Bioelectricity and channels (L1-4)

Question 1

What is the cell membrane made up of?

Answer 1

42% lipids
55% proteins
3% carbohydrates

Question 2

What is the ionic composition (cations and anions) of blood plasma, interstitial fluid, and intracellular fluid?

Answer 2

Blood plasma has high sodium with low potassium, and very low calcium conc (approx 2mM). The interstitial fluid is very similar to blood plasma. Intracellular fluid has a very low sodium, calcium (approm 1microM) and high potassium. This means there is a high driving force for sodium and calcium to enter the cell.
Plasma and ISF both contain high levels of chloride with lower carbonate and protein (there is more protein in plasma tho). High chloride outside means chloride goes into cells (except in chloride secreting cells like upper epithelia). There are high levels of protein and sulphate in cells.

Question 3

How does transport across the cell membranes differ for different types of molecules?

Answer 3

Lipid soluble materials e.g. oxygen and CO2. Can move across via diffusion with no help e.g. in the lungs. Water is only small and only has a small charge so can also move freely across (osmosis)
Small molecules and ions have to be transported using transport proteins bc they’re too charged to get through the hydrophobic inner part of the lipid
Large molecules are transported into the cells by endocytosis and moved using vesicles

Question 4

What are the different types of transporters found in membranes?

Answer 4

Channels - pores, can open and close, molecules diffuse through.
Carriers - facilitated diffusion, driven by the electrochemical gradient of 1 protein, so it carries the other (no ATP needed)
Pumps - powered by ATP (ATPase) e.g. sodium potassium transporters - primary active transport proteins

Question 5

What are the different types of transport?

Answer 5

Active transport:
When energy is needed for transport due to the absence of a gradient of trying to go against an electrochemical gradient.
Active transport has a low turnover (no more than 100x per sec) Energy is harvested by the hydrolysis of ATP
Passive Transport
Follows a gradient
Uses carriers or channels for large and charged molecules.
Much faster than pumps (about 1000x per sec depending on saturation)
The max rate is reached when all the saturation of substrate s so high that all the transporters are being used up. If there is no carrier, a small amount of diffusion may still take place but it is not efficient enough to sustain the cell.
Uniporters may still undergo conformational changes even if only transporting one thing one way.
Carriers are usually gated and can be very selective but this varies

Question 6

How does a patch clamp test work and what is it used for?

Answer 6

Discovered by Nehr and Sakman in the 1980s
Revolutionised physiology and won a Nobel prize.
1 put a small silver wire (chlorinated) in a tiny glass pipette. The wire is attached to a reference electrode
2. seal the pipette onto a specific part of the membrane (with specific channels) to measure the current
It allows you to look at the ion channel function in different conditions in real time directly. You get a graph showing the current (in picoamps usually) so you can identify when they are open and closed.
You can make transgenics with different mutations in the channels to see how it effects it compared to wild types.
You can also send pulses through the pipette to open up the cell and record the current flow of the whole cell at once (voltage clamp)

Question 7

How do you find the total current carried by a population of channels?

Answer 7

I= N x Po x g x (Vm-Ei)
(total current = number of channels times open probabilit x signle channel conductance x membrane potential - equilibrium potential

Question 8

How is transport regulated?

Answer 8

The number of channels via membrane shuttling, open probability via phosphorylation, calcium or G proteins. Potential difference via activating, inhibiting or changing channels to change gradient.

Question 9

How are channels categorised?

Answer 9

Via their structures into families. Structures identified by growing the channel protein crystals and then using x-ray diffraction analysis.
The first potassium channel crystal structure was discovered in 1998 (won a Nobel prize in 2003) KcsA (bacterial) was found to be homologous with Kir family in mammals. Found to have 4 interlocking subunits that form a pore down the middle.

Question 10

How can you measure the potential difference of a membrane?

