Berg Tymoczko Stryer Biochemistry Seventh Edition Chapter 13 Membrane Channels Pumps Copyright 2012 by W H Freeman and Company cid 190 Membranes transport involve many proteins Concepts Pumps Carriers Channels cid 190 Mechanisms of pump transport SERCA ATPase pump for Ca2 MsbA ABC pump for lipid transport cid 190 Mechanisms of carrier transport Antiport symport Lactose permease symport cid 190 Mechanisms of channel transport Na K channels and action potential K channel structure and function Channel gating mechanisms Voltage gating K channel Ligand gating Ach receptor Transport across membranes Ability of a molecule to cross a membrane depends on 1 The membrane permeability to the molecule 2 The presence of appropriate transporter and an energy source For uncharged membrane permeable molecules chemical concentration gradient determines spontaneous movement across membrane GTrans RT ln c2 c1 R gas constant T temperature mol mol For charged molecules one need to consider an additional electrical potential term GTrans RT ln c2 c1 ZF V Z charge of the molecule F Faraday constant V charge gradient potential for all ions Na2 For all ions Na2 GTrans indicates if the transport is passive GTrans 0 or if it requires energy input e g ATP and is thus active GTrans 0 3 classes of proteins for transports across membranes 1 Membrane pumps 2 Membrane carriers 3 Membrane channels For primary active transport When GTrans 0 Drive thermodynamic uphill reactions with ATP as free energy source Different pumps use different strategy for transport and use of ATP For secondary active transport When GTrans 0 Drive thermodynamic uphill reactions using the chemical gradient of one molecule to drive the transport of a second molecule against its own gradient No use of ATP For passive transport Provide a selective pore through which ion can flow rapidly When GTrans 0 No use of ATP Can be sensitive to membrane polarization Membranes pumps Involved in primary active transport of molecules often ions against their conc gradient Need an energy source ATP often the source of energy for this process sometimes process driven by light Pumps have ATPase activity hydrolyze ATP to transfer phosphate group to their own residues e g Asp Free energy of ATP induces large conformational changes in pumps both phosphorylation dephosphorylation Interconversion between 2 conformations open close provides unidirectional pumping of ions Pump are important to maintain steady state concentration of ions in cells Regulation of Ca2 conc important for cell signaling Regulation of Na K conc important for membrane potential SERCA ATPase Ca2 pumps Sarcoplasmic reticulum Ca2 SERCA ATPase important to rapidly remove excess Ca2 after Ca2 triggered muscle contraction relaxation phase Action potential Sarcoplasmic reticulum SERCA pumps Ca2 back into SER after muscle contraction Reconstitute Ca2 reserves for next action potential and next contraction Works against concentration of Ca2 so need ATP P type ATPase P for phosphorylated intermediate Modified from Wray Burdyga Physiol Rev 90 113 178 2010 SERCA ATPase Ca2 pumps continued 2 Ca2 transported for 1 ATP hydrolyzed Structure 10 transmembrane helices 3 cytosolic domains A P N P domain Phosphorylated on Asp351 N domain Binds Nucleotide ATP A domain Actuator that induces conformational changes ATP binding and ADP dissociation induce two large conformational changes that result in disruption of Ca2 binding sites in transmembrane domain and Ca2 release A SERCA ATPase Ca2 pumps continued Binding of ATP Asp phophory lation Ca2 x2 bind Induce fit binding of ATP Release of Ca2 Release of ADP SERCA reset Evert back to initial conforma tion Phospho aspartate hydrolyzed Conformat ional change ABC transporter pumps Good example of pumps where coupling of ATP to conformational changes differ significantly from P ATPase for a similar transport mechanism ABC ATP Binding Cassette 1 transmembrane domain and 2 ABC 1 molecule transported for 2 ATP hydrolyzed Can work for transport in out cell eukaryotes or out in cell prokaryotes Works against concentration gradients so need ATP Interconvertion between open and close form in the absence of ATP Binding of specific substrate induces steric fit than enhances affinity for ATP MsBa lipid flippase for LPS ABC transporter pumps continued Substrate binding Equilibrium stabilizes weak between open close state close state Reset of pump by ATP hydrolysis ABC interaction lead to conformational changes substrate release Increased affinity for ATP ATP binding induces strong interaction between ABC A look at membrane carriers Involved in secondary active transport of ions Use concentration gradient rather than ATP for conformational changes Secondary active transport coupling a thermodynamically unfavorable reaction e g transport against conc gradient with a favorable one e g transport with conc gradient COTRANSPORT Antiporter Symporter Transport of 2 solutes in Transport of 2 solutes in opposite directions same direction Uniporter Transport of a single solutes following conc gradient An example of symport membrane carrier Lactose permease in E coli Use proton H gradient down their conc gradient to drive the entry of lactose against it conc gradient H Lactose Proton binds Glu 269 Protonated form favors lactose binding H Lactose Reset to initial state Loss of proton inside cell Lactose release Inside cell Change in conformation Membrane ion channels Involved in passive transport of ions e g Na K down concentration gradients when GTrans 0 Fastest transporters 1000 folds faster than pumps or carriers Close to diffusion rates of ions Different ion channels use similar transport mechanisms yet are highly selective for specific ions Channels can work because of ion concentration gradients generated by membrane pumps in live cells Na and K ion channels are particularly important for signal communication in the nervous systems Sequential triggering of Na and K ion channels generates action potential nerve impulse Na ion channel Payandeh et al Nature 2011 475 353 358 K ion channel Action potential are generated in neurons by membrane depolarization involving Action potential Na and K ion channels Na channels More open Na channels close K channels open K channels close Na channels open Ion channels can be studied by patch clamping Isolation of a single channel with a thin pipette Recording changes in current upon opening and closing A p t n e r r u C Time ms Structure of K channel