uptake. This means that GluT (Brain) has a high Vm. Moreover, GluT (Brain) must have a high affinity for glucose in order to continuously uptake it. Therefore, GluT (Brain) has a low Km. Because the brain cannot go without food supply, then GluT (Brain) must be more active and have a higher affinity for glucose than GluT-1 (Erythrocytes). Therefore, GluT (Brain) has a higher Vm and smaller Km than GluT-1 (Erythrocytes).

When multiple ions can cross the membrane, the Goldman equation allows the calculation of the membrane potential that results from these different ions.Why is the resting membrane potential at -70mV much closer to the equilibrium membrane potential of K+ (-100mV) than that of Na+ (+60mV)?What in the membrane causes this difference?

Because the membrane is much more permeable to K+ than to Na+, K+ contributes much more in the Goldman equation. This is because there are many more always-open K+ channels on the membrane of the cell and very few open Na+ channels

A motor neuron sends an action potential (AP) to the neuromuscular junction. The Goldman equation allows us to understand how this action potential develops (g represents the conductance of the membrane to each ion. Define the conductance of a membrane for a given ion and how it can change during an action potential.

Highly specific Ion channels that can open and close

During development or learning, changes in the internode length (the distance between two nodes of Ranvier) may be used to tune the conduction speed of myelinated axons. There is an optimal distance. What would be the effect on conduction speed if the distance would be shortened much below the optimum length (more nodes per axons)?

The AP would become very slow

a) Acetylcholine binds to the ionotropic acetylcholine receptor at the membrane of muscle cells at the neuromuscular junction. This is a ligand-gated Cation channel permeable to K+, Na+ and Ca++, but not Cl-. What is the consequence of opening this channel for the muscle cell's membrane potential? Explain.

Depolarization; although both Na+ and K+ get across, Na+ is much further away from its equilibrium potential (Nernst equation) and thus many more Na+ will get into the cell thasn K+ will leak out: The membrane potential will be mid-way between Na+ (+60mV) and K+ (-100mV), i.e. around -20mV: this is enough to trigger and AP since it is above the threshold.

Imagine that you are investigating a specific membrane protein in an organism. How would you test your hypothesis that they are "static" (do not diffuse freely)? Describe the main experimental technique, how this technique works, and what would you expect if the proteins are static vs. if they diffuse freely.

I would perform a Fluorescence Recovery After Photobleaching (FRAP) experiment. The experiment is done by fluorescently labeling a cell, bleaching a portion of it with a laser, and then recording its rate of fluorescence recovery. If the proteins are static, we expect the FRAP graph to show a drop in fluorescence after the bleaching process and then remain at that low fluorescence intensity. If the proteins diffuse freely, we expect the FRAP graph to show a drop in fluorescence after the bleaching process but increase back to the pre-bleaching fluorescence intensity over time.

During development or learning, changes in the internode length (the distance between two nodes of Ranvier) may be used to tune the conduction speed of myelinated axons. There is an optimal distance. What would be the effect on conduction speed if the distance would be lengthened to e.g. 4 times the normal length (many fewer nodes per axons)?

If too far apart, the depolarization that reaches the next node of Ranvier would be too low, below the threshold and the AP would not propagate

b) Myelinated axons carry the action potential much faster than unmyelinated axons. Suggest two parameters of the myelinated neurons that could speed up the propagation up to 100m/s.

Increasing the distance between two nodes of Ranvier (as long as it still allows an AP to develop) - Increasing the diameter of the axon

The Nernst equation allows the calculation of the equilibrium membrane potential of a specific ion. Explain how it represents an 'equilibrium'

It represents an equilibrium between the chemical (or concentration) gradient and the electrical gradient inside and outside the cell.

c) The GABA receptor agonist (the opposite of antagonist!) Benzodiazepin is used as a sleeping pill. Explain why.

