Membrane Transport Proteins PDF

Cellular Biology & Homeostasis MEMBRANE TRANSPORT VP Summer 2023 Clara Camargo, DVM MEMBRANE TRANSPORTLEARNING OBJECTIVES 1. List characteristics that affect permeability of molecules 2. Recognize membrane proteins and their topology 3. Provide examples of common membrane proteins 4. List importa...

Cellular Biology & Homeostasis MEMBRANE TRANSPORT VP Summer 2023 Clara Camargo, DVM MEMBRANE TRANSPORTLEARNING OBJECTIVES 1. List characteristics that affect permeability of molecules 2. Recognize membrane proteins and their topology 3. Provide examples of common membrane proteins 4. List important differences between ion channels and transport proteins (carriers) 5. What are channels and how are they activated? 6. Explain facilitated diffusion across the plasma membrane 7. Explain primary, secondary, and tertiary active transport MEMBRANE TRANSPORT- how do cells organize what comes in and what goes out? Extracellular space Cystinuria in dogs - FIY • Cystinuria – presence of cystine crystals in the urine. Can lead to obstruction of urine flow, bladder and kidney stones. Cystine crystals in urine • 3 types of cystinuria related to genetic mutations • Mutation affect transport protein responsible for reabsorbing cystine (specially in kidney and intestine) • Cystine crystals tend to form in acidic urine (recall amino acid properties!) – cystine become insoluble in water and precipitate (forming crystals). Cystine stones Cystinosis in humans Nature structural and molecular biology Structural and mechanical insights into cystinosin FYI Research Gate: Cellular model for the reabsorption of amino acids across a proximal kidney tubule cell. MEMBRANE TRANSPORT • However, cells have to transfer molecules and ions across their membranes in order to maintain the homeostasis • 15-30% of all membrane proteins are transport proteins MEMBRANE PERMEABILITY AND TRANSPORT • • The interior of the lipid bilayer is hydrophobic, thus the passage of most polar molecules is restricted. This allows cells to maintain concentrations of solutes in its cytosol that differ from those in the extracellular fluid and in each of the intracellular compartments CYTOSOL (ICF): liquid matrix surrounding organelles CYTOPLASM: All of the materials inside a cell except for the cell nucleus 6 MEMBRANE TRANSPORT TRANSPORT PROTEINS: • transfer specific water soluble (hydrophilic) molecules across the plasma membrane MEMBRANE TRANSPORT The smaller the molecule and the less strongly associated with water → the more rapidly the molecule diffuses across the membrane MEMBRANE POTENTIAL Membrane potential (resting membrane potential = the membrane potential of an unstimulated cell) • A difference in the electrical charge on the two sides of a membrane due to a slight excess of positive ions over negative ones on one side and a slight deficit on the other MEMBRANE POTENTIAL • The resting membrane potential of cells is the result of an active transport (electrogenic) and a passive diffusion, as follows: • Na +,K +-ATPase pumps Na+ out of the cell and draws K+ ions into the cell • K+ tends to diffuse out of the cell through potassium channels to reach an equilibrium whereas negatively charged ions (phosphates and proteins) stay inside the cell • The interior of the cell will turn more negative • (-70 to -90 mV) MEMBRANE TRANSPORT The electrochemical gradient of a charged solute affects its transport (Electrochemical gradient = combination of membrane potential and concentration gradient of the solute) MEMBRANE TRANSPORT PROTEINS Proteins can associate with the plasma membrane in different ways (recall, proteins contain hydrophilic and hydrophobic regions) 1) Single alpha helix; 2) Multiple alpha helices 3) Rolled-up beta sheet (beta barrel) 4) Attached only to one layer (with one hydrophobic face) 5) Attached to the membrane by a covalently bound lipid chain 6) Via an oligosaccharide 7) and 8) attached to other proteins MEMBRANE TRANSPORT PROTEINS- TOPOLOGY Proteins can associate with the plasma membrane in different ways (recall, proteins contain hydrophilic and hydrophobic regions) 1) Single alpha helix; 2) Multiple alpha helices 3) Rolled-up beta sheet (beta barrel) 4) Attached only to one layer (with one hydrophobic face) 5) Attached to the membrane by a covalently bound lipid chain 6) Via an oligosaccharide 7) and 8) attached to other proteins TRANSPORT PROTEINS TRANSPORTERS SHARE COMMON STRUCTURAL FEATURES: • They typically consist of 10 or more alpha helices that span the membrane (transmembrane domains) • Substrate binding sites are located midway through the membrane • They show two different states: • inward-open OR • outward-open conformation • The binding sites are accessible by passageways from only one side of the membrane at one time • They would be able to work in the reverse direction if ion and solute gradients were adjusted TRANSPORT PROTEINS Absorption by enterocytes of the monosaccharide products of carbohydrate digestion. Most membrane proteins cross the lipid bilayer in an alpha-helical conformation GLUT = glucose transporter, Na+/glucose cotransporter SGLT SGLT-1 = sodium (Na+)-dependent glucose cotransporter Glucose transporter GLUT 15 TRANSPORT PROTEINS Na+/Ca2+ exchanger (NCX) Bidirectional transporter → 1 x Ca2+ out of the cell and 3 x Na+ into the cell. Na+/Ca2+ exchange inhibitors: a new class of calcium regulators https://pubmed.ncbi.nlm.nih.gov/17896959/ - FYI MEMBRANE TRANSPORT The Na+, K+-ATPase is present in the plasma membrane of almost all animal cells and maintains Na+ and K+ concentration differences accross the plasma membrane