Control of Cells Flashcards
What are the components that make up a cell membrane?
Lipids = 42% weight Proteins = 55% weight Carbohydrates = 3% weight
What are the three categories of ion channels?
Carries, pumps and gated channels
What is the role of carriers?
Facilitate movement using passive diffusion
Binds ions/solutes causing a conformational change
Requires a concentration gradient
What is the role of pumps?
Use active transport to move ions against their concentration gradient
What is the role of gated channels?
Cylinder protein with a gated pore so not always open critical for physiology
What are the two driving forces for the movement of ions?
Potential (charge) and chemical (concentration)
What is the turnover of active transport?
<100/second - not very fast as molecules move down their concentration gradient and ATP has to be hydrolysed first so phosphate must be present
Where are sodium potassium pumps not found?
Red blood cells in dogs
What is the structure of sodium potassium atpase?
4 subunits - 2 alpha and 2 beta
What can block sodium potassium atpase?
Nitric oxide
What is meant by a secondary active transporter?
A carrier which relies on the activity of an ATPase in order to work eg sodium potassium atpase works to keep IC low
What is the turnover of a carrier?
10^2 to 10^3 molecules per second
What are the three classifications of carrier?
Uniporter, symporter and antiporter
What is the turnover of gates ion channels?
10^6 to 10^8 ions per second
Who developed the patch clamp technique?
Nehr and Sakman
What is the patch clamp technique used for?
To directly see or measure ion channels opening and closing in a cell
How is a patch clamp test carried out?
A glass pipette is used which contains a silver electrode and a salt solution of a known concentration
Another electrode is in an experimental bath
The glass pipette touches the back of the cell - when sucked it seals the membrane (cell attached configuration)
This allows the function of a single ion channel to be measured
If the membrane is broken the current through all channels can be measured (whole cell configuration)
What is the equation used to work out total current?
I = N x Po x g x (Vm-Ei) I = current N = no. of channels Po = open probability g = single channel conductance Vm = membrane potential Ei = Equilibrium potential of ion
How can ion channels be regulated?
Number of channels at membrane The open probability of the channel - affected by phosphoryl, calcium and g proteins etc The potential (Vm - Ei)
What is the general structure of a voltage gated potassium channel?
6 transmembrane spanning domains in each of the 4 subunits
What is the general structure of a kir protein?
2 transmembrane spanning domains in each of the 4 subunits
What is the general structure of a voltage gated sodium channel?
24 transmembrane spanning domains in 1 subunit
beta 1 and 2 regulate the ion channel
What is KcsA?
A bacterial K+ channel homologous to the kir mammalian family
How was Vm measured before the patch clamp technique was developed?
A glass electrode is filled with solution (1m KCl) and a silver electrode is also used with respect to zero Vm
The diameter of the tip used is much smaller than that used in patch clamping (1x 10^-6) and is very sharp
This measures Vm but does not tell us what causes it
What is the EC concentration of sodium?
150mM
What is the IC concentration of sodium?
15mM
What is the EC concentration of potassium?
5 mM
What is the IC concentration of potassium?
150mM
What is the EC concentration of A-?
0mM
What is the IC concentration of A-?
65mM
What is the nernst equation?
Eion = RT/zF x log [ion} out / [ion] in R = gas constant T = Temperature in kelvin z = charge of ion F = Faradays constant
Usually RT/zF = 61/Z
What is the nernst potential for potassium?
-91mV
What is the nernst potential for sodium?
+61mV
What is the relative permeability of sodium?
1
What is the relative permeability of potassium?
50-75
What is the Goldmann’s equation?
Vm = 61/z x log p[Na]out + p[K]in / p[Na]out + p[K]in
What happens if the proximal tubule is exposed to phenylalanine and why?
