Membrane Transport - Ion Channels and Pumps 2 PDF

Membrane Transport - Ion channels and pumps 2 Dr Dawn Jones  Ion pumps Transporters working in active transport are called pumps Transport is energetically unfavourable, so an energy source is required  Transporters switch between two different...

Membrane Transport - Ion channels and pumps 2 Dr Dawn Jones  Ion pumps Transporters working in active transport are called pumps Transport is energetically unfavourable, so an energy source is required  Transporters switch between two different conformations Pumps work by alternating access to the substrate binding pocket from one side of the membrane and the other, driven by energy input How is the energy provided?  Energy sources for pumps Secondary Primary transport transport  Primary transporters Driven by ATP hydrolysis Substrate moving up ATP ! ADP+ Pi electrochemical Energetically favourable reaction gradient (ATP hydrolysis) is coupled to an energetically unfavourable reaction (movement of a substrate up its electrochemical gradient) ATP-driven (primary) transporters The Na+-K+ pump Also called Na+-K+ ATPase Pumps Na+ out of and K+ into cells Sets up Na+ and K+ gradients needed for action potentials This pump uses a large proportion of our cells’ energy needs! Belongs to a family of proteins called P- type pumps, because they are phosphorylated during transport  Cyclic nature of Na+/K+ pump 1. Na+ ions bind activating the ATPase 2. ATPase splits off terminal PO4 group from ATP and transfers it in a high energy bond to the pump itself – the pump is phosphorylated 3. Conformational change releases Na+ to outside and exposes K+ binding site 4. Extracellular K+ binds triggering (i) loss of the high- -energy PO4group--dephosphorylated pump and (ii)conformational change to starting form 5. K+ ions now released into cytosol, K+ site is lost and Na+ site reforms and pump is ready to work Cycle takes ~10ms! 3 binding sites for Na+ only 2 for K+. again.  Clinical insight Digitalis inhibits the Na+-K+ pump Digitalis, derived from the foxglove plant, is a potent inhibitor of the Na+-K+ pump It prevents dephosphorylation of the pump during the reaction cycle Blocking the pump raises the intracellular [Na+], which increases intracellular [Ca2+], causing the heart to pump harder Traditionally used to treat congestive heart failure Has been used as a treatment for hundreds of years in the UK!  Ca2+ ATPase (SERCA) is another P-type ion pump Pumps Ca2+ into the sarcoplasmic reticulum (ER of muscle cells), driven by ATP hydrolysis Mechanism is fairly well understood, as we have structures for the 2 major conformations of the protein (solved by x-ray crystallography) called E1 and E2 E1 E2 Ca2+ ATPase in E1 conformation Transmembrane domain – 10 TM a-helices with 2 Ca2+ ions bound A domain – actuator domain P domain – phosphorylation domain. Aspartate 351 is phosphorylated in reaction cycle N domain – nucleotide binding domain. ATP binds and hydrolyses here Ca2+ ATPase in E2 phosphorylated (E2-P) conformation Structure solved with aspartate 351 phosphorylated Conformation has switched to E2 Mechanism of the Ca2+ ATPase ATP-driven (primary) transporters  ABC transporters Contain domains called ATP-binding cassettes (ABC) 2 transmembrane domains + 2 ABC domains  ABC transporters Switching between 2 conformations causes substrate transport Binding and hydrolysis of ATP at ABC domains causes conformational change Substrate released on other side of membrane Substrate binds  Clinical insight Multidrug resistance in cancer Tumour cells often develop resistance to anti-cancer drugs Due to expression of an ABC transporter called MDR or P-glycoprotein Pumps a wide range of substrates, including drugs out of cells Structure of the mouse MDR protein was solved in 2009 Knowing the structure may help scientists to find inhibitors to prevent drug resistance  Clinical insight Cystic fibrosis Cystic fibrosis is an autosomal recessive inherited disease Causes frequent lung infections and digestive problems Caused by defects in an ABC transporter called cystic fibrosis transmembrane conductance regulator (CFTR) Acts as a chloride ion channel, regulated by ATP binding and hydrolysis  Energy sources for pumps Secondary Primary transport transport  Secondary (coupled) transporters Driven by movement of an ion down its electrochemical gradient Ion moving down Substrate moving up electrochemical electrochemical gradient gradient  Secondary (coupled) transporters Two types: Antiporters – ion and substrate molecule move in opposite direction Symporters – ion and substrate move in same direction  Na+-glucose symporter Glucose can be moved into cells against its concentration gradient Driven by the Na+ electrochemical gradient, set up by the Na+-K+ ATPase Na+-glucose symporter  Glucose transporter allows glucose to leave down its concentration gradient The two glucose--transporting proteins (Sglt1, Glut1) are kept in separate parts of the cell membrane by tight junctions – more in cell junction lectures.  Na+-glucose symporter How much glucose can be pumped into the cell?? DG for Na+ electrochemical gradient = -14.5 kJ.mol-1  Na+-glucose symporter How much glucose can be pumped into the cell?? DG for Na+ electrochemical gradient = -14,500 J mol-1 Amount of energy available to pump glucose = 14,500 J mol-1 Energy required to move glucose = cin ΔG = RT ln + ZFΔV cout Glucose is not charged cin so Z = 0 ΔG = RT ln cout  Summary – ion channels and pumps Ion channels allow movement of ions down their electrochemical gradient (passive transport) The structure of K+ channels has been well studied and explain selectivity for K+ Ion channels can be voltage gated or ligand gated Ligand-gated ion channels are important in action potentials Pumps are transporters that move molecules against their electrochemical gradient ATP-driven pumps (primary transporters) include P-type pumps and ABC transporters Secondary transporters use the energy of an electrochemical gradient of an ion to move another molecule against its concentration gradient