L3 - Control of Intracellular pH

Question 1

Why is it important to control intracellular pH?

Answer 1

pH is a logarithmic scale
Proteins act to buffer changes in H+
- Change in protein charge = change in conformation = change in function

Question 2

An increase in H concentration leads to

Answer 2

Decrease in pH

Compensate by removing H - alkalinisation

Question 3

A decrease in H concentration leads to

Answer 3

Increase in pH

Compensate by adding H - acidification

Question 4

How to measure intracellular pH - microelectrodes

Answer 4

1. Measure voltage difference between V1 and V2
   - V1 contains H+ sensitive resin
   - V2 is a standard electrode
2. Change in voltage is proportional to change in pH
3. The electrodes are calibrated with pH standards
   - Use two different pH and plot against recorded voltage
   - Draw a straight line through the two point

Question 5

Microelectrode advantages and disadvantages

Answer 5

Good for - big cells, nerves, muscles and oocytes

Bad for - small epithelial cells

Question 6

How to measure intracellular pH - fluorescent indicators

Answer 6

1. Cells loaded with a lipid soluble inactive form of indicator (active form cannot get through membrane)
2. Inside the cell it is converted to the active form (negatively charged)
3. Indicator is excited with light of a specific wavelength
4. The amount light emitted at a second wavelength is measured
5. Fluorescence is proportional to the intracellular pH

Question 7

Fluorescent indicator is calibrated inside the cell

Answer 7

Membrane is permeabilised with a proton ionophore and the bath solution pH is changed
In the presence of the ionophore - bath pH = intracellular pH

Question 8

Control of intracellular pH - buffering

Answer 8

Buffering power - amount of strong base that must be added to a solution in order to raise the pH by a given amount
Buffering by proteins/amino acids
- If pH increases - COOH donates H+
- If pH decreases - NH2 receives H+

Question 9

Buffers

Answer 9

Moderate effects of an acid/alkali load by reversibly consuming or releasing protons
Act to minimise the magnitude of pH changes
Cannot reverse changes in pH, any recovery is due to the presence of acid loading/extrusion mechanisms

Question 10

Control of intracellular pH - acid extrusion

Answer 10

Sodium protein exchanger - Na+ in and H+ out

Action of the exchanger relies on the inward Na gradient created by the Na/K pump

Question 11

Effect of pH on sodium protein exchanger (NHE) activity

Answer 11

High activity at acidic pH
When pH is more alkaline than the exchanger setpoint, the exchanger is inactive
Allosteric modification – protons other than the one being transported bind to the NHE protein – the conformational change increases exchanger activity

Question 12

NHE1 role

Answer 12

Housekeeping function in regulation of pH and control of cell volume
Inhibited by low concentrations of amiloride and its analogue EIPA

Question 13

Where is NHE1 found?

Answer 13

Basolateral membrane of epithelial cells

Question 14

Control of intracellular pH - acid loading

Answer 14

Chloride bicarbonate exchanger – Cl- in and HCO3 out

H2O + CO2 H2CO3 HCO3- + H+

Question 15

Effect of pH on chloride bicarbonate exchanger activity

Answer 15

Low activity at acidic pH

Activity increases as pH becomes alkaline

Question 16

Anion exchanger family are inhibited by?

Answer 16

All isoforms inhibited by the drug DIDS

The exchange of Cl and HCO3 is independent of Na

Question 17

AE1 role

Answer 17

In red blood cells, some in kidney – responsible for chloride/hamburger shift