Membrane transport and signaling Andy Howard Introductory Biochemistry
Membrane transport and signaling Andy Howard Introductory Biochemistry Tuesday 1 October 2013 Membrane Transport; Signaling 10/01/2013
Membranes: transport and signaling n We’ll complete our discussion of the ways that things move across membranes, and then talk about the way that signals pass across membranes. 10/01/2013 Membrane Transport; Signaling p. 2 of 47
What we’ll discuss n Membrane transport Neutral molecules n Transporting charges n Pores & Channels n Passive Transport n Active Transport n Moving large molecules n n Signal transduction General Principles n G proteins n Adenylyl cyclase n Inositolphospholipid signaling pathway n Sphingolipid messages n Receptor tyr kinases n 10/01/2013 Membrane Transport; Signaling p. 3 of 47
Transmembrane Traffic: Types of Transport Type Protein Carrier Diffusion No Facilitated Yes diffusion* Passive Yes transport Active^ Yes Saturable w/substr. No No Movement Rel. to conc. Down Energy Input? No No Yes Down No Yes Up Yes * accomplished primarily through pores and channels ^ two kinds: primary and secondary 10/01/2013 Membrane Transport; Signaling p. 4 of 47
Cartoons of transport types n From accessexcellence. org 10/01/2013 Membrane Transport; Signaling p. 5 of 47
Thermodynamics of passive and active transport • • If you think of the transport as a chemical reaction Ain Aout or Aout Ain It makes sense that the free energy equation would look like this: Gtransport = RTln([Ain]/[Aout]) More complex with charges; see eqns. 9. 1 through 9. 2 10/01/2013 Membrane Transport; Signaling p. 6 of 47
Example n n n Suppose [Aout] = 145 m. M, [Ain] = 10 m. M, T = body temp = 310 K Gtransport = RT ln[Ain]/[Aout] = 8. 325 J mol-1 K-1 * 310 K * ln(10/145) = -6. 9 k. J mol-1 So the energies involved are moderate compared to ATP hydrolysis 10/01/2013 Membrane Transport; Signaling p. 7 of 47
Charged species n n Charged species give rise to a factor that looks at charge difference as well as chemical potential (~concentration) difference Most cells export cations so the inside of the cell is usually negatively charged relative to the outside 10/01/2013 Membrane Transport; Signaling p. 8 of 47
Quantitative treatment of charge differences n n n Membrane potential (in volts J/coul): Y = Yin - Yout (there’s an extra in eqn. 9. 4) Gibbs free energy associated with difference in electrical potential is Ge = z. F Y where z is the charge being transported and F is Faraday’s constant, 96485 JV-1 mol-1 Faraday’s constant is a fancy name for 1. 10/01/2013 Membrane Transport; Signaling p. 9 of 47
Faraday’s constant n n n Relating energy per mole to energy per coulomb: Energy per mole of charges, e. g. 1 J mol-1, is 1 J / (6. 022*1023 charges) Energy per coulomb, e. g, 1 V = 1 J coul-1, is 1 J / (6. 241*1018 charges) 1 V / (J mol-1) = (1/(6. 241*1018)) / (1/(6. 022*1023) = 96485 So F = 96485 J V-1 mol-1 10/01/2013 Membrane Transport; Signaling p. 10 of 47
Total free energy change n n n When charges move, we typically have both a chemical potential difference and an electrical potential difference so Gtransport = RTln([Ain]/[Aout]) + z. F Y Sometimes these two effects are opposite in sign, but not always 10/01/2013 Membrane Transport; Signaling p. 11 of 47
Pores and channels n Transmembrane proteins with central passage for small molecules, possibly charged, to pass through n n Rod Mac. Kinnon Bacterial: pore. Usually only weakly selective Eukaryote: channel. Highly selective. Usually the Gtransport is negative so they don’t require external energy sources Gated channels: n n Passage can be switched on Highly selective, e. g. v(K+) >> v(Na+) 10/01/2013 Membrane Transport; Signaling p. 12 of 47
Gated potassium channels n n n Eukaryotic potassium channels are gated, i. e. they exist in open or closed forms When open, they allow K+ but not Na+ to pass through based on ionic radius (1. 33Å vs. 0. 95Å) Some are voltage gated; others are ligand gated 10/01/2013 Membrane Transport; Signaling p. 13 of 47
Protein-facilitated passive transport n All involve negative Gtransport n n n Uniport: one solute across Symport: two solutes, same direction Diagram courtesy Saint-Boniface U. Antiport: two solutes, opposite directions Proteins that facilitate this are like enzymes in that they speed up reactions that would take place slowly anyhow These proteins can be inhibited, reversibly or irreversibly 10/01/2013 Membrane Transport; Signaling p. 14 of 47
Kinetics of passive transport n n Michaelis-Menten saturation kinetics: v 0 = Vmax[S]out/(Ktr + [S]out) We’ll derive that relationship in the enzymatic case in a later chapter Vmax is velocity achieved with fully saturated transporter Ktr is analogous to Michaelis constant: it’s the [S]out value for which half-maximal velocity is achieved. 10/01/2013 Membrane Transport; Signaling p. 15 of 47
