Molecular Biology of the Cell

Transcription

1 Alberts Johnson Lewis Morgan Raff Roberts Walter Molecular Biology of the Cell Sixth Edition Chapter 11 Membrane Transport of Small Molecules and the Electrical Properties of Membranes Copyright Garland Science 2015 Phospholipids Spontaneously Form Bilayers 1

2 The prohibition of free edges leads to closing in on itself CHAPTER CONTENTS PRINCIPLES OF MEMBRANE TRANSPORT TRANSPORTERS AND ACTIVE MEMBRANE TRANSPORT CHANNELS AND THE ELECTRICAL PROPERTIES OF MEMBRANES 2

3 Introduction PRINCIPLES OF MEMBRANE TRANSPORT Protein-Free Lipid Bilayers Are Impermeable to Ions 3

4 Protein-Free Lipid Bilayers Are Impermeable to Ions Depending on how fast it can be dissolved in lipid bilayers Small Hydrophobic Nonpolar PRINCIPLES OF MEMBRANE TRANSPORT There Are Two Main Classes of Membrane Transport Proteins: Transporters and Channels 4

5 There Are Two Main Classes of Membrane Transport Proteins: Transporters and Channels Molecules /s Molecules /s ConformaEonal change ConformaEonal change PRINCIPLES OF MEMBRANE TRANSPORT Active Transport Is Mediated by Transporters Coupled to an Energy Source 5

6 Passive v.s. active transport? [sugar] [ion] - chemical gradient [sugar]? [ion] - electrochemical gradient V m =-200mV For uncharged molecules: Passive: move from higher concentration to lower concentration Active: need energy input in order to move from lower concentration to higher concentration Active Transport Is Mediated by Transporters Coupled to an Energy Source 6

7 Active Transport Is Mediated by Transporters Coupled to an Energy Source TRANSPORTERS AND ACTIVE MEMBRANE TRANSPORT Introduction 7

8 Introduction Passive transport mediated by uniporter Introduction 8

9 facilitated diffusion The differences between passive diffusion and facilitated diffusion: 1. Transporting rate 2. Certain V max : limited number of transporters presented on the membrane. 3. Specificity: K m K M =1.5 mm Blood glucose is 5 mm, 77% of the maximal rate K M =20 mm After a meal, blood glucose increase from 5 mm to 14 mm, GLUT1 or GLUT2 expressing cells double their glocose uptake? Introduction Active transport Ion concentration gradients ATP Light/redox Bacteriorhodopsin/ Cytochrome C oxidase Energy sources 9

10 TRANSPORTERS AND ACTIVE MEMBRANE TRANSPORT Active Transport Can Be Driven by Ion-Concentration Gradients Three types of transporters: Uniporter, Symporter and antiporter 1.4 min 10

11 Active Transport Can Be Driven by Ion-Concentration Gradients Three types of transporters Secondary active transporter In many cases, they share similar 3D structure Mechanism of glucose transport fueled by Na + gradient Animal plasmamembrane: Using Na + gradients Plants, Yeasts, Bacteria: Using H + gradients 11

12 17/10/17 Transporters are built from (inverted) repeats Inverted repeat MFS: Direct repeat Transporter can works in the reverse direction TRANSPORTERS AND ACTIVE MEMBRANE TRANSPORT Antiporters 12

13 Antiporters in the Plasma Membrane Regulate Cytosolic ph Metabolic will make cytosol acidic, use Na + in /HCO 3 - in /Cl- out /H+ out exchanger and Na + in /H+ out increase cytosolic ph, and Band 3 to balance out their reaction H + /H + Important for CO 2 circulation Carbonic anhydrase: HCO 3 - CO 2 + OH - TRANSPORTERS AND ACTIVE MEMBRANE TRANSPORT An Asymmetric Distribution of Transporters in Epithelial Cells Underlies the Transcellular Transport of Solutes 13

14 1.4 min An Asymmetric Distribution of Transporters in Epithelial Cells Underlies the Transcellular Transport of Solutes Tight junction: 1. Prevent glucose diffusion 2. Help define membrane domains 14

15 TRANSPORTERS AND ACTIVE MEMBRANE TRANSPORT There Are Three Classes of ATP-Driven Pumps There Are Three Classes of ATP-Driven Pumps Form phosphoprotein 15