Answer 10

You use a similar method to the patch clamp test, except the wire is potassium rather than silver and the electrode goes through the membrane rather than just resting on top.
You can determine the Vm due to the unequal distribution and selective movement of certain ions e.g. sodium, potassium and anions

Question 11

What contributes to the membrane potential?

Answer 11

ATPase directly contributes to about 20% of the potential and indirectly is responsible for the potential caused by intracellular sodium and potassium. The other 80% comes from the electrochemical gradient of potassium

Question 12

Explain the contribution of potassium channels to the membrane potential

Answer 12

80% if the potential comes from the electrochemical gradient of potassium. There is a high conc of potassium inside the cell and a low conc outside due to the pump - therefore theres a high conc of potassium inside the cell and a low conc outside due to the pump, therefore theres a high driving force to move potassium out of the cell due there being a big electrochemical gradient. Anions can’t cross the membrane so they remain high inside. This is important otherwise no charge is generated.
IC potassium moves to the outside, taking its positive charge and making the inside more negative. The negative inside now ‘pulls’ the positively charged potassium back. Eventually this force will become balanced with the chemical gradient and form an equilibrium. So overall theres no net movement of potassium.

Question 13

What is the Nernst equation and what is it used for?

Answer 13

Used to measure at what point this equilibrium is reached (for one ion).
The Nernst potential (Eion) of an ion is the point where the concentration gradient and potential gradient is balanced so therefore there is no net movement and no current. Eion = RT/zf x Ln x ion in/ ion out
When there is only passive movement of potassium, the Nernst potential of potassium - so for K it would be -90.1mV but in vivo its about -70mV because of the other ions

Question 14

What is the contribution of sodium channels to membrane potential?

Answer 14

There is a large amount of extracellular sodium because its being pumped out by ATPase. Therefore, theres a high driving force for sodium into the cell - so the inside stays negative. So its the opposite to potassium. The Nernst potential for sodium is 61 mV, however obvs the with the potassium this means theres a massive discrepancy and in vivo its about -70mV

Question 15

How does the Nernst potential equate to channels being open or closed?

Answer 15

The closer the Nernst potential of an ion is to the membrane potential, the more permeable the membrane is to that ion, so at rest potassium channels are open (because -90mV is closer to -70mV than +60mV) so the sodium channels are mostly closed

Question 16

What is the Goldmann equation and what is it used for?

Answer 16

Similar to the Nernst except its used when multiple ions are involved. - more relevant for in vivo situations.

Question 17

didnt know how to turn this into a question so just learn it

Answer 17

• Change in membrane potential is linked to change in a permeability of electrogenic transport e.g. voltage gated sodium transport or sodium coupled co-transport (with amino acids etc)
• When a particular ion channel is opened in the membrane, it moves the potential towards the Nernst for that ion (because its permeability increases)
• Amino acid-sodium cotransport is similar to sodium glucose co-transport, AAs move due to the driving force of sodium
• Sodium moves into the cell which causes a slight initial depolarisation but its small so the transporter can do its job without effecting the resting potential of the membrane.
• The slight depolarisation is corrected by potassium moving out.

Question 18

Why is it important to control intracellular pH?

Answer 18

pH in on a log scale (pH = -log10[H+ conc]
therefore, a small change in pH is actually a big change in hydrogen ions. This drastic change in protons can have a serious effect on the cell because proteins are altered by pH - e.g. changes their charge, which alters its conformation which changes function.

Question 19

How can you measure intracellular pH?

Answer 19

1 - using microelectodes
This method is goo for measuring large cells like oocytes, muscles or nerves. 2 microelectrodes are inserted into the cell. 1 (v2) is measuring the current produced by the movement of all molecules across the membrane and the other (V1) is coated in a proton selective resin so it is measuring the current produced by movement of all molecules EXCEPT protons. You can then subtract and work out potential difference cue to protons - then calibrate electrodes using known pHs to make a standard curve and work out pH (the change in voltage is proportional to the change in pH)
2. Using florescent indicators
Better for smaller cells because you don’t have to damage them as much. Indicator used has to be lipid soluble to enter the cell. When inside enzymes convert indicator into its active form (becomes negatively charged). Indicator can be excited with light at a certain wavelength. The amount of florescence in the cell is proportional to intracellular pH - you can also then permeabilise the cell with a proton ionophore so they leave the cell and the pH of the bath is changed - you can then excite the fluorescence in the bath and measure that instead.