It would bind to and activate the GABA receptor leading to Cl- influx, 'hyperpolarization, and lower neuron activation in general

Voltage-gated K+ channels open when the membrane is depolarized: How is it then possible that the membrane hyperpolarizes (presumably because many K+ channels are open) before returning to the resting potential at the end of the action potential while the channels should be closed?

K+ channels open and close slower than Na+ channels do. During hyperpolarization, all Na+ channels are closed but some K+ channels are still open, which is what causes the drop in membrane potential below the resting membrane potential.

Gated channels, that are necessary for the creation of the AP, are only present at the nodes of Ranvier. If these channels were anchored at regularly spaced intervals located where the nodes of Ranvier normally are, could the Action Potential jump from one location to the next WITHOUT the myelin sheath?

Not really.....This would be an issue as the AP would not be 'maintained' between two locations and the charges would be diluted where the myelin was supposed to be. This would severely interfere with the movement of charges towards the next pseudo-node of Ranvier. (This is what happens when MS patients lose their myelin and the AP cannot be conducted properly )

What are saturated vs. unsaturated fatty acids? In which food products do you find them? The saturation (saturated vs. unsaturated) of fatty acid tails of phospholipids affects "membrane fluidity" of the plasma membrane; explain why

Saturated fatty acids have no double bonds and are found in butter. Unsaturated fatty acids have one or more double bonds and are found in oil. The more unsaturated a phospholipid fatty acid tail is, the more fluid the membrane is. This occurs because the kinks formed by the double bonds produce less Van der Waals interactions which decreases the stability of the membrane. The decreased stability will then increase the fluidity of the membrane.

Is TEA also a poison and if so, how does it act?

TEA blocks voltage gated K+ channels, which will slow down the repolarization process and increase the duration of action potentials. This makes TEA a poison because it will end up prolonging action potentials that are meant to be quick and interfere with important bodily processes, such as heart rhythm or brain function.

How does TTX cause paralysis of animals that eat pufferfish (fugu)?

TTX blocks voltage-gated Na+ channels, which can cause paralysis when the blocked channels are in the animals' central and/or peripheral nervous system. By blocking the voltage-gated Na+ channels, TTX prevents cells from initiating and propagating action potentials.

c) In myelinated axons, voltage-gated Na+ channels are located exclusively at the node of Ranvier. In Multiple Sclerosis, auto-antibodies destroy the myelin, rendering the axon naked. However, the Na+ channels remain at the location of the former nodes of Ranvier. Why does this lead to severe effects on the propagation of the AP, much more than a simple slow-down of the propagation of the AP?

The AP can't 'jump' past the areas where myelin used to be because there are no voltage gated channels and they're no longer insulated by membrane

Propagation of an AP from one heart cell to the next occurs through gap junctions between heart cells. Explain why this allows the contraction of the heart muscle to move slowly (nearly 1s) from the tip of the heart towards its top, and thus to expel the blood through the aorta

The AP propagates from one cardiomyocyte to the next but the current has to go through the gap junctions that have a low conductance. This slows down significantly the AP (it is like propagation through a very tiny axon). Thus, it can take a good fraction of a second for the AP to reach the top of the ventricule: the ventricule contracts slowly from bottom to top

The action potential propagates down the axon. Explain why it does not decay

The AP propagates through local depolarization. As soon as it reaches the threshold, an AP will develop with the same height as the previous one: the signal is sustained as it propagates.

In a cell, Chloride concentration outside of a cell is 150mM, similar to Na+ (150mM) while the concentration inside the cell is 15mM (like 15mM for Na+). Use the Nernst equation to calculate the equilibrium membrane potential of Na+ and Cl- (NO NEED FOR A CALCULATOR, Log10 of 1=0; Log10 of 10=1; Log10 of 100= 2; Log10 of 1000=3 etc). Since the intra/extracellular concentrations of Na+ and Cl- are similar, do they have a very similar equilibrium membrane potential?

