lec 5. control of intracellular pH Flashcards
methods of measuring intracellular pH
Microelectrodes and fluorescent indicators
Microelectrodes
two microelectrodes are inserted into a cell. One measures total Vm while the other is proton sensitive and measures Vm - proton. If you subtract these you get the Vm due to protons which is proportional to the pH.
control - previously calibrate electrodes with pH6 and pH8 to create slope
Fluorescent Indicators
You load a cell with inactive fluorescent indicator which becomes activated in the cell. Then shine a light of a specific wavelength on it and measure the wavelength of the light that is emitted which is proportional to the pH.
control - introduce proton ionophores to allow intracellular [H+] = extracellular [H+], and measure the fluorescence to get a signal for a known pH.
Buffering
minimises change in pH to prevent damage to cell by reversibly consuming to releasing protons
(cannot reverse changes in pH)
Buffering power
amount of acid/base needed to drop/raise pH by a certain amount
Buffering when too alkali
when pH is too high
release an H+ from COOH to make it COO
Buffering when too acidic
when pH is too low
add H+ to NHH to make NH3
Acid Extrusion
when pH is too acidic, NHE removes H+ from the cell
Properties of NHE
- requires Na/K ATPase to reduce intracellular Na+
- as pH lowers, it works harder (proton binds to regulatory allosteric site)
NHE1 housekeeping function is to regulate pH and control cell volume. It is inhibited by amiloride and EIPA
Acid Loading
when pH is too alkali, Cl/HCO3- exchanger removes HCO3- (bicarb) from the cell
Properties of Cl/HCO3- Exchanger
- part of the anion exchanger family (AE)
- independent of Na+
- as pH increases, it works harders
- AE family is inhibited by stilbene derivate drugs DIDS
Steady State pH
when the rate of acid extrusion equals the rate of acid loading, this gives the resting intracellular pH