Velocity versus [S]out Vmax = 0. 5 m. M s-1 Ktr = 0. 1 m. M 10/01/2013 Membrane Transport; Signaling p. 16 of 47
1/v 0 versus 1/[S]out 10/01/2013 Membrane Transport; Signaling p. 17 of 47
Primary active transport n n Energy source is usually ATP or light Energy source directly contributes to overcoming concentration gradient n n Bacteriorhodopsin: light energy used to drive protons against concentration and charge gradient to enable ATP production P-glycoprotein: ATP-driven active transport of many nasties out of the cell 10/01/2013 Membrane Transport; Signaling p. 18 of 47
Secondary active transport n n n Active transport of one solute is coupled to passive transport of another Net energetics is (just barely) favorable Generally involves antiport n n Bacterial lactose influx driven by proton efflux Sodium gradient often used in animals 10/01/2013 Membrane Transport; Signaling p. 19 of 47
Complex case: Na+/K+ pump n n n Typically [Kin] = 140 m. M, [Kout] = 5 m. M, [Nain] = 10 m. M, [Naout] = 145 m. M. ATP-driven transporter: 3 Na+ out for 2 K+ in per molecule of ATP hydrolyzed 3 Na out: 3*6. 9 k. Jmol-1, 2 K in: 2*8. 6 k. Jmol-1 = 37. 9 k. J mol-1 needed, ~ one ATP 10/01/2013 Membrane Transport; Signaling Diagram courtesy Steve Cook p. 20 of 47
What’s this used for? n n Sodium gets pumped back in in symport with glucose, driving uphill glucose transport That’s a separate passive transport protein called Glu. T 1 10/01/2013 Membrane Transport; Signaling Diagram courtesy Steve Cook p. 21 of 47
How do we transport big molecules? n n Proteins and other big molecules often internalized or secreted by endocytosis or exocytosis Special types of lipid vesicles created for transport 10/01/2013 Membrane Transport; Signaling p. 22 of 47
Receptor-mediated endocytosis n n n Bind macromolecule to specific receptor in plasma membrane Membrane invaginates, forming a vesicle surrounding the bound molecules (still on the outside) Vesicle fuses with endosome and a lysozome Inside the lysozyome, the foreign material and the receptor get degraded … or ligand or receptor or both get recycled 10/01/2013 Membrane Transport; Signaling p. 23 of 47
Example: LDL-cholesterol Diagram courtesy Gwen Childs, U. Arkansas for Medical Sciences 10/01/2013 Membrane Transport; Signaling p. 24 of 47
Exocytosis n n n Materials to be secreted are enclosed in vesicles by the Golgi apparatus Vesicles fuse with plasma membrane Contents released into extracellular space 10/01/2013 Membrane Transport; Signaling Diagram courtesy Link. Publishing. com p. 25 of 47
Transducing signals n n Plasma membranes contain receptors that allow the cell Image courtesy to respond to chemical stimuli Nobelprize. org that can’t cross the membrane from 1994 P&M award to Gilman & Bacteria can detect chemicals: Rodbell if something useful comes along, a signal is passed from the receptor to the flagella, enabling the bacterium to swim toward the source 10/01/2013 Membrane Transport; Signaling p. 26 of 47
Multicellular signaling n Hormones, neurotransmitters, growth factors all can travel to target cells and produce receptor signals 10/01/2013 Membrane Transport; Signaling Diagram courtesy Science Creative Quarterly, U. British Columbia p. 27 of 47
Extracellular Signals n n n Internal behavior of cells modulated by external influences Extracellular signals are called first messengers 7 -helical transmembrane proteins with characteristic receptor sites on extracellular side are common, but they’re not the only receptors 10/01/2013 Membrane Transport; Signaling Image courtesy CSU Channel Islands p. 28 of 47
Internal results of signals n n Intracellular: heterotrimeric G-proteins are the transducers: they receive signal from receptor, hydrolyze GTP, and emit small molecules called second messengers Second messengers diffuse to target organelle or portion of cytoplasm Many signals, many receptors, relatively few second messengers Often there is amplification involved 10/01/2013 Membrane Transport; Signaling p. 29 of 47
Roles of these systems n n n n Response to sensory stimuli Response to hormones Response to growth factors Response to some neurotransmitters Metabolite transport Immune response This stuff gets complicated, because the kinds of signals are so varied! 10/01/2013 Membrane Transport; Signaling p. 30 of 47
G proteins (G&G § 32. 4) n n Transducers of external signals into the inside of the cell These are GTPases (GTP GDP + Pi) GTP-bound protein transduces signals GDP-bound protein doesn’t Heterotrimeric proteins; association of b and g subunits with a subunit is disrupted by complexation with hormone-receptor complex, allowing departure of GDP & binding of GTP 10/01/2013 Membrane Transport; Signaling p. 31 of 47