The Nernst equation leads to +60mV for Na+ but -60mV for Cl since it is a negative ion and n= - 1

2. Explain why phospholipids spontaneously assemble into closed membranous structures when ultrasonicated in aqueous solutions. What is the difference between a micelle and a liposome?

The amphipathic lipids of the membrane can interact with water in order to protect the hydrophobic components from coming into contact with the aqueous region. Micelles are made up of a single lipid later and liposomes are a double lipid layer (like in a cell membrane)

You measure the resting membrane potential of two cell types from the same organism in the same conditions and discover that one type of cells has a resting membrane potential at -65mV while the other has a different resting potential at -80mV. What could be the reason for this observation?

The cells with the -80mV resting membrane potential have more K+ ion channels open than the cells with the -65mV resting membrane potential.

d) In voltage gated K+ channels, the passing K+ ion is closely held in the center of the channel by charges on amino acid residues that replace the dipoles of the molecules of water that hydrate the ion in solution. how do these channels only allow K+ through and not Na+?

The energy of dehydration of K+ is compensated by the energy of the charges in the channel that hold the K+ and replace the water Na+ is too small for the charges in the channel to hold the ion and replace the water: It cannot be dehydrated: It will not make it into the channel because It cannot be dehydrated.

The equilibrium membrane potential of Cl- is very close to the resting membrane potential of the cell, which is -60mV. How much does Cl- contribute to setting the resting membrane potential? Why?

The equilibrium membrane potential of Cl- is -60mV, almost equal to the resting membrane potential at -70mV. However, there are NO Cl- channels open on the membrane at rest and the Cl conductance is zero ( gCl=0): In the Goldman equation that includes Cl-, since gCl-=0, Cl- plays no role and therefore Cl does not contribute

What allows the unidirectional propagation of the AP from the cell body of the motor neuron to the neuromuscular junction?

The hyperpolarization of the membrane after the AP makes it more difficult to reach the threshold AND voltage gated Na channels are inactivated and cannot go through their positive feedback activation

What would happen to the resting membrane potential if you were to open many Chloride channels in the membrane? Can you imagine other consequences for the cell?

The membrane potential will now be strongly influenced by Cl, but will remain at -60mV. However, it will take a lot more opening of Na+ channels to change this membrane potential because the membrane potential will be 'clamped' to -60mV and it will be very difficult to depolarize it and thus to trigger an action potential

During development or learning, changes in the internode length (the distance between two nodes of Ranvier) may be used to tune the conduction speed of myelinated axons. There is an optimal distance. How could the internode length affect the action potential propagation?

The shorter the distance, the smaller the jump, the slower the speed of propagation

c) Predict the effect of a mutation that eliminates the positively charged N-terminus "ball" of the voltage gated Na+ channel.

The voltage-gated Na+ channels would remain open, leading to a longer depolarization. This would also eliminate the refractory period

b) The resting membrane potential is -70mV. Explain how it is established and which channels contribute to this resting membrane potential.

There are many more always-open channels K+ than Na+ channels that bring the resting membrane potential (-70mV) closer to the equilibrium membrane potential of K+ (-100mV)

e) Although multiple epsp's in dendrites can lead to an action potential on the axon, this does not happen on the dendrites. Imagine a molecular mechanism that prevents an action potential to be generated on the dendrites yet allows it on the axon.

There are only voltage gated channels on the axon and not the dendrites

Why is it important to have a threshold before voltage gated Na+ channels open?

There is membrane potential fluctuation even when there is a small stimulus. So, having a threshold allows cells to save energy by differentiating between this normal membrane potential fluctuation and when the signal needs to be fired.

Why are there always synaptic vesicles anchored at the membrane of the axon terminal?

