Cellular Processes

Diffusion, channels and transporters

Cellular Membranes

Two main roles

• Allow cells to isolate themselves from the environment, giving them control of intracellular conditions

• Help cells organize intracellular pathways into discrete subcellular compartment, including organelles

Membrane Structure

Lipid bi-layer: phospholipids, primarily phosphoglycerides

Other lipids

Sphingolipids: alter electrical properties

Glycolipids: communication between cells

Cholesterol: increase fluidity while decreasing permeability

Membrane Proteins

Can be more than half of the membrane mass

Two main types• Integral membrane proteins – tightly bound to the

membrane, either embedded in the bilayer or spanning the entire membrane

• Peripheral proteins – weaker association with the lipid bilayer

We will discuss membrane proteins that allow for flow of ions or for transport of molecules

Membrane permeability

Lipid bilayer

Membrane proteins we will discuss

Membrane Transport

Three main types

• Passive diffusion

• Facilitated diffusion

• Active transport

Passive Diffusion

Lipid-soluble molecules (alcohol, CO2)

No specific transporters are neededNo energy is neededDepends on concentration gradient

High lowSteeper gradient results in higher rates

Gradients can be chemical, electrical or both depending on the nature of the molecule

e.g., Membrane potential – electrical gradient across a cell membrane

Facilitated Diffusion

Hydrophilic moleculesProtein transporter is needed - UniporterNo energy is neededDepends on concentration gradientExamples: amino acids, nucleosides,

sugars (glucose)

Facilitated Diffusion, Cont.

Three main types of proteins1. Ion channels – form pores, channel has to be open

a) Open/close in response to a membrane potential

b) Open via specific regulatory molecules

c) Regulated through interactions with subcellular proteins

2. Porins – like ion channels, but for larger molecules

Cool stuff: aquaporin allows water to cross the plasma membrane – 13 billion H2O molecules per second! But, as pointed out by T. Todd Jones that is only 0.000000000000018 ml of water.

3. Permeases – function more like an enzyme. Binds the substrate and then undergoes a conformation change which causes the carrier to release the substrate to the other side. Ex. Glucose permeases

Facilitated Diffusion, Cont.

Facilitated Diffusion - Uniporter

GLUT1 – mammalian glucose transporterUses concentration gradient of glucose to drive transportCan work in reverseUsed by most mammals

Electrical Gradients

All transport processes affect chemical gradients

Some transport processes affect the electrical gradient

Electroneutral carriers: transport uncharged molecules or exchange an equal number of charged particles

Electrogenic carriers: transfer a charge, e.g., Na+/K+ ATPase exchanges 3Na+ for 2K+

Membrane Potential

Difference in charge inside and outside the cell ↔ electrochemical gradient

Active transporters establish this gradientTwo main functions

Provide cell with energy for membrane transportAllow for changes in membrane potential used by

cells in cell-to-cell signaling

Can be determined by Nernst equation and Goldman equation

Nernst equation

Used to calculate the electrical potential at equilibrium

Recall: ΔG = RTln([Xi]/[Xo]) + zFEm

Chemical component + electrical component

At equilibrium: zFEm = RTln([Xo]/[Xi])

Equilibrium potential is:

Ex = (RT/zF) ln [Xo]/[Xi]where R – gas constant, T = absolute temperature (Kelvin),

z = valence of ion, F – Faradays constant

Example: K+ out: 0.01 M; K+ in: 0.1 M; T = 22oC

So, EK+ = (1.9872*295)/(1*23062) ln (0.01/0.1)

= -58 mV at 22oC

Nernst equation

Each ion has a different potential given the difference in concentration gradients.

Must have pores or channels to create potential!

Nernst equation and ion concentrations

Differences in Nernst potential reflect differences in chemical gradients!

We will discuss the protein pumps that are necessary to maintain these gradients.

Active Transport

Protein transporter is neededEnergy is requiredMolecules can move from low to high

concentration

Active Transport, Cont.

Two main types: distinguished by the source of energyPrimary active transport – uses an

exergonic reaction ie ATPSecondary active transport – couples the

movement of one molecule to the movement of a second molecule

Primary Active Transport

Hydrolysis of ATP provides energy

Three types• P-type: pump specific ions, e.g., Na+, K+, Ca2+

• F- and V-type: pump H+ • ABC type: carry large organic molecules, e.g.,

toxins

P-class pumps: Na+/K+ ATPase pump

• pumps 2 K+ in and 3 Na+ out• uses ATP as energy source• Blocked with poisons like ouabain or digitalis• Potential built up in the Na+ ions will be used by many different

processes i.e. cotransporters, neuronal signaling etc.