Question 20

What are the factors involved in controlling pH?

Answer 20

Buffering , acid loading and acid extrusion.
Acid extrusion involves removing protons from the cell, therefore increasing the pH - via a sodium proton exchanger (NHE). Acid loading involves adding more protons to the cell, therefore decreasing the pH - uses a chloride/bicarb exchanger. Each of these systems balances to eventually give a resting pH.

Question 21

What is a buffer and how do they work?

Answer 21

A pH buffer is any system that moderates the effect of an acid or alkali load by reversibly consuming or releasing protons. The buffering system acts to minimise pH change and help protect the cell from damage. Buffering power is defined as the amount of strong base that must be added to a solution in order to raise the pH by a given amount. They cannot prevent the pH change, they just minimise its magnitude. (any recovery of pH is done acid loading/extrusion)

Question 22

How do proteins help with buffering?

Answer 22

Buffering from proteins relies on the ability of COOH and NH2 groups on amino acids to donate or receive protons. If pH increases, H+ is released from COOH to help counteract it. If pH decreased, the NH2 can receive protons to help reduce pH
Also, there are various residues on proteins e.g. lysine and aspartine which can also accept/donate. HOWEVER, when you alter a protein’s charge, its shape also changes

Question 23

How does acid extrusion work?

Answer 23

Done by an NHE transporter. Its an anti port that moves sodium into the cell and protons using the sodium electrochemical gradient. (the gradient is set up by the sodium/potassium ATPase. It has a set point )like an on/off switch) so when pH is more alkali than the set point, the exchange becomes more inactive, so less protons are removed. When pH is too low, NHE becomes more active through allosteric modification -where protons other than being transported bind to it, leading to a conformational change which speeds it up.

Question 24

What is the structure of NHE1?

Answer 24

NHE1 is a housekeeping gene its primary roles are of regulation of pH and control of cell volume. Its inhibited by ‘low’ concentrations of amiloride (potassium sparring diuretic) Its made up of 12 transmembrane spanning domains - the junction between 4 and 5 is though to be linked to transport. It has a large calcium calmodulin site on its C terminus which is important in regulation.

Question 25

What is the difference between NHE1 and NHE3?

Answer 25

NHE3 is the isoform of NHE1, its found in the apical membrane of the proximal tuble - its more involved in bicarb reabsorption than pH regulation

Question 26

Explain how acid loading works.

Answer 26

Done by th CL/HCO3 exhanger. Its in the anion exchange family (AE)
Its direction of transport is usyally inwards movement of Cl and bicarb out, however its direction can be reversed in some cells e.g. RBCs (what causes the hamburger shift)
Removal of HCO3- causes acidification because of the relationship between bicarb and H+. The activity of this exchanger is modulated by pH - it has a low activity at an acidic pH (becasue no H+ is needed in the cell) and its activity is increased at a higher pH due to allosteric modification

Question 27

What is the AE family like?

Answer 27

there are 4 subtypes. All isoforms are inhibited by the stilbene derivative drug DIDS which makes cross links between 2 closely located lysienes. They have 14 transmembranes spanning domains. TMSD no.8 is thought to be critical in the ion translocation pathway. A KO out AE in the kidney causes renal tubule acidosis (

Question 28

What is the resting pH of a cell?

Answer 28

The point at which acid loading and acid extrusion are equal leading the a balanced pH (seen as an overlap on the graph)

Question 29

What is the conc of sodium inside and outside the cell at normal conditions?

