Lecture 12-Transport across biological membranes III (April 1).pdf

LECTURE 12 W24 Passive Transport (Facilitated Diffusion) 2: Erythrocyte Anion Exchange Protein (AE1, or Band 3 in erythrocytes) (chloride-bicarbonate exchanger) Part of a larger family of anti-port proteins member of the AE family of proteins, found in all cell types: AE1 (erythrocytes), AE2 (liver)...

LECTURE 12 W24 Passive Transport (Facilitated Diffusion) 2: Erythrocyte Anion Exchange Protein (AE1, or Band 3 in erythrocytes) (chloride-bicarbonate exchanger) Part of a larger family of anti-port proteins member of the AE family of proteins, found in all cell types: AE1 (erythrocytes), AE2 (liver), AE3 (brain, heart, retina); also, plants and microorganisms Anti-port – one-for-one exchange of Cl− for HCO3− (so also electro-neutral) Physiological role – involved in shuttling of CO2 from the tissues to the lungs via the blood CO2 has a low water solubility, so waste CO2 from tissues is converted to the more soluble HCO3− form for transport in blood to lungs the enzyme that converts CO2 to HCO3- is in the cytosol of the red blood cell at tissues: – CO2 enters cell by diffusion; carbonic anhydrase catalyses conversion to HCO3− (equilibrium constant = 1, reversible) – HCO3− exits cell via Band 3 down its gradient in exchange for Cl− from blood at lungs: – HCO3− re-enters cell via Band 3 down its gradient, Cl− leaves – HCO3− converted to CO2, which diffuses out to blood, and is exhaled Lehninger Figure 11-32 The Band 3 Transporter (AEP) performs electroneutral co-transport The coupling of Cl- and HCO3- is obligatory AE is a co-transport system. In the absence of Cl-, HCO3transport cannot be carried out AEP is a dimeric integral protein spanning the membrane 14 times Commun Biol 5, 1372 (2022). https://doi.org/10.1038/s42003-022-04306-8 Two types of active transport Primary Active Transport: Solutes transport directly coupled to an exergonic reaction Secondary Active Transport 1. Primary active transport creates a gradient of one solute 2. The exergonic downhill movement of this solute is coupled to the endergonic movement of another up its concentration gradient Unlike with passive transport, active transport results in accumulation of solutes above the equilibrium concentration Transport energy can be derived from: ▪ ▪ ▪ ▪ Absorption of sunlight Oxidation reactions Hydrolysis of ATP Simultaneous flow of another chemical species down its electrochemical gradient Primary Active Transport 1. P-Type ATPases (“P” for phosphorylation) A family of cation transporters that are reversibly phosphorylated by ATP Phosphorylation gives rise to a conformational change leading to the movement of ions across the membrane Examples of P-type ATPases All P-type pumps have similar structures and mechanisms Integral proteins with 8-10 membrane spanning sections in a single polypeptide Conserved Asp that undergoes phosphorylation Sensitive to inhibition by phosphate mimic Vanadate Sarcoplasmic/endoplasmic reticulum Ca+2 ATPase (SERCA) pump Along with the plasma membrane Ca+2-ATPase pump (PMCA), SERCA plays an important roles in maintaining cytoplasmic Ca+2 level below 1μM. The maintenance of this low [Ca+2] is important for cell signalling. Both transporters are uniporters of Ca+2 ions. SERCA pumps Ca+2 from the cytoplasm to the sarcoplasmic reticulum. Plasma membrane Ca+2-ATPase pumps Ca+2 out of the cell. SERCA pump Each catalytic cycle moves two Ca+2 ions across the membrane and converts ATP to ADP + Pi. E1: the two Ca+2 binding sites are exposed on the cytoplasmic side of ER and binds Ca+2 with high affinity ATP binding and the phosphorylation of Asp converts E1 conformation to E2 E2: Exposes the Ca+2 binding site on the lumenal side of the membrane and reduces the affinity to Ca+2 Release Ca+2 to the lumen Na+K+ ATPase of the plasma membrane Uses a variation of the mechanism used by SERCA Couples the phosphorylation-dephosphorylation of the Asp to the movement of 3 Na+ out and 2 K+ in across the plasma membrane against their electrochemical gradient-electrogenic co-transport Maintains low Na+ and high K+ in the cell relative to the extracellular fluid, creating a net separation of charge - inside negative, outside positive leading to a membrane potential of -50 to -70 mV Up to 30% of energy (ATP) used by cells go towards inhibited by ouabain and maintaining cytosolic Na+ and K+ concentrations. digitoxigenin (foxglove) In a nerve cell this can be up to 70%. energy source for (40 kg ATP required per day for a person just sitting secondary active around!) transport Homework Mechanism of action of some important drugs How does Digitoxigenin, an inhibitor of Na+/K+ ATPase act as a drug to stimulate cardiac contraction (Increase cardiac output)? 