Organization of ATPases

Transcription

1 The Primary Active Transporter II: The ATPase Objectives: Organization P type with NPA domains Proton pumps of the rotary V type ATPase 1 Organization of P type, solute transport, found in plasma membranes and organelles. V type, solute transport, found originally in eukaryotic vacuoles. F type, ATP producers, driven by the proton gradient generated by oxidative phosphorylation (mitochondria) or photosynthesis (chloroplasts). A type, ATP producers, found in Archaea with function like F type. E type, cell surface enzymes, hydrolysing a range of NTPs (ATP, GTP, UTP, etc). 2 1

2 The P type ATPase 3 Organization 4 2

3 Activity 5 The Na + K + pump 6 3

4 Voltage dependence of charge movement (A) and of decay rate constant (B) during transient, strophanthidinsensitive currents (inset in A). Holding potential, 40 mv; temperature, 35 C. A, inset: superimposed sample records of strophanthidin sensitive currents elicited by pulses to 60, 20, 20, and 60 mv; graph, time integral [Q(V)] of current transients initiated by each pulse, plotted against pulse potential (V). Open, Charge measured directly from the records; Filled, charge estimated by extrapolation of the exponential current decay. Only on charge movement is plotted. Off and on charge movements appear to be the same. The smooth curve, derived from the Boltzmann relation. B: rate constants of exponential fits to transient currents plotted against membrane potential: Open, rate constants of on transients; Filled, rate constants of off transients, at 240 mv, following repolarization from 260 mv, V,180 mv 7 ATP dependence Recording of patch current in response to superfusion of the patches with solutions containing different MgATP concentrations. At the beginning and the end of the current recording, a 10 mv voltage pulse was applied to test the seal resistance. Excised patch was from a rat ventricular myocyte. Temperature was 24 C. 8 4

5 Activation of Na + K + pump current by [Na + ] i 9 Activation of Na + K + pump current in guinea pig ventricular myocytes at 0 mv by [K + ] o at high [Na + ] o 10 5

6 ATP hydrolysis speed in different Na + /K + concentrations 11 Early effects of Na/K pump inhibition on the action potential of a guinea pig ventricular myocyte. Within 10 s after application, ouabain, a specific inhibitor of the Na/K ATPase, prolongs the action potential at 90% repolarization (APD90) and raises the action potential plateau. Note the depolarization of the resting sarcolemma. Temperature 35 C. 12 6

7 The Na + K + pump Electrogenic Dependent on Na + and K + in internal or external medium, respectively Blocked by cardiac glycosides (digoxin, ouabain, strophanthidin, etc.) Also blocked by oligomycin B, palytoxin, etc. Requires metabolic energy Contributes to the rest membrane potentials. 13 Cardiac Glycosides Inhibit the Na + /K + pump, Depolarize cardiac myocytes, Raise intracellular Ca ++, Improve stroke volume and Reduce distention of the heart. 14 7

8 Mutations of ATP1A2 gene encoding α subunit of the Na + /K + ATPase 15 Mutations in P cause impairs in various tissues especially those metabolically active. 16 8

9 Structure of the Na + K + pump Crystal structure of the sodium pump with a bound sodium ion. The sodium pump is a protein complex consisting of α and β subunits, and a FXYD protein. Three sodium ion binding sites (I III) are identified in the transmembrane region. [Nature 2013, 502: ] 17 General features of the P IIc ATPase structure Ten transmembrane segments are represented in an unfolded disposition, but they actually form a bundle around the 3 central 4th, 5th, and 6th transmembrane segments. Color coding shows the actuator (A; orange) domain, which is constituted by the NH2 terminal segment preceding the 1st transmembrane segment and the intracellular loop between the 2nd and 3rd transmembrane segments. It contains the TGES motif that is involved in the dephosphorylation step. The phosphorylation (P; blue) domain is made up of the proximal and distal parts of the large intracellular loop between the 4th and 5th transmembrane segments. In its initial section, it contains the DKTGT motif with the aspartyl residue D376, on which the phosphate of ATP is transiently transferred. The nucleotide (N; green) domain is made up of the main part of the large intracellular loop and forms a kind of pocket for ATP binding. The subunit has a single transmembrane segment, a short intracellular NH2 terminal, and a large extracellular domain with several glycosylation sites (3 are shown). In this scheme the subunit transmembrane segment is located close to the 7th and 8th transmembrane segments because the early part of the extracellular domain of the subunit is known to interact with a conserved SYGQ motif located in the relatively large extracellular loop between the 7th and 8th transmembrane segments. The Na K ATPase and subunits may also associate with a third subunit belonging to the FXYD protein family. 18 9

10 Integrated scheme of the Na K ATPase functional cycle ntegrated scheme of the Na K ATPase functional cycle Left: the main steps of the cation transport cycle The circular arrow in the center shows the 19 State dependence of Na K ATPase poisoning ntegrated scheme of the Na K ATPase functional cycle Left: the main steps of the cation transport cycle The circular arrow in the center shows the 20 10

11 The H + /K + ATPase The H + /K + ATPase belongs to the P type ATPase superfamily. The H + /K + ATPase transports one H + out of the cell in exchange for one K + into the cell. The process consumes a high energy phosphate in ATP during each transport cycle. 21 The Sarco/Endoplasmic Reticulum Ca 2+ ATPase (SERCA) 22 11

