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Cellular Processes Diffusion, channels and transporters Cellular Processes Diffusion, channels and transporters

Cellular Membranes Two main roles • Allow cells to isolate themselves from the environment, 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: 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 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 permeability Lipid bilayer

Membrane proteins we will discuss Membrane proteins we will discuss

Membrane Transport Three main types • Passive diffusion • Facilitated diffusion • Active transport Membrane Transport Three main types • Passive diffusion • Facilitated diffusion • Active transport

Passive Diffusion ¬ Lipid-soluble molecules (alcohol, CO 2) ¬ No specific transporters are needed Passive Diffusion ¬ Lipid-soluble molecules (alcohol, CO 2) ¬ No specific transporters are needed ¬ No energy is needed ¬ Depends on concentration gradient ¬High low ¬Steeper 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 molecules ¬Protein transporter is needed - Uniporter ¬No energy is needed Facilitated Diffusion ¬Hydrophilic molecules ¬Protein transporter is needed - Uniporter ¬No energy is needed ¬Depends on concentration gradient ¬Examples: amino acids, nucleosides, sugars (glucose)

Facilitated Diffusion, Cont. Three main types of proteins 1. Ion channels – form pores, Facilitated Diffusion, Cont. Three main types of proteins 1. 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

Facilitated Diffusion, Cont. 2. Porins – like ion channels, but for larger molecules Cool Facilitated Diffusion, Cont. 2. Porins – like ion channels, but for larger molecules Cool stuff: aquaporin allows water to cross the plasma membrane – 13 billion H 2 O molecules per second! But, as pointed out by T. Todd Jones that is only 0. 000000018 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 - Uniporter GLUT 1 – mammalian glucose transporter ¬Uses concentration gradient of Facilitated Diffusion - Uniporter GLUT 1 – mammalian glucose transporter ¬Uses concentration gradient of glucose to drive transport ¬Can work in reverse ¬Used by most mammals

Electrical Gradients ¬All transport processes affect chemical gradients ¬Some transport processes affect the electrical 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 3 Na+ for 2 K+

Membrane Potential ¬Difference in charge inside and outside the cell ↔ electrochemical gradient ¬Active Membrane Potential ¬Difference in charge inside and outside the cell ↔ electrochemical gradient ¬Active transporters establish this gradient ¬Two main functions ¬Provide cell with energy for membrane transport ¬Allow 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]) Nernst equation Used to calculate the electrical potential at equilibrium Recall: ΔG = RTln([Xi]/[Xo]) + z. FEm Chemical component + electrical component At equilibrium: z. FEm = RTln([Xo]/[Xi]) Equilibrium potential is: Ex = (RT/z. F) 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 = 22 o. C So, EK+ = (1. 9872*295)/(1*23062) ln (0. 01/0. 1) = -58 m. V at 22 o. C

Nernst equation Each ion has a different potential given the difference in concentration gradients. 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! 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 needed ¬Energy is required ¬Molecules can move from low Active Transport ¬Protein transporter is needed ¬Energy is required ¬Molecules can move from low to high concentration

Active Transport, Cont. Two main types: distinguished by the source of energy ¬Primary active Active Transport, Cont. Two main types: distinguished by the source of energy ¬Primary active transport – uses an exergonic reaction ie ATP ¬Secondary 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 Primary Active Transport Hydrolysis of ATP provides energy Three types • P-type: pump specific ions, e. g. , Na+, K+, Ca 2+ • 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 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 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 Ca 2+ ions out for P-class pumps: Ca 2+ ATPase pump • pumps 2 Ca 2+ 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 Ca 2+ ATPase causes a protein conformational change and Ca 2+ transported across membrane • Ca 2+ binding sites on outside are low affinity and Ca 2+ 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 Ca 2+ binding sites change from high to low affinity.

P-class pumps: Ca 2+ ATPase pump cont. • In muscle cells the Ca 2+ P-class pumps: Ca 2+ ATPase pump cont. • In muscle cells the Ca 2+ ATPase is the major protein found in the membrane of the sacrcoplasmic reticulum (SR) • 80% of the protein in the SR is the Ca 2+ ATPase • SR is a storage site for Ca 2+ that is release to drive muscle contraction • Ca 2+ ATPase will remove excess Ca 2+ 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 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 (p. H 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 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 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 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-/HCO 3 - exchanger in the red blood cell Example of a Cotransporter

Membrane Potential and Na+ ¬ Animal cells are more negative on the inside than Membrane Potential and Na+ ¬ Animal cells are more negative on the inside than on the outside (~ -80 to -70 m. V) ¬ 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 m. V. 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-transporters Na+/glucose co-transporter • Used by cells in the intestine to Membrane Potential and Co-transporters Na+/glucose co-transporter • Used by cells in the intestine to transport glucose against a large concentration gradient • This is a symporter: both in the same direction • ΔG for 2 Na+ is -6 kcal/mol

Membrane Potential and Co-transporters 3 Na+/Ca 2+ antiporter • Important in muscle cells • Membrane Potential and Co-transporters 3 Na+/Ca 2+ antiporter • Important in muscle cells • Maintains the low intracellular concentration of Ca 2+ • Plays a role in cardiac muscle [Ca 2+]i = 0. 0002 m. M and [Ca 2+]o = 2 m. M • ΔG = RTln (2/0. 0002) = 5. 5 kcal. mol • ΔG = z. FEm = 2(23062)(0. 070 Volts) = 3. 3 kcal/mol • Total = 8. 8 kcal/mol ►So must transport 3 Na+ in for 1 Ca 2+ out

Co-transporters HCO 3 -/Cl- antiporter • Regulate p. H • Carbon dioxide from respiration: Co-transporters HCO 3 -/Cl- antiporter • Regulate p. H • Carbon dioxide from respiration: CO 2 + H 2 O ↔ H 2 CO 3 ↔ H+ + HCO 3 - in the presence of • carbonic anhydrase (enzyme) • Note: ~80% of the CO 2 in blood is transported as HCO 3 -. • This is generated by red blood cells (RBC) • RBC have a protein (AE 1) and this is the HCO 3 -/Clantiporter • Pumps 1 X 109 HCO 3 - every 10 msec. • Clears the CO 2 and Cl- transport ensures that there isn't a build up of electrical potential

Membrane Potential and Co-transporters HCO 3 -/Cl- antiporter Membrane Potential and Co-transporters HCO 3 -/Cl- antiporter

Co-transporters Other transporters that regulate p. H Na+/H+ antiporter • Remove excess H+ when Co-transporters Other transporters that regulate p. H Na+/H+ antiporter • Remove excess H+ when cells become acidic Na+HCO 3 -/Cl- co-transporter • HCO 3 - is brought into the cell to neutralize H+ in the cytosol: HCO 3 - + H+ ↔ H 2 O + CO 2 in the presence of carbonic anhydrase • Driven by Na+: Couples the influx of HCO 3 - and Na+ to an efflux of Cl-

Co-transporters Exchangers are regulated by internal p. H and increase their activity as the Co-transporters Exchangers are regulated by internal p. H and increase their activity as the p. H in the cytosol falls