Transport across cell membranesFor the same reason that flip-flop is rare, the permeability of water, small hydrophilic molecules and ions is low.

So, specific transport mechanisms are needed.

Also, the differential distribution of ions and other molecules (e.g. glucose)serves as a driving force (stored energy)

So, pumps are needed to maintain these gradients.

But obviously large and polarmolecules are found in cells.

How do they get in??

Researchers need experimental systems in whicha particular membrane-transport protein predominates.

1. Heterologous expression2. Liposomes

GLUT1 increases gluc. transportand is specific for D glucose

Facilitated transport (aka facilitated diffusion) occurs with the same change in free energy as diffusion.

But it would go, due to activation energy.

So, think of the channel or transporter as a catalyst.

3 things distinguish a uniport from diffusion:

1. The rate of facilitated transport is higher than diffusion2. Facilitated transport is specific3. Facilitated transport has a Vmax

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

Pay attention to these arrow heads

12 glucose binding triggers conformational change.23 glucose now facing the cytoplasm3—4 glucose can be released to cytoplasm45 glucose dissociation triggers return to original conformation

How can this continue to run?? Answ: hexokinase

GLUT1 High to Low (RBC)12 helices2% of RBC proteinSpecific for D glucose

In summary:

1. The membrane is differentially permeable2. Three kinds of transport3. Transport can be studied in isolated systems4. Uniport transport differs from diffusion in many ways5. Transporters follow rules of enzymes

Typical ion concentrations in cells and blood

cells (mM) blood (mM)K+ 139 4Na+ 12 145Cl- 4 116Ca++ <0.0002 2

So, how is this maintained??

Intracellular ion environmentsand membrane potential

-70 mV = 200,000 V/cmHigh tension lines is 200,000/km

Membrane potential is maintained by pumpsBut set by leak channels

Membrane potential drives the transport reactions on the membrane

Intracellular ion environmentsand membrane potential

Read but don’t memorize the Nearnstexplanation on p 587

Rather follow along my explanation of how resting potentials are established.

Intracellular ion environmentsand membrane potential

The distribution of ions on both sidesof the impermeable barrier is set like thecell and blood.

No ion flow possible so V=0

Now, open a Na+ selective channel

Na+ move left until positive charges buildon the left and negative charges on rightV= -59 mV with respect to the right (out)

Now, open a K+ selective channel

Same except V= -59mV with respect to the cytoplasm.

Question: How did the original concentration differences become established?

Answer: pumps!!

P-class: heterotetramer, alpha is phosphorylated, Na+/K+ ATPasealso Ca++ ATPase (SR of muscles), H+ATPase (lysozomes)

F- and V-class: 3 TM subunits, 5 extrinsic, no phosphointermediate, V types in plant vacuoles and lysozomes, F types in bacteria and mitochondria, F pumps usedfor ATP synthesis, V pumps for acidification

ABC superfamily: heterotetramer, 100’s of members, transport sugars,amino acids, peptides, CFTR

We’ll use the Ca++ ATPase as an example to study mechanism. F and ABC later

1. 2 Ca++ bind, note two E1’s 2. Binding induces a conformational change that permits phosphorylation.3. P form causes the flip, note the change from high to low energy P bond4. The flip changes the Ca++ binding site to low affinity.5. Return to E1.

Na+/K+ ATPase maintains the intracellular Na+ and K+ in cellsRequired: classic experiment 15.1

Evidence that this pump is responsible for coupled K+/Na+ movement:1. Oubain blocks the ATPase and Na+/K+ movement2. Liposome reconstitution demonstrated Na+/K+ exchange

3. The mechanism is similar to the Ca++ ATPAse but not exactly the samea. Coupled transportb. Phosphate hydrolysis drives “K+ in” conformation

Required: Media Connections, Biological Energy Conversions

[Na+]out ~400 mM[K+]out ~ 4-20 mM

[Na+]in ~12-50 mM[K+]in ~400 mM

Km Na+ ~ 0.6 mM

Km K+ ~ 0.2 mM

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.

Next scenario:

How do you move glucose through a cell?

Polarized epithelial cell

Gut and kidney epithelial cells must pump glucose up their gradients (low to high)into the cells. Energy stored in the membrane potential drives this transport.

2 Na+ cotranspost with 1 glucose. This permits transport against a steep gradient.