16 TRANSPORTERS AND ACTIVE MEMBRANE TRANSPORT A P-type ATPase Pumps Ca 2+ into the Sarcoplasmic Reticulum in Muscle Cells Structure of the Sarcoplasmic Reticulum Ca 2+ Pumps in Muscle Cells 16

17 The pumping cycle of the sarcoplasmic reticulum Ca 2+ pump 10-7 M (resting cells) 10-6 M (contracting cells) Calcium binding è 1. Close the pathway to cytosol 2. Phosphate transferred to aspartate high-affinity binding 10-2 M low-affinity binding ADP replaced with ATP è 4. Open the pathway to SR lumen ~90% of integral protein in SR membrane TRANSPORTERS AND ACTIVE MEMBRANE TRANSPORT The Plasma Membrane Na + -K + Pump Establishes Na + and K + Gradients Across the Plasma Membrane 17

18 The function of the Na + -K + Pump 1.4 min The function of the Na + -K + Pump Animal Cell devotes more than one third of its ATP to fuel this pump 18

19 TRANSPORTERS AND ACTIVE MEMBRANE TRANSPORT ABC (ATP-binding cassettes) Transporters Constitute the Largest Family of Membrane Transport Proteins Small-molecule transport by typical ABC transporters Importing or exporting Exporting To extracellular space or ER 19

20 A small section of the double membrane of Gram-negative bacteria, e.g. E. coli 1. In E. coli, 78 genes (5% of the total genes) encode ABC transporters 2. The variety of substrates is great and includes inorganic ions, amino acids, mono- and polysaccharides, peptides, lipids, drugs and even proteins The auxiliary transport system associated with transport ATPase in bacteria with double membrane 20

21 Multiple drug resistance (MDR) protein, also call P-glycoprotein 1. Resistant to multiple anticancer drugs (MDR) 2. Chloroquine (malaria drug) resistance 3. Cystic fibrosis transmembrane conductance regulator protein (CFTR) ATP-gated Cl - channel 2.2 min CHANNELS AND THE ELECTRICAL PROPERTIES OF MEMBRANES Aquaporins Are Permeable to Water But Impermeable to Ions 21

22 The role of aquporins in fluid secretion in exocrine glands Another example: epithelial cells of kidney Water pass through the pore of aquaporin in a single file From 1.4 min, total 2 min 22

23 Why do aquoporins transport only water, but not ions or proton? 1. Energy cost of dehydrating ion is too high 2. Two Asn in the center prevent proton to get past Carbonyl group Hydrophobic Hydrophilic Proton diffuse, using molecular relay mechanism that required the making and breaking of hydrogen bonds between adjacent water molecules CHANNELS AND THE ELECTRICAL PROPERTIES OF MEMBRANES Ion Channels Are Ion-Selective and Fluctuate Between Open and Closed States More than 100 types of channels Electrical excitability of muscle Electrical signaling of neuron Leaf closing response of mimosa 23

24 Ion Channels Fluctuate Between Open and Closed States Ion selectivity The gating of ion channels 24

25 CHANNELS AND THE ELECTRICAL PROPERTIES OF MEMBRANES The Membrane Potential in Animal Cells Depends Mainly on K + Leak Channels and the K + Gradient Across the Plasma Membrane The Membrane Potential in Animal Cells Depends Mainly on K + Leak Channels and the K + Gradient Across the Plasma Membrane 25

26 The Membrane Potential in Animal Cells Depends Mainly on K + Leak Channels and the K + Gradient Across the Plasma Membrane Nongated ion channels and the resting membrane potential 26

27 E K =(RT/ZF)*ln([K] o /[K] c ) (Nernst equation) R:gas constant, cal/degree.mol T:absolute temperature F:Faraday constant, cal/mol.v At 20 cytosol 150 mm 15 mm K + : E K (V)=(0.059/Z)*log([K] o /[K] c ) E K (mv)=(59/z)*log([k] 0 /[K] c ) E K (mv)=(59/1)*log(15/150) E K (mv)= -59mV E Na =(RT/ZF)*ln([Na] o /[Na] c ) (Nernst equation) cytosol 15 mm 150 mm R:gas constant, cal/degree.mol T:absolute temperature F:Faraday constant, cal/mol.v At 20 Na + : E Na (V)=(0.059/Z)*log([Na] o /[Na] c ) E Na (mv)=(59/z)*log([na] 0 /[Na] c ) E Na (mv)=(59/1)*log(150/15) E Na (mv)= +59mV 27

