Bioelectricity and channels (L1-4) Flashcards
(35 cards)
What is the cell membrane made up of?
42% lipids
55% proteins
3% carbohydrates
What is the ionic composition (cations and anions) of blood plasma, interstitial fluid, and intracellular fluid?
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.
How does transport across the cell membranes differ for different types of molecules?
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
What are the different types of transporters found in membranes?
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
What are the different types of transport?
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
How does a patch clamp test work and what is it used for?
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)
How do you find the total current carried by a population of channels?
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
How is transport regulated?
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.
How are channels categorised?
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.
How can you measure the potential difference of a membrane?
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
What contributes to the membrane potential?
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
Explain the contribution of potassium channels to the membrane potential
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.
What is the Nernst equation and what is it used for?
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
What is the contribution of sodium channels to membrane potential?
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
How does the Nernst potential equate to channels being open or closed?
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
What is the Goldmann equation and what is it used for?
Similar to the Nernst except its used when multiple ions are involved. - more relevant for in vivo situations.
didnt know how to turn this into a question so just learn it
- 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.
Why is it important to control intracellular pH?
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.
How can you measure intracellular pH?
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.
What are the factors involved in controlling pH?
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.
What is a buffer and how do they work?
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)
How do proteins help with buffering?
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
How does acid extrusion work?
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.
What is the structure of NHE1?
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.
