1. Questions regarding Biomembranes or Antibodies????????

2. FRAP
Cell fusion
Liposomes
Immunoprecipitation
SDS PAGE
GFP fusions
Western Blots
Polyclonal Ab
Protein fractionation
Micelles
Detergent extractions
Monoclonal Ab

3. Intracellular
5-15 millimolar sodium’
140 millimolar potassium
Less than micromolar calcium
Extracellular
145 millimolar sodium
5 millimolar potassium
1 millimolar calcium
Cells are electrically neutral although the charges are not evenly distributed.

4. The gradients are established by transporters and pumps.
Selectivity

5. Bilayers (not cell membranes) are a billion times
Memorize this.
Bilayers (not cell membranes) are a billion times
more permeable to water than to sodium

6. (Facilitated diffusion)
Solutes cross membranes by either passive or active transport. Here’s how:
(Facilitated diffusion)
You already read about bacteriorhodopsin,

7. Concentration and electrical
gradients drive the movement/direction.
e.g. Glucose transport-bidirectional,
electrical is irrelevant
Add the sarcoplasmic reticulum- Calcium pump

8. An example of active transport: Calcium pump on the sarcoplasmic reticulum

9. Passive: A Uniport, Glucose Carrier

10. Km D-glucose 1.5 mM; L-glucose 3000 mM

11. GLUT1 High to Low (RBC)
12 a helices
2% of RBC protein
Specific for D glucose
Pay attention to these arrow heads
12 glucose binding triggers conformational change.
23 glucose now facing the cytoplasm
3—4 glucose can be released to cytoplasm
45 glucose dissociation triggers return to original conformation
How can this continue to run?? Answ: hexokinase

12. Good example: potassium

13. Three common ways to couple energy release with transport:

14. P type
Calcium ATPases
sodium-potassium pump
V-type
ATP production- more on this later
ABC Transporters
lots in bacteria
MDR proteins in selected cancer cells
malaria- chloroquine pump CF
yeast mating factor
class I MHC immune response

15. Pumps move solutes against their electrochemical gradient-
obviously they require energy

16. across the plasma membrane. Operates constantly.
A major use of ATP in cells is setting the sodium and potassium gradients
across the plasma membrane. Operates constantly.
Large electrochemical
Balanced electrochemical

17. 10 milliseconds!!

18. [Na+]out ~400 mM [K+]out ~ 4-20 mM [Na+]in ~12-50 mM [K+]in ~400 mM
E1’ E1 E2
[Na+]in ~12-50 mM
[K+]in ~400 mM E2
Km Na+ ~ 0.6 mM
Km K+ ~ 0.2 mM

19. Na+/K+ ATPase maintains the intracellular Na+ and K+ in cells
Evidence that this pump is responsible for coupled K+/Na+ movement:
Ouabain blocks the ATPase and Na+/K+ movement
Liposome reconstitution demonstrated Na+/K+ exchange
The mechanism is similar to the Ca++ ATPase but not exactly the same
Coupled transport and phosphate hydrolysis drives “K+ in” conformation

20. Coupled transporters can use the energy stored in gradients.

22. Polarized epithelial cell

24. two

25. AE1 protein, a Cl-/HCO3- antiporter is crucial to
CO2 transport in RBC
i.e an anion transporter. No net charge movement.
Concentration only.
Movement of CO2 from peripheral tissues
(systemic capillaries) to lungs.
Carbonic anhydrase in blood converts CO2
to water soluble bicarbonate/
. i.e CO2 is loaded into cells and
carbonic acid is pumped out.
Release of CO2 in the lungs because O2
drives carbonic anhydrase in reverse.
CO2 in the lungs moves into RBC
with Cl- exchange.