28 The ionic basis of a membrane potential Figure The ionic basis of a membrane potential. A small flow of inorganic ions through an ion channel carries sufficient charge to cause a large change in the membrane potential. 1. Na + K + pump to generate K + gradient 2. K + leak channel to mediate K + influx and generate membrane poteneal Only 1/100,000 total number of K flowing out is enough to generate 100 mv CHANNELS AND THE ELECTRICAL PROPERTIES OF MEMBRANES The Three-Dimensional Structure of a Bacterial K + Channel Shows How an Ion Channel Can Work 28

29 The Three-Dimensional Structure of a Bacterial K + Channel Shows How an Ion Channel Can Work 1.5 min The Three-Dimensional Structure of a Bacterial K + Channel Shows How an Ion Channel Can Work 29

30 K + specificity of the selectivity filter in a K + channel A model for the gating of a bacterial K + channel Cytosolic side Like diaphragm ( 光圈 ) 30

31 CHANNELS AND THE ELECTRICAL PROPERTIES OF MEMBRANES Mechanosensitive Channels Protect Bacterial Cells Against Extreme Osmotic Pressures In hypotonic conditions, cell Swells, mechanosensitive channel activated, small molecular leak out Auditory hair cell in human cochelea 1.3 min 31

32 The structure of Mechanosensitive Channels CHANNELS AND THE ELECTRICAL PROPERTIES OF MEMBRANES Voltage-Gated Cation Channels Generate Action Potentials in Electrically Excitable Cells 32

33 Voltage-Gated Cation Channels Generate Action Potentials in Neuron 3.2 min A typical vertebrate neuron 33

34 Structure models of voltage-gated Na + channels How do Voltage-Gated Cation Channels Generate Action Potentials? Green line: in the absence of Na + channel 34

35 The propagation of an action potential along an axon CHANNELS AND THE ELECTRICAL PROPERTIES OF MEMBRANES The Use of Channelrhodopsins Has Revolutionized the Study of Neural Circuits Channelrhodopsins: photosensitive ion channels that open in response to light 35

36 Optogenetics: Use light flashes to activate specific neurons From 2.1 min CHANNELS AND THE ELECTRICAL PROPERTIES OF MEMBRANES Myelination Increases the Speed and Efficiency of Action Potential Propagation in Nerve Cells 36

37 Myelination Related desease: Multiple sclerosis Where the Na + channels are concentrated Advantages: 1. Action potential travel much fast 2. Save metabolic energy by confining the excitation at node of Ranvier CHANNELS AND THE ELECTRICAL PROPERTIES OF MEMBRANES Patch-Clamp Recording Indicates That Individual Ion Channels Open in an All-or- Nothing Fashion 37

38 The technique of Patch-Clamp Recording Patch-Clamp Recording Indicates That Individual Ion Channels Open in an All-or-Nothing Fashion 38

39 CHANNELS AND THE ELECTRICAL PROPERTIES OF MEMBRANES Transmitter-Gated Ion Channels Convert Chemical Signals into Electrical Ones at Chemical Synapses Transmitter-Gated Ion Channels Convert Chemical Signals into Electrical Ones at Chemical Synapses 1.5 min 39

40 A chemical synapse Neurotransmitter-gated channels (ionotropic receptors) CHANNELS AND THE ELECTRICAL PROPERTIES OF MEMBRANES Chemical Synapses Can Be Excitatory or Inhibitory Excitatory neurotransmitters (acetylchloline, glutamate and serotonin) open Na + or Ca ++ channels Inhibitory neurotransmitters (γaminobutyric acid GABA and glycine) open Cl - or K + channels Two types of neurotransmitter receptors Ionotropic receptors: fast Metabotropic receptors: slow, but longlasting ² G-protein coupled receptors 40

