115:408/512 - Molecular Biology & BiochemistryLecturer: Bob Niederman A317 Nelson BiologyLabs, Busch Campus, Tel: 445-3985; E-Mail:[email protected]#1 - Membrane Transport March 1, 20051) Overview -- transport maintains cellular levels of solutessufficient to meet metabolic needs by using various membrane-localized non-specific pores and specific carrier proteins(permeases)2) Passive diffusion -- equalizes transmembrane concentrations ofuncharged molecules; for charged molecules, depends on theircharges and transmembrane concentrations, transportedspecies provides driving force.3) Facilitated diffusion -- mediated by intramembrane proteinfacilitators: e.g., erythrocyte glucose and anion transporters;show saturation kinetics, solute flows in thermodynamicallyfavored direction.4) Active transport systems-- energy-driven mediators ofselective membrane permeability.a.) ATP-driven transport processes:(i) Ca2+ transport by Ca2+-ATPase -- carries Ca2+ from musclecell cytosol to sarcoplasmic reticulum, inducing musclerelaxation; X-ray structure shows distinct membrane,nucleotide binding, actuator and phosphorylation domains(phosphorylation state alters conformation between E1 (Ca2-binding) E2 (Ca2+-releasing) states .(ii) Na+,K+-ATPase drives cation transport -- plasmamembrane Na+ pump, maintains cellular [Na+ and K+] andNa+ and K+ gradients that drive amino acid, sugar andnucleotide transport; structurally and mechanisticallyhomologous to Ca2+-ATPase.(iii)Gastric H+,K+-ATPase -- maintains H+ gradient @pH 1.0 instomach lumen, facilitating digestion; electrically neutralflow of H+ of into stomach lumen, balanced by K+ flow backinto cell via K+ channel(iv) Multidrug-resistance transport ATPases promoteresistance to chemotherapeutic agents and multiple drugresistance.b.) Light-driven active transport performed bybacteriorhodopsin, a light-transducing H+ pump:Uses photon as energy source to transport H+ acrossmembrane against electrochemical H+ gradient, resulting inaltered affinities for H+ on opposite sides of membrane;Establishes H+ gradient that drives transport and ATPsynthesis.c.) Ion-gradient driven processes -- H+, Na+, other cation andanion gradients drive the transport of amino acids andsugars by symport (ion and solute move in same direction)or antiport (movement in opposite directions) mechanisms.5) Specialized membrane pores:a.) Porins -- outer membrane of Gram-negative bacteria andmitochondria -- solutes move by bulk H2O flow.b.) Aquaporins (AQPs) -- in tissues with intrinsically high H20permeability, form constitutive H20-selective channel forosmotically-directed H20 flow; found in kidney tubules,plants, yeast, bacteria;c.) Gap junctions -- in mammalian cell membranes -- connectadjacent cells for passive flow of small molecules, ions,mediating cellular metabolic and electrical coupling.Reading: Garrett, R. H. & Grisham, C. M. (2005) Biochemistry, 3rdEdition, Chapter 9, pp. 284-306.Membrane Transport• Passive diffusion• Facilitated diffusionRBC glucose transporterRBC anion transporter• Active transport systemsATP drivenLight drivenIon-gradient driven• Specialized membrane poresPorinsAquaporinsGap junctionsPassive Diffusion of an Uncharged Species Across the Membrane• Transport of uncharged species solelydependent upon concentrations (C1 =high, and C2 = low ) on two sides ofmembrane• Driven by concentration differenceacross membrane (C2 < C1 )• No net solute accumulation at equil-ibrium (C2 = C1 , !G = 0 ).• Passively transported species diffusein bilayer in absence protein carrier,except when permeability coefficientis high (e.g., aquaporins as carriers intissues with intrinsically high H2Opermeability) where passive diffusionis not the whole story• (1) !G = chemical potential (freeenergy) difference = G2- G1; R = gasconstant (~2 cal/•mol); and T=absolutetemp (•K)Passive Diffusion of a Charged Species Across the Membrane• The passive diffusion of a chargedspecies depends not only upon itsconcentration, but also on its netcharge (Z) and on the membranepotential (!" )• Note that side 2 has higherchemical potential than side 1.• But, since Z = -1, this electricalterm makes a negative !Gcontribution:ZF!"<0; (F =Faraday’s constant (charge of 1mol of e-))• Thus, transported species providesits own driving force and passesspontaneously across the bilayer.• (2) !G = G2 - G1 = RTln C2 / C1 +ZF!"Facilitated Diffusion -- Mediated by IntramembraneProtein Facilitators (Carriers)• Facilitate S movement (!G<0)• Protein facilitates transport-- has a specific soluteaffinity, increasing thetransport rate• Solute flows in thermo-dynamically favoreddirection• Uptake shows saturationkinetics• Solute may be phosphoryl-ated, rapidly metabolizedGlucose transporter (erythrocytes) -- 55-kDa integral mem-brane protein, 12 #-helical TMS, trimer as active form, 3helix bundle as hydrophilic solute channel, reduced expres-sion in diabetics (Family of facilitative sugar transporters --GLUT1-GLUT12)1Anion Transporter (Erythrocytes)• 95-kDa protein with14 TMS• Catalyzes one-for-oneCl-/HCO3- exchange• Collects HCO3- in res-piring tissue, releasesCl-• Exchanges HCO3- forCl- in lungsA model for the arrangement of the anion transport protein inthe membrane, based on hydropathy analysis.Active Transport -- Energy-Driven SoluteMovement Against Concentration Gradient• Variety of active transport systems -- mainmediators of selective membrane permeability• Energy source and transport machinery arecoupled• Energy sources -- ATP, light, ion gradientsATP-Driven Active Transport Processes• Sarcoplasmic reticulum (SR) Ca2+-ATPase• Plasma membrane Na+,K+-ATPase• Gastric H+,K+-ATPase• Multidrug-resistance (MDR) transport ATPase• Cystic fibrosis transmembrane regulator(CFTR) (ATP-driven Cl- channel)Ca2+-ATPase -- Calcium Transport in Muscle Cells• Acts as a Ca2+ pump -- (“relaxing factor”) maintains lowCa2+ levels (~0.01 µM) in resting muscle cell cytosol• Ca2+-ATPase (110 kDa protein) located in sarcoplasmicreticulum (SR) membrane• P-type ATPase (Single multifunctional polypeptide chain)• Transports Ca2+ into SR lumen (inside), inducing musclerelaxation (resting state)• Pumps 2 Ca2+/ATP hydrolyzed, autophosphorylatedduring catalysis• Ca2+ stores released from sarcoplasmic reticulum duringmuscle contraction via Ca2+ channel (raises cytosoliclevels to ~10 µM)2Crystal Structure of Sarcoplasmic