2. V-Type ATPases (V for vacuolar) Involved in acidifying various intracellular compartments Vacuoles of fungi and higher plants maintained at a pH of 3-6 (cytoplasm 7.5) Acidification of lysosomes, endosomes, the Golgi complex, secretory vesicles in animals V0- Proton channel V1-ATP binding site and ATPase activity Structure is similar to F-type ATPase 3. F-type ATPases (F-energy coupling factors) Catalyses the uphill transport of H+ powered by ATP hydrolysis F0 (integral)- (o for inhibition by oligomycin) – Pathway for H+ F1 peripheral- (First factor identified) – uses ATP energy to drive H+ up its gradient In the presence of a sufficiently large H+ gradient, can work in reverse direction to synthesise ATP –more appropriately called ATP synthases Synthesise ATP in mitochondria and chloroplasts and in bacteria and archaea Proton gradient created by oxidation of reduced cofactors or by sunlight 4. ABC ATPases (“ABC” for ATP-binding cassette) The ABC superfamily has a common generic structure ~48 ABC transporters in human; ~80 in E. coli Many are found in the plasma membrane but also found in ER, mitochondria and lysosomes All members have two ATP binding domains (cassettes) (hence the name)-NBD and 2 transmembrane domains, each with 6 transmembrane helices Could be monomers or dimers The NBD is conserved across different ABC transporters. 7 sub-families (ABCA-ABCG) Actively transports (against a concentration gradient) a wide variety of compounds (e.g., amino acids, peptides, proteins, metal ions, various lipids, bile salts as well as drugs) The inward facing and the outward facing forms interconvert to move substrates across the membranes, driven by ATP hydrolysis ~1:1 stoichiometry for ATP hydrolysed: number of molecules transported Some ABC transporters are specific for a given substance while some others are not Table 11-2 Some ABC Transporters in Humans Gene(s) Role/characteristics Text reference ABCA1 Reverse cholesterol transport; defect causes Tangier disease Fig. 21-47 ABCA4 Only in visual receptors, recycling of all-trans-retinal Fig. 12-19 ABCB1 Multidrug resistance P-glycoprotein 1; transport across blood-brain barrier ⸻ ABCB4 Multidrug resistance; transport of phosphatidylcholine ⸻ ABCB11 Transports bile salts out of hepatocytes Fig. 17-1 ABCC6 Sulfonylurea receptor; targeted by the drug glipizide in type 2 diabetes Fig. 23-27 ABCG2 Breast cancer resistance protein (BCRP); major exporter of anticancer drugs p. 396 ABCC7 CFTR (Cl₋ channel); defect causes cystic fibrosis Box 11-2 multidrug transporter (MDR1) = human ABC transporter with very broad substrate specificity – encoded by the ABCB1 gene – removes toxic compounds – responsible for resistance of tumors to drugs Physiological role of ABC drug efflux pumps 15 ABC proteins are involved in drug efflux They play an important role in protecting organisms from toxicity of both exogenous (diet, toxicants) and endogenous molecules The specific tissues that are protected depend on the pattern of expression and activity of each protein The three clinically most important transporters involved in drug efflux are: – P-glycoprotein (MDR1; ABCB1) – Multidrug resistance-associated protein (MRP1; ABCC1) – Breast cancer resistance protein (BCRP; ABCG2) Multidrug transporter (MDR1) encoded by the ABCB1 gene In placental membrane and the blood brain barrier protects the fetus and brain from harmful compounds Also responsible for the resistance shown by some tumors to anti cancer drugs MDR1 pumps the chemotherapeutic drugs doxorubicin and vinblastin out of cells Overexpression of MDR1 is associated with treatment failure of liver, colon and kidney cancers Breast Cancer Resistance Protein (BCRP) encoded by the ABCG2 gene Overexpressed in breast cancer cells & confers resistance to anticancer drugs Inhibitors of these transporters can increase the efficacy of anti cancer drugs The Cystic Fibrosis Transmembrane conductance Regulator Protein (CFTR) An ABC protein that is an ion channel (Cl-) Regulated by ATP hydrolysis but does not have the pumping function of other ABC transporters Transports Cl- across plasma membrane when both NBDs have ATP bound. Channel closes when ATP on one of the NBDs is hydrolysed to ADP + Pi. Further regulated by phosphorylation of several Ser in the R domain. The three states of the CFTR protein The Cystic Fibrosis Transmembrane conductance Regulator Protein (CFTR) The most common mutation in CF (>90%) is a deletion of Phe at position 508 (F508del) leading to the incorrect folding of the protein. Reduced Cl- ion transport across plasma membrane of epithelial cells lining airways, digestive tract etc. Reduced Cl- transport results in reduced export of water from cells leading to thick, dehydrated mucus that prevents the removal of bacteria by the movement of cilia. Frequent bacterial infection of the airways lead to progressive damage. Less common mutation such as G551D (Gly to Asp) results in correctly folded protein targeted to the membrane but defective in Cl- transport ABC Transporters in microbes ABC transporters in E. Coli and other bacteria such as those used to import vitamin B12 are thought to be the precursors of MDRs in animals ABC transporters can confer antibiotic resistance in pathogenic bacteria Is a serious public health issue – target for novel drug development