Answer 29

Extracellular sodium is 145mM and intracellular sodium is 15mM

Question 30

Explain the importance of regulating intracellular sodium

Answer 30

E.g. in the thick ascending limb
- Reabsorption of NaCl in preference to water
- this creates the transepithelial osmotic gradient
- If intracellular sodium conc is raised, then NaCl reabsorption is inhibited. The transepithelial osmotic gradient is dissipated, which leads to diuresis (increased water in the urine) and increased sodium and CL- in the urine (because it can’t be reabsorbed back into the blood)
E.g. the excitable cell
- Under normal conditions, the equilibrium potential for sodium is +60mV, yet the membrane potential is -70mV.
If intracellular sodium was increased from 15-45mM, there would be a decrease in the inward chemical gradient. The equilibrium potential for sodium would become about +30mV due to an increase in the electrical gradient. Therefore, it would take longer for the potential to develop, meaning there would be problems with propagation of the AP (slower conduction)

Question 31

How is intracellular sodium controlled?

Answer 31

Governed by the Na/K ATPase. It extrudes 3 sodium and uptakes 2 potassium, which is coupled by the hydrolysis of 1 ATP. The rate of transport depends on the metabolic state of the cell (How much of each substrate is available). The pump is inhibited by cardiac glycosides (oubain and digoxin)
Action of the pump maintains a low IC sodium and high IC potassium. It is also responsible for setting up the membrane potential due to causing an electrical gradient.

Question 32

What are the levels of calcium under normal conditions and how are they maintained? why is this important?

Answer 32

EC calcium is about 1mM and IC is 100nM (10000x less). Caclium regulation is important because it is an important secondary messenger involved in many signalling pathways. E.g. pancreatic Acinar cell for secretion. The equilibrium potential of calcium is approx 120mV so the gradients are extremely in favour of calcium entering the cell - but its kept low by the Na/Ca exchanger, and Ca ATPase

Question 33

How does the sodium calcium exchanger work?

Answer 33

Normally exchanges extracellular sodium for intracellular calcium, but there is a 10000 fold difference in calcium from inside to outside and only a 10 fold difference in sodium, so really, the transporter should be going the other way - but this is not the case due to the exchanger being electrogenic. The stiochemistry is 3 Na:1Ca, therefore the net sodium gradient is magnified. the effect of the 10 fold gradient is cubed. The exchange is a member of the SCL8 gene family, which is part of a much bigger CaCA superfamily. It has 9 transmembrane domains with an intracellular C terminal. It works by undergoing a conformational change to open and close it.

Question 34

What are the different types of Calcium ATPases?

Answer 34

Members of the p-type ATPases (also includes the Na/K pump. Cells contain 3 types of Ca pumps.
1. PMCA - plasma membrane caclium pump, these act to pump calcium across the plasma membrane and out of the cell
SERCA- Sacroplasmic/endoplasmic reticulum calcium ATPases - act to pump calcium out of the cytoplasm into organelles which act as calcium stores
SPCA- Ca pumps found in the golgi, they also transport magnesium.

Question 35

What are the different types of pathways involved in calcium signalling?

Answer 35

Plasma membrane pathways

1. VOCCs - voltage operated calcium channels found in excitable cells - activated by depolarisation
2. ROCCs - receptor operated Ca channels - found in secretory cells and nerve terminals - activated by binding of an agonist E.g. NMDA receptors
3. Mechanically activated Ca Channels - found in many cells and respond to cell deformation (stretch mediated)
4. SOCCs - store operated Ca channels - they’re activated following a depletion of calcium stores.

Store pathways:
There are 2 classes of calcium channels in store membranes
1. IP3 receptors - this channel is activated following the binding of IP3. In most cell types
2. Ryanodine receptors - low concentrations of ryanodine activate the channel - higher concentrations inhibit it. The channel is also stimulated by caffiene, but the natural activator is cADP ribose. These channels tend to be found in excitable cells.
When a receptor is activated, this causes PLC to bind with PIP2. This splits into IP3 and DAG. The IP3 activates the release of calcium stores. This release of calcium opens the SOCC, so more calcium can enter the cell.