P-class pumps: Na+/K+ ATPase pump

• Na+ binding sites switch from high affinity on inside to low affinity on outside to allow for binding of Na+ on inside and release of Na+ ions on outside.

• K+ binding sites with from high affinity on outside to low affinity on inside

P-class pumps: Ca 2+ ATPase pump

• pumps 2 Ca2+ ions out for every 1 ATP molecule used• Uses ATP to drive Ca 2+ out against a very large concentration gradient• Internal Ca 2+ binding sites have a very high affinity • Energy transfer from ATP to the aspartate of the Ca2+ ATPase causes a protein conformational change and Ca2+ transported across membrane• Ca2+ binding sites on outside are low affinity and Ca2+ is released• The transfer of energy from the ATP to the pump triggers a conformational change that moves the protein and allows the translocation of Ca 2+ across the membrane• At the same time the Ca2+ binding sites change from high to low affinity.

P-class pumps: Ca 2+ ATPase pump cont.

• In muscle cells the Ca2+ ATPase is the major protein found in the membrane of the sacrcoplasmic reticulum (SR)

• 80% of the protein in the SR is the Ca2+ ATPase • SR is a storage site for Ca2+ that is release to drive

muscle contraction• Ca2+ ATPase will remove excess Ca2+ from the cytoplasm

and pump it into the lumen of the SR

V-class pumps: proton pumps

• Transport H+ only• Found in lysosomes, endosomes and plant vacuoles• Transport H+ ions to make the lumen or inside of the

lysosome acidic (pH 4.5 - 5.0)

• Many of these pumps are paired with Cl- channels to offset the electrical gradient that is produced by pumping H+ across the membrane.

V-class pumps: proton pumps

• H+ is transported into the lysosome • Cl- flows in to keep a balance. Why?

Secondary Active Transport

Use energy held in the electrochemical gradient of one molecule to drive another molecules against its gradient

Antiport or exchanger carrier: molecules move in opposite directions

Symport or cotransporter carrier: molecules move in the same direction

Secondary Active Transport

• Uniporter: One molecule. Amino acids, nucleosides,sugars• Symporter/cotransporter: movement in the same directions.

Na+/glucose cotransporter in the intestine• Antiporter/Exchanger: Cl-/HCO3

- exchanger in the red blood cell

Example of a Cotransporter

Membrane Potential and Na+

Animal cells are more negative on the inside than on the outside

(~ -80 to -70 mV)Mostly due to K+ ions (inside >

outside) created via Na+ /K+ pump, K+ leak channels and anions inside the cell (proteins etc)

Remember K+ will move down its concentration gradient

Nernst potential for K+ is - 80 to -70 mV.

Why is this important???Transport of Na+ down the

chemical gradient and the electrical gradient. Makes Na+ a powerful co-transporter!

Favors movement of Na+ into the cell

Membrane Potential and Co-transportersNa+/glucose co-transporter

• Used by cells in the intestine to transport glucose againsta large concentration gradient

• This is a symporter: both in the same direction• ΔG for 2 Na+ is -6 kcal/mol

Membrane Potential and Co-transporters3 Na+/Ca2+ antiporter

• Important in muscle cells• Maintains the low intracellular concentration of Ca2+

• Plays a role in cardiac muscle [Ca2+]i = 0.0002 mM and [Ca2+]o = 2 mM

• ΔG = RTln (2/0.0002) = 5.5 kcal.mol• ΔG = zFEm = 2(23062)(0.070Volts) = 3.3 kcal/mol• Total = 8.8 kcal/mol►So must transport 3 Na+ in for 1 Ca2+ out

Co-transporters

HCO3-/Cl- antiporter

• Regulate pH• Carbon dioxide from respiration:

CO2 + H2O ↔ H2CO3 ↔ H+ + HCO3- in the presence of

• carbonic anhydrase (enzyme)• Note: ~80% of the CO2 in blood is transported as HCO3

-. • This is generated by red blood cells (RBC)• RBC have a protein (AE1) and this is the HCO3

-/Cl- antiporter

• Pumps 1 X 109 HCO3- every 10 msec.

• Clears the CO2 and Cl- transport ensures that there isn't a build up of electrical potential

Membrane Potential and Co-transporters

HCO3-/Cl- antiporter

Co-transporters

Other transporters that regulate pH

Na+/H+ antiporter• Remove excess H+ when cells become acidic

Na+HCO3-/Cl- co-transporter

• HCO3- is brought into the cell to neutralize H+ in the

cytosol: HCO3- + H+ ↔ H2O + CO2 in the presence of

carbonic anhydrase• Driven by Na+: Couples the influx of HCO3

- and Na+ to an efflux of Cl-

Co-transportersExchangers are regulated by internal pH and increase

their activity as the pH in the cytosol falls