They contain neurotransmitters that have to accumulate to sustain multiple action potential.

d) Neurons in our brain integrate the inputs of thousands of neurons whose axons synapse on their dendritic trees. Some of these synapses are excitatory (e.g. Glutamatergic synapses) while others are inhibitory (GABAergic synapses). Explain how this neuron integrates information received on the dendrites to decide to fire (or not!) an action potential on the axon: Explain what generates epsp (excitatory postsynaptic potentials, which are graded potentials) and ipsp (inhibitory postsynaptic potential), and how they are integrated. Please keep your answer to one or two sentences.

Upstream neurotransmitter release triggers epsps and ipsps that are graded membrane potentials, which are then added and subtracted over time and space before they reach the threshold for the voltage gated channel at the axon hillock

The structure of all bio-membranes depends on the chemical properties of phospholipids, whereas the function of each specific bio-membrane depends on the specific proteins associated with that membrane. Explain this statement. Include the 3 ways phospholipids can vary:

Variation among phospholipids can be caused by differences in their fatty acid tail lengths, fatty acid tail saturation, and polar head groups. Different transporter proteins can be embedded in biomembranes allowing the passage of different materials through the membrane giving these membranes different functions. The structure of phospholipids depends on their chemical properties, since the hydrophobic components must be on the interior and the hydrophilic components are on the exterior of the membrane

The Km of the Glucose transporter in red blood cells is lower than that of the liver transporter. Explain the meaning of Km and Vm and what these values mean for the properties of the transporter in a given tissue.

Vm is the maximum speed at which transport occurs across the membrane. It relates to the number of transporters in the membrane. Km is the concentration of a solute at 1/2 Vm. It relates to the affinity of the solute to its respective transporter. The low Km of the transporter in red blood cells allows them to get glucose even when blood glucose is low. The high Km of the transporter in liver cells only efficiently imports glucose when there is an excess of blood glucose

The Synapse: What is the role of Ca++ channels at the presynaptic terminal of an axon? When do these channels open?

Voltage gated, allow entry of Ca++, changing its concentration and leading to the release of vesicles in response to AP (depolarization)

a) At equilibrium, ΔGconcentration = ΔGelectric for a given ion. Explain in words what this means and how this allows you to write the Nernst equation to calculate the equilibrium membrane potential of each ion.

When there is a concentration gradient of an ion (eg Na+), they move to the lower concentration. However, this leads to the movement of + charges that prevent further + (Na+ ) ions to get in. The Nernst equation allows us to calculate this accumulation of charges, i.e. the membrane potential created by the movement of ions when the equilibrium is reached between the electric and the chemical gradients. This means that ΔE (the electric potential) = RT/nF ([conc]out / [conc]in).

What about Hyperkalemia, where there is higher K+ in the blood: What is its effect?

With higher K+ in the blood, there is a higher concentration of K+ outside the cells. This means that both the resting and the K+ equilibrium membrane potentials will increase. With the membrane potentials being higher, it will be easier for cells to reach their excited state since the distance between the resting membrane potential and the Na+ equilibrium membrane potential is smaller than normal.

There are conditions that alter the resting membrane potential of excitable cells (neurons and muscle) can have a profound impact on their proper functioning. Hypokalemia is a state in which there is a lower than normal amounts of K+ in the blood. What would be the effect of this condition on the resting membrane potential?

With less K+ in the blood, there is a lower concentration of K+ outside the cells. This means that both the resting and K+ equilibrium membrane potentials will decrease. With the membrane potentials being lower, it will be more difficult for cells to reach their excited state since the distance between the resting membrane potential and the Na+ equilibrium membrane potential is greater than normal.

Absorption of Lysine (amino acid) from the lumen of the intestine to the cytosol of intestine epithelial cells is mediated by a symporter that uses a favorable gradient of Na+ . If the Na+ gradient was inverted (high in cytosol, low in lumen), could a lysine/Na+ antiporter transport lysine from the lumen to the cytosol? Explain briefly

Yes, because an antiporter functions by moving two molecules in opposite directions across the membrane. Therefore, a Lysine/Na+ antiporter can transport Lysine into the cytosol by using the inverted Na+ gradient and moving Na+ into the lumen.