It moves towards the nernst potential as it is co transported with sodium
Describe the ion channels in the epithelial cells lining the thick ascending limb of the loop of Henle
Basolateral membrane: sodium potassium atpase CLKB allows chlorine to exit cell Apical membrane: NKCC (sodium potassium 2 chloride) Potassium recycling channels
Describe the model of the sodium potassium atpase
1) Sodium binds to the sodium binding point
2) ATP phosphoryaltes the pump
3) Conformational change occurs so sodium leaves the cell
4) K binds to the new binding siteand dephosphorylation causes another conformational change
What can Na/K atpase be inhibited by?
cardiac glycosides such as oubain and digoxin
What is the exctracellular concentration of calcium?
1mM (1,000,000nM)
What is the intracellular concentration of calcium?
100nM
What is ECa?
+120mV
What are the two main ways to keep intracellular calcium low?
Na/Ca exchanger
Ca ATPase
How does the Na/Ca exchanger work?
It moves 3 sodium in for 1 calciumm out
This makes the effect of the 10 fold gradient cubed (sodium)
What gene family is the Na/Ca exchanger part of?
SLC8
What are the three types of Ca ATPases in cells?
PMCA - plasma membrane calcium pumps act calcium across the cell membrane
SERCA - Ca pumps found on the sarcoplasmic reticulum or endoplasmic reticulum which pump calcium out of the cytoplasm into organelles to act as calcium stores
SPCA - Ca pumps on golgi apparatus
What are mechanically activated calcium channels?
Found in many cells and respond to cell deformation eg stratch activated channels
What are the two classes of calcium channels in calcium store membranes?
IP3 receptors
Ryanodine receptors
What are ryanodine receptors?
Receptors that respond to low concentrations of ryanodine (high concentrations inhibit? or caffeine. Tend to be found in ecxitable cells
What is the relationship between calcium release from stores and SOCC?
IP3 receptor is acivated
This causes PLC (phospholipase C to be activated)
This activates PIP2
This causes calcium to be released from stores and calcium to enter the cell
How is pH calculated?
pH = -log[H+]
What could be the effects of changes in pH?
Change in protein charge, conformation and function
How is pH measured using electrode?
Two electrodes used - V1 and V2
V1 has a sensitive H+ resin
V2 is a normal electrode and measures the potential difference of all ions whereas V1 excludes H+
The voltage difference between V1 and V2 is measured
The change in voltage is proportional to pH
Calibration with two solutions with a known pH must be done first
Using electrodes to measure pH is suitable for which cell types?
Big cells, nerves, muscles or Xenopus oocytes
The electrodes are to big to use in epithelial cells
How is pH measured using fluorescent indicators?
Cells are loaded with a lipid soluble inactive form of an indicator - it is converted to the active form inside the cell
The indicator is excited with light at a specific wavelength
The wavelength emitted is measured
The indicator is calibrated inside the cell. The membrane is permealised with a proton ionophore and the pH of the bath solution is changed
What is a proton ionophore?
An ionophore allows the pH in and out the cell to be the same
What are the three main ways of controlling intracellular pH?
Buffering
Acid extrusion
Acid loading
How is buffering power defined?
The amount of strong base that must be added to a solution in order to raise the pH by a given amount
Which channel is used in acid extrusion?
Na/H exchanger
What is the role of Na/H exchangers?
Move Na into the cell and H+ out of the cell
Its action relies on the inward gradient of sodium
Activity is stimulated at a setpoint - this is when the pH is acidic
Describe the allosteric modicification of Na/H exchangers
Protons bind to allosteric sites ( not the transport site) which leads to a conformational change which increases the activity of the protein
NHE1 can be inhibited by what?
Low concentrations of amoloride and its analogue EIPA (EIPA works on sodium exchangers but not sodium channels)
Where is NHE1 found?
The basolateral membrane of epithelial cells
Which channel is used in acid loading?
The Cl/HCO3 exchanger
The Cl/HCO3 exxhanger belongs to which family?
The AE family
How does the Cl/HCO3 exchanger work?
It causes Cl to leave the cell and HCO3 to enter the cell. This works as removing HCO3 increases the relative concentration of H+ = acidification
What are Cl/HCO3 exchangers inhibited by?
The AE family are all inhibited by the stilbene derivative drigs DIDs
Where are the Cl/HCO3 exchangers found?
Red blood cells, some in the kidney