12 A model of how the SERCA moves Ca SERCA and PMCA P type with N, P and A domains. Stoichiometry of Ca ++ /ATP is 2. Stoichiometry of H + /Ca ++ is 2. They have Ca ++ affinity higher than troponin

13 The V type ATPase 25 Proton Pumps The proton pumps, also called V, are ATP driven transporters that function to both acidify intracellular compartments and to transport protons across the plasma membrane

14 Role of intracellular V ATPase in membrane trafficking, endocytosis, and secretion. Extracellular ligands are internalized by receptor mediated endocytosis and trafficked to the sorting endosome. Acidification of the endosome by the V ATPase allows for release of the ligand and the recycling of the receptor back to the membrane. Budding of endosomal carrier vesicles and multivesicular bodies are also dependent on the acidic environment. Lysosomal proteins are synthesized in the trans Golgi network and are trafficked to the lysosome via the mannose 6 phosphate receptor. Acidification of the late endosome allows for the release of the lysosomal proteins and recycling of the man 6 P receptor back to the Golgi. In the lysosome acidification activates cathepsins and other degradative enzymes. The ph gradient created by the V ATPase in secretory vesicles drives the uptake of neurotransmitters and other molecules to be secreted. The V0 domain, has also been proposed to play an important role in membrane fusion.(b) 27 Function of plasma membrane V 28 14

15 Role of intracellular V ATPase in the entry of envelope viruses and toxins. Envelope viruses and bacterial toxins such as diphtheria toxin enter the cell via endocytosis where they are trafficked to the sorting endosome. The low ph generated by the V ATPase causes the viral coat to fuse with the endosomal membrane releasing the viral m RNA into the cytoplasm. The acidic environment also induces the diphtheria toxin B chain (shown in green) to form a pore in the membrane that facilitates the entry of the A chain (shown in red) into the cytoplasm. 29 Function of Intracellular V : Receptor mediated endocytosis, Intracellular membrane trafficking, Prohormone processing, Protein degradation, Neurotransmitter uptake, as well as Infection and inflammation responses

16 Function of Plasma Membrane Proton Pumps: Urinary acidification, Bone resorption, Sperm maturation and Tumor metastasis. 31 Structural Model of the Vacuolar (H + ) ATPase (V ATPase). Electron cryomicroscopy (cryo EM) structure of the yeast V ATPase with known crystal structures of individual subunits from S. cerevisiae and T. thermophilus fitted into the map. The V 1 cytosolic domain, made up of subunits A H, is involved in ATP hydrolysis, while the integral V 0 domain, made up of subunits a, d, e, c, and c, conducts protons across the membrane. ATP hydrolysis in the A 3 B 3 complex drives central stalk movement (subunits D, F, and d), which in turn rotates the proteolipid ring to allow proton transport across the membrane through hemichannels located in subunit a. Subunits E, G, C, and H, and the N terminal domain of subunit a, make up the peripheral stalks and serve to tether V 1 to V 0. Note that subunit e is absent from the preparation used for this cryo EM structure owing to its loss during detergent extraction [Nature 32521: ]. 16

17 33 The proton V ATPase Made of a large multi subunit complex. A peripheral domain (V1) responsible for rotary movement by hydrolysis of ATP An integral domain (V0) that carries out proton transport by sequential protonation and deprotonation of glutamic acid residues

18 Glutamate Arg 735 A. ATP hydrolysis by the V1 domain drives the rotation of a rotor composed of the D, F, d, and proteolipid subunits (c, c0, and c00). Rotation of the proteolipid ring past the a subunit drives the movement of protons from the cytoplasmic to the luminal side of the membrane. Two peripheral stalks, the EGC and EGH subcomplexes, hold the A3B3 hexamer stationary with respect to a. B. Schematic representation of the helical arrangement in the proteolipid ring showing the unique orientation of the proteolipid subunits embedded in the membrane. The view is from the cytoplasmic side of the membrane. C. Mechanism of proton translocation in the V0 domain. Only the membrane integral C-terminal domain of a (yellow) and the proteolipid ring (blue) are shown. The unprotonated form of the proteolipid s essential glutamate residue enters the incoming hemi-channel on a. A proton from the cytoplasmic side of the membrane (the top in this diagram) enters the channel and protonates the exposed glutamic acid residue. As the proteolipid ring rotates, the now neutral glutamic acid is exposed to the hydrophobic lipid bilayer. As the ring rotates, the glutamic acid approaches the a subunit again, and interacts with a secondhemi-channel on the luminal side of the membrane. Residue Arg-735 on a (shown in green) interacts with the glutamic acid, changing its pka and promoting the 35 deprotonation and release of the proton into the luminal side of the membrane through the hemi-channel. Mechanism Hydrolysis of an ATP molecule leads to a rotary movement of the V1 domain by 120. The rotation exposes a glutamic acid residue in the V0 domain to the cytoplamic side. The glutamate is protonated. A rotary movement by 60 covers the glutamate in the cytoplamic side and exposes it to the extracellular/luminal side. Meanwhile, the glutamate is exposed to Arg 735, leading to a change in its pka and deprotonation. The protonation and deprotonation of the glutamic acid residue complete a cycle to transport two protons per ATP, or stoichiometry is

19 Similarity of F and V type as proton pumps 37 ATP is produced by F ATPase but not V ATPase

20 Although structurally similar to the F type ATPase, the V type ATPase does not have the capability of reverse operation to produce ATP using ph gradients