From: Silbernagl and Despopoulos. Color Atlas of Physiology TWO MAIN CLASSES OF MEMBRANE TRANSPORT PROTEINS: CHANNELS AND TRANSPORTERS (CARRIERS) • Channels form pores for specific solutes (ions, water, ammonia)  They interact with the solute much more weakly compared to transporters • Transporters bind the specific substrate (solute) to be transported and undergo a series of conformational changes that alternately expose solute-binding sites on one side of the membrane and then to the other to transfer the solute across it 18 GATED ION CHANNEL TYPES Examples: Channel/K+ Channel Nicotinic Ach-receptor Mechanosensitive channels Mechanosensitive channels: convert mechanical stimuli to chemical or electrical signals thereby modulating sensation. Skin: sensing vibration, pressure sensation, stretch, touch, and light touch Veins: blood pressure Cells: osmotic pressure + K LEAK CHANNEL The vestibule and the selectivity filter • Channels are not gated, K⁺ flows through concentration gradient • In the vestibule (chamber), the ions are hydrated • In the selectivity filter, they have lost their water and oxygens of the carbonyl groups and the channel accomodate the dehydrated solutes • Since Na+ is smaller than potassium, it can not be succesfully accomodated and will not be recognized in the filter AQUAPORINS: SPECIFIC WATER CHANNELS • Facilitate osmotic water flow • Cells that secrete high amounts of water (such as those lining ducts of exocrine glands, mammary gland, sweat glands) OR • Cells that reabsorb high volumes of water (in the kidney)  express aquaporins on plasma membrane making water movement more efficient MEMBRANE TRANSPORT Aquaporins Some aquaporins are hormone-responsive and play an important role in the formation of a concentrated urine in animals Anti-diuretic hormone (ADH) stimulates AQPs in the collecting ducts Water deficit  ↑ extracell. osmolarity  activation of osmoreceptors (hypothalamus)  ADH secretion (post. pituitary)  ↑ water permeability in collecting ducts. From: Klein. Cunningham‘s Texbook of Veterinary Physiology https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3774486/ MEMBRANE TRANSPORT - Aquaporins What about fish??? Figure: modification of work by Duane Raver, NOAA FYI Aquaporin evolution in fishes https://www.frontiersin.org/articles/10.3389/fphys.2011.00044/full MEMBRANE TRANSPORT - Transporters Each transporter can have one or more specific binding sites for its solute (substrate) • Outward-open state: binding site for solutes is exposed to the outside • Occluded state: binding sites are not accessible • Inward-open state: binding sites exposed to the inside TRANSPORT CAN BE ACTIVE OR PASSIVE Passive transport down a concentration gradient occurs spontaneously by diffusion (through the plasma membrane or channels or passive transporters). No energy required. Active transport requires energy as it moves solutes against their concentration gradients  it is always mediated by transporters PASSIVE - SIMPLE DIFFUSION • Here, small or non charged molecules simply dissolves in the phospholipid bilayer, diffuses across it, and then dissolves in the aqueous solution at the other side of the membrane. • No membrane proteins are involved, and the direction of transport is determined simply by the relative concentrations of the molecule inside and outside of the cell • The net flow of molecules is always down their concentration gradient—from a compartment with a high concentration to one with a lower concentration of the molecule PASSIVE - FACILITATED DIFFUSION • It involves the movement of molecules in the direction determined by their relative concentrations inside and outside of the cell • No external source of energy is provided, so molecules travel across the membrane in the direction determined by their concentration gradients and, in the case of charged molecules, by the electric potential across the membrane • It differs from passive diffusion in that the transported molecules do not dissolve in the phospholipid bilayer • Their passage is mediated by proteins (channels or carriers) that enables large and polar (charged) molecules to cross the membrane without directly interacting with its hydrophobic interior FACILITATED DIFFUSION GLUT transporters GLUT 5 – Fructose GLUT 2 – Glucose, fructose, galactose FACILITATED DIFFUSION VS SIMPLE DIFFUSION • Simple diffusion and channelmediated transport rates are directly proportional to the solute concentration • Carrier-mediated transport is saturable (recall: enzyme kinetics and inhibition of activity – binding site) ACTIVE TRANSPORT • Uses energy or a gradient generated by another active transporter • Active transporters can be classified according to the direction of transport as well as the use of energy Direction Uniport Symport Antiport Energy Primary active Secondary active Tertiary active MEMBRANE TRANSPORT ACCORDING TO THE TRANSPORT DIRECTION • Uniporters: transport of only one molecule (H⁺ ATPase) • Symporters: coupled transporters of 2 molecules in the same direction (SGLT cotransporter) • Antiporters: transport of a second molecule in the opposite direction (Na⁺/Ca⁺² NCX exchanger; Na⁺/K⁺ ATPase) ACCORDING TO THE ENERGY SOURCE Primary active (Na+, K+ ATPase) Na+ Secondary active (Na⁺, H⁺ exchanger, NHE) H+ Tertiary active (Proton/peptides co-transporter, PEPT) Lumen Cytosol ATP ADP+Pi K+ Na+ Secondary active • is driven by a gradient that was generated by a primary active transporter H+ Dipeptides Tripeptides Tertiary active • is driven by a gradient that was generated by a secondary active transporter Clara Camargo, DVM [email protected] Thank you