Regulation of cellular pH Flashcards
What is the pH of the plasma?
7.4
What is the pH inside the cell?
7-7.1
What scale is pH? (equation)
What does this mean?
Logarithmic scale:
pH = -log[H+]
1 small change in H+ has a LARGE change in pH
How do proteins buffer pH?
1) H+ binds to protein - changes the protein charge
2) Conformational change in the protein
3) Change in protein function
Why can increase in H+ concentration potentially have disastrous effects?
H+ binds to proteins and changes their charge and therefore shape and FUNCTION
What 2 methods can be used to measure intracellular pH?
1) Microelectrodes
2) Fluorescent indicators
Which method is good for measuring pH of large cells?
Describe this method
Microelectrodes:
1) 2 voltage electrodes - measure ion current across the membrane
2) Measure voltage difference between V1 and V2
3) Change in voltage is proportional to pH
How are the electrodes in the microelectrode technique calibrated?
1) Buffer at pH 6 and measure voltage
2) Buffer at pH 8 and measure voltage
3) Construct a curve
How can voltage be converted into pH?
Using the curve constructed:
pH = (V-offset) / slope
Which method is good for measuring pH of small cells?
Describe this method
Fluorescent indicators:
1) Cells loaded with LIPID SOLUBLE, INACTIVE form of the indicator
2) Inside the cell - the inactive form is converted into an active form
3) Indicator is excited with light of a specific wavelength and the amount of EMITTED fluorescence light is measured (emits at a different wavelength)
4) Fluorescence is proportional to intracellular pH and [H+]
In the fluorescent indicator method to measure pH, why is the cell loaded with an INACTIVE form of the indicator?
It is positively charged - so it can cross the membrane
In the fluorescent indicator method to measure pH, how is the inactive form of the indicator turned into the active form?
What does this cause?
Inactive form is cleaved
Active form has a negative charge - is TRAPPED inside the cell
How is the fluorescent indicator method calibrated?
At the end of the experiment:
- Proton ionophore which calibrates pH inside of the cell with the pH outside
- When change pH outside of the cell, pH inside the cell becomes the same
- Look at the signal intensity when the pH changes
What 3 things are involved in the control of intracellular pH?
1) Buffering
2) Acid extrusion
3) Acid loading
What is the relationship between acid extrusion and acid loading in a cell under normal conditions?
What do these systems do?
They are balanced
These systems act to REVERSE pH changes
What is ‘buffering power’?
The amount of strong base that must be added to a solution in order to raise the pH by a given amount
What buffers inside the cell?
How?
Proteins
Reversibly consume or release protons
Using their COOH and NH2 groups
What can buffers do and what can’t they do?
Can MINIMISE pH changes
Cant REVERSE or PREVENT pH changes
What happens if pH increases (in regards to buffers)?
COOH in the protein buffer releases a H+
COO-
What happens if pH decreases (in regards to buffers)?
NH2 in the protein buffer accepts a H+
NH3+
Which transport protein performs acid extrusion and what does this channel rely on?
Na/H exchanger (Na in, H out)
Relies upon the inward Na gradient created by Na/K ATPase
What is allosteric modification?
- H+ which aren’t being transported out of the exchanger BIND to the exchanger
- Leading to a conformational change of the exchanger and amplification of activity
- Increased acid extrusion
At normal pH conditions, what does the Na/H exchanger work on?
The Na gradient
What is the structure of the Na/H exchanger?
- 12 transmembrane domains
- Between transmembrane 4 and 5 is the Na+ and H+ binding and exchange region
- Large intracellular C domain which is the regulating region
What is the name for the Na/H exchanger?
NHE1
What function does NHE1 have?
Housekeeping function:
- Regulates pH inside the cell and cell volume
What is NHE1 (Na/H exchanger) inhibited by?
Amiloride and EIPA (analogue of amiloride)
Which transport protein is involved in acid loading?
How does this work?
Cl/HCO3 exchanger:
- HCO3- out and Cl into the cell
- Removal of HCO3- leaves H+ behind, causing acidification
What is the name of the Cl/HCO3 exchanger?
AE (anion exchanger)
What is an example of an anion exchanger?
Band 3
When is AE active?
At alkaline pH
Is AE a target of allosteric modification?
Yes
Does the AE rely on Na+?
No
What is the structure of an AE?
14 transmembrane domains
In AE, which transmembrane span is important in the transport pathway?
8
How many subtypes are there of AE?
4
What inhibits all isoforms of AE?
DIDs
What is another form of Na/H exchanger?
Does this have a housekeeping role?
NHE3
No
What happens when calmodulin binds to the C terminal of NHE1?
Shifts the set point in the alkaline direction
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