41 CHANNELS AND THE ELECTRICAL PROPERTIES OF MEMBRANES The Acetylcholine Receptors at the Neuromuscular Junction Are Excitatory Transmitter-Gated Cation Channels Permeable to K +, Na + and Ca ++, but Ca ++ is too low, and K + is at equilibrium. So, it mainly mediate Na + influx, cause depolarization, and then the muscle contract. The Acetylcholine Receptors at the Neuromuscular Junction Are Excitatory Transmitter-Gated Cation Channels Free acetylcholine is cleaved by acetylcholinesterase 41

42 CHANNELS AND THE ELECTRICAL PROPERTIES OF MEMBRANES Many Psychoactive Drugs Act at Synapses Curare, targeting acetylcholine receptors on skeletal muscle cells, to relax muscles during operation Ambien, sleeping pills, bind to and activate GABA receptor (an inhibitory Cl - receptor) Neuromuscular Transmission Involves the Sequential Activation of Five Different Sets of Ion Channels 1. Voltage-gated Ca ++ channel 2. Acetylcholine receptors 3. Voltage-gated Na + channel 4. Voltage-gated Ca ++ channel in transverse tubules 5. Ca ++ -pump in SR Cytosolic Ca ++ increase, and then muscle cell contract. 42

43 Single Neurons Are Complex Computation Devices Neuronal Computation Requires a Combination of at Least Three Kinds of K + Channels in the initial segment for encoding 1. Delayed K + Channels: Reploarize the membrane after each action potential to prepare the cell to file again Voltage-gated, but slow kinetics, open during the falling phase of the action potential when Na+ channels are inactive, make the membrane back to the K+ equilibrium potential which make Na+ channels rcovered from their inactivated state Repolarization of the membrane also closes the delayed K+ channels 43

44 Neuronal Computation Requires a Combination of at Least Three Kinds of K + Channels in the initial segment for encoding 2. Rapidly inactivating K+ channel: The frequence of the firing has to reflect the intensity of the simulation. And, below a threshold level of stimulation, the cell will not fire It also opens when the membrane is depolarized, But with specific voltage sensitivity and kinetics of inactivation Figure The magnitude of the combined postsynaptic potential (PSP) is reflected in the frequency of firing of action potentials. The mix of excitatory and inhibitory PSPs produces a combined PSP at the initial segment. A comparison of (A) and (B) shows how the firing frequency of an axon increases with an increase in the combined PSP, while (C) summarizes the general relationship Neuronal Computation Requires a Combination of at Least Three Kinds of K + Channels in the initial segment for encoding 3. Ca +2 activating K + channel: Adaptation: decrease the response of the cell to an unchanging. Prolonged stimulation will increase cytosolic Ca 2+ via voltage-gated Ca 2+ channel, and then activate Ca +2 activating K + channel. Make the membrane harder to depolarize Feel a light touch on the shoulder and yet ignore the constant pressure of our clothing 44

45 CHANNELS AND THE ELECTRICAL PROPERTIES OF MEMBRANES Long-Term Potentiation (LTP) in the Mammalian Hippocampus (for Learning) Depends on Ca 2+ Entry Through NMDA- Receptor Channels Some synapses in the hippocampus show a striking form of synaptic plasticity with repeated use: whereas occasional single action potentials in the presynaptic cells leave no lasting trace, a short burst of repetitive firing causes long-term potentiation (LTP ), such that subsequent single action potentials in the presynaptic cells evoke a greatly enhanced response in the postsynaptic cells. The effect lasts hours, days, or weeks, according to the number and intensity of the bursts of repetitive firing Long-Term Potentiation (LTP) in the Mammalian Hippocampus Depends on Ca 2+ Entry Through NMDA-Receptor Channels LTP occurs on any occasion when a presynaptic cell fires (once or more) at a time when the postsynaptic membrane is strongly depolarized (either through recent repetitive firing of the same presynaptic cell or by other means). The NMDA-receptor channels are doubly gated, opening only when two conditions are satisfied simultaneously: glutamate must be bound to the receptor, and the membrane must be strongly depolarized. The second condition is required for releasing the Mg2+ that normally blocks the resting channel 45

46 Long-Term Potentiation (LTP) in the Mammalian Hippocampus Depends on Ca 2+ Entry Through NMDA-Receptor Channels 46