GTP G protein cycle n n Inactive GDP a b Ternary complex g disrupted by binding of b receptor complex g Ga-GTP interacts with effector enzyme GTP slowly hydrolyzed away Then Ga-GDP reassociates with b, g 10/01/2013 Membrane Transport; Signaling Active a GTP H 2 O Pi a GDP Inactive p. 32 of 47
Adenylyl cyclase n n c. AMP and c. GMP: second messengers Adenylyl cyclase converts ATP to c. AMP n n n Integral membrane enzyme; active site faces cytosol c. AMP diffuses from membrane surface through cytosol, activates protein kinase A Protein Kinase A (PKA) phosphorylates ser, thr in target enzymes; action is reversed by specific phosphatases 10/01/2013 Membrane Transport; Signaling Cyclic AMP p. 33 of 47
Modulators of c. AMP n n n Caffeine, theophylline inhibit c. AMP phosphodiesterase, prolonging c. AMP’s stimulatory effects on protein kinase A Hormones that bind to stimulatory receptors activate adenylyl cyclase, raising c. AMP levels Hormones that bind to inhibitory receptors inhibit adenylyl cyclase activity via receptor interaction with the transducer Gi. 10/01/2013 Membrane Transport; Signaling p. 34 of 47
Inositol-Phospholipid Signaling Pathway n n n 2 Second messengers derived from phosphatidylinositol 4, 5 -bisphosphate (PIP 2) Ligand binds to specific receptor; signal transduced through G protein called Gq Active form activates phosphoinositidespecific phospholipase C bound to cytoplasmic face of plasma membrane 10/01/2013 Membrane Transport; Signaling PIP 2 p. 35 of 47
PIP 2 chemistry n n Phospholipase C hydrolyzes PIP 2 to inositol 1, 4, 5 trisphosphate (IP 3) and diacylglycerol Both of these products are second messengers that transmit the signal into the cell 10/01/2013 Membrane Transport; Signaling p. 36 of 47
IP 3 and calcium n n n IP 3 diffuses through cytosol and binds to a calcium channel in the membrane of the endoplasmic reticulum The calcium channel opens, releasing Ca 2+ from lumen of ER into cytosol Ca 2+ is a short-lived 2 nd messenger too: it activates Ca 2+-dependent protein kinases that catalyze phosphorylation of certain proteins 10/01/2013 Membrane Transport; Signaling p. 37 of 47
Calcium homeostasis & IP 3 Courtesy Oulu Univ. , Finland 10/01/2013 Membrane Transport; Signaling p. 38 of 47
Diacylglycerol and protein kinase C n n n Diacylglycerol stays @ plasma membrane Protein kinase C (which exists in equilibrium between soluble & peripheralmembrane form) moves to inner face of membrane; it binds transiently and is activated by diacylglycerol and Ca 2+ Protein kinase C catalyzes phosphorylation of several proteins 10/01/2013 Membrane Transport; Signaling p. 39 of 47
Control of inositolphospholipid pathway n n Figure courtesy Motifolio. com After GTP hydrolysis, Gq is inactive so it no longer stimulates Plase C Activities of 2 nd messengers are transient n n IP 3 rapidly hydrolyzed to other things Diacylglycerol is phosphorylated to form phosphatidate 10/01/2013 Membrane Transport; Signaling p. 40 of 47
The big picture Courtesy bmj. com 10/01/2013 Membrane Transport; Signaling p. 41 of 47
Sphingolipids give rise to 2 nd messengers n Some signals activate hydrolases that convert sphingomyelin to: n n sphingosine-1 -P, and ceramide Each of these modulates a second messenger 10/01/2013 Membrane Transport; Signaling p. 42 of 47
Interconversions Courtesy AOCS Lipid Library 10/01/2013 Membrane Transport; Signaling p. 43 of 47
n n n Fates of sphingolipid products Sphingosine inhibits Protein Kinase C Ceramides activate a protein kinase and a protein phosphatase Sphingosine-1 -P can activate Phospholipase D, which catalyzes hydrolysis of phosphatidylcholine; products are 2 nd messengers 10/01/2013 Membrane Transport; Signaling Phospholipase D Streptomyces with phosphatidyl choline bound PDB 2 ZE 4 54 k. Da monomer 2. 5Å p. 44 of 47
ligands Receptor tyrosine kinases n n n exterior Tyr kinase monomers Most growth factors function via a pathway that involves these enzymes In absence of ligand, 2 nearby tyr kinase molecules are separated Upon substrate binding they come together, form a dimer 10/01/2013 Membrane Transport; Signaling p. 45 of 47 interior
Autophosphorylation of the dimer n n P P Enzyme catalyzes phosphorylation of specific tyr residues in the kinase itself; so this is autophosphorylation Once it’s phosphorylated, it’s activated and can phosphorylate various cytosolic proteins, starting a cascade of events 10/01/2013 Membrane Transport; Signaling p. 46 of 47
Insulin receptor n n n Insulin binds to an a 2 b 2 tetramer; binding brings b subunits together Each tyr kinase (b) subunit phosphorylates the other one The activated tetramer can phosphorylate cytosolic proteins involved in metabolite regulation 10/01/2013 Membrane Transport; Signaling Sketch courtesy of Davidson College, NC p. 47 of 47
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