Imagine you grow cancer cells under normal conditions but you add labeled inorganic phosphate to the media. a) Which glycolytic intermediate would be first labeled? b) If you also want to inhibit the incorporation of inorganic phosphate to glycolytic intermediaries using a chemical inhibitor: would you use a compound that inhibits ATP/ADP interconversion, or an inhibitor of NAD+/NADH interconversion? Please explain why.

a) 1,3-Bisphosphoglycerate (1,3BPG) b) If you inhibit ATP you'll stop the entire glycolytic process not this specific incorporation of inorganic phosphate at step 6. NAD/NADH interconversion is coupled with the incorporation of inorganic phosphate and that's why using an inhibitor of NAD/NADH interconversion will inhibit the incorporation of inorganic phosphate.

5. The electron transport chain (ETC) is a critical step in the metabolism of carbon. a) Is this a cytoplasmic or a mitochondrial process? If cytosolic, in which side of the ER does it happen, if mitochondrial, where in the mitochondria? b) What is the energy source that fuels this process? c) Describe how the outcome of this process helps produce ATP.

a) Inner Mitochondrial membrane. With inputs from the matrix and pumping protons out to the inter-membrane space. b) energy from the transfer of high-energy electrons c) Energy from high-energy electrons is used to pump protons into the intermembrane space. These protons are then pumped back into the matrix by ATP synthase and the energy generated by doing so is harnessed by ATP synthase

6. Cholera is a potentially lethal disease in many areas of the world. A main symptom is diarrhea, which causes severe dehydration. The toxin produced by cholera binds to and constitutively activates (works all the time) the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, which leads to ATP-mediated efflux of chloride ions into the small intestine. Negative chloride ions flood the small intestine lumen from the surrounding cells followed by Na+ and other positive ions that compensate for the negative charge. a) Why does this cause the movement of water from the blood to the intestine (dehydration) and then diarrhea? b. Erythrocytes have glucose transporters (GluT1) that allow glucose to move down its concentration gradient. What kind of transporter is this? Why is it preferential for red blood cells to receive glucose before liver cells? c) This is achieved because liver cells (where glucose is turned into glycogen for long-term storage when energy is abundant) express a different Glucose transporter (GluT2). Is GluT1's Vmax and Km higher or lower than liver GluT2? d) A cell infected with a pathogen may attempt to prevent the spread of the foreign entity by undergoing programmed cell death (apoptosis). The cell will also signal phagocytic cells to engulf it in order to prevent inflammation from the corpse. How do they do this with reference to their phospholipid bilayer?

a) Osmolarity: since Cl and Na+ move to the intestine, water follows the ions into the small intestine leading to loss of water from the blood (dehydration) and diarrhea b) Passive transporter. In the presence of low glucose, cells that need energy (erythrocytes) must get it before cells that store energy (Liver) c) The liver enzyme GlutT2 has a lower affinity (higher Km) and a slower/lower Vmax than GluT1. d) The differences between the outer and inner membrane layers of the bilayer get scrambled: Phosphatidyl-Serine is the "eat me signal" to phagocytes and get displayed on the surface.

1. Imagine you discover a new microorganism that seems to be some type of prokaryote. After some experiments you determine that this organism has a lipid membrane with integral proteins. a) What steps would you take to determine if these proteins can diffuse on the membrane or if they are anchored and static? Please describe the experimental rationale, and setup. b) Plot the results that you would expect if the membrane would be static (not fluid) and if it would be fluid. Remember to label the axes and explain succinctly your data.

a) perform a FRAP experiment to analyze membrane fluidity. First, radiolabel a microorganism cell with GFP, then bleach a portion of it with a laser beam and measure how long it takes for fluorescence to return. b) If the proteins are static, then the fluorescence would remain at the low intensity after bleaching