Membrane Channels and Pumps

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Chapter 13

• Membrane Channels and Pumps

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Membrane Channels and Pumps

• Lipid bilayer impermeable to ions and polar
  molecules

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Transport of Molecules

• Two factors determine whether a molecule will
  cross a membrane
• (1) permeability of molecule in lipid bilayer
• Lipophilic molecules
• Simple diffusion High to Low
• Increase in entropy powers transport across the
  membrane
• (2) availability of an energy source
• Charged or polar molecules

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Membrane Proteins

• Membrane proteins confer permeability
• Protein Transporters to cross membrane
• Channels
• Pumps

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Types of Transport

• Active Transport
• Pumps
• Thermodynamically uphill transport of ions or
  molecules
• Requires an input of energy (ATP or light energy)
• Passive Transport (Facilitated Diffusion)
• Channels
• Thermodynamically downhill transport of ions

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Types of Transport

• Active Transport
• ATP-driven pumps
• P-type ATPase
• ATP-Binding Cassette
• Secondary Transporters
• E. coli lactose transporter
• Passive Transport (Facilitated Diffusion)
• Ion Channels
• Gap Junctions

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Expression of Transporters

• Each cell type expresses a specific set of
  transporters in its plasma membrane
• Transporters determines
• Ionic composition inside cell
• Substrates taken-up from cells environment
• Types of biochemical reactions within a cell

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Free Energy Stored in Concentration Gradients

• Z - electrical charge of transported species
• ?V potential across membrane
• F Faraday Constant (96.5 kJ/V-mol) or (23.1
  kcal/V)

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Free Energy Stored in Concentration Gradients

• Transport of an uncharged molecule from
• c1 1 x 10-3 M to
• c2 1 x 10-1 M
• at 25oC (298 K)

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Free Energy Stored in Concentration Gradients

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Free Energy Stored in Concentration Gradients

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Free Energy Stored in Concentration Gradients

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Types of Transport

• Active Transport
• ATP-driven pumps
• P-type ATPase
• ATP-Binding Cassette
• Secondary Transporters
• E. coli lactose transporter
• Passive Transport (Facilitated Diffusion)
• Ion Channels
• Gap Junctions

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Active Transport

• ATP-Driven Pump
• P-type ATPase
• ATP-Binding Cassette
• Secondary Transporters
• E. coli lactose transporter

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Active Transport

• ATP-Driven Pump
• P-type ATPase
• ATP-Binding Cassette
• Secondary Transporters
• E. coli lactose transporter

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P-type ATPase

• Form key phosphorylated intermediate
• ATP hydrolysis
• Phosphoryl group linked to a specific aspartate
  residue in ATPase
• Aspartate residue conserved
• ATP hydrolysis coupled to orientation change and
  affinity change in ion-binding site

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P-type ATPases

• Na-K ATPase
• H-K ATPase
• Gastric
• Pumps H into stomach to lower pH below 1.0
• Ca2 ATPase
• Pumps Ca2 from cytoplasm into sarcoplasmic
  reticulum of muscle cells

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Ca2 ATPase

• Sarcoplasmic reticulum of muscle cells
• Integral Membrane Protein
• 80 of sarcoplasmic reticulum membrane protein
• Role in muscle contraction
• Removal of Ca2 from cytosol
• 0.1 ?M in cytosol 1.5 mM SR

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P-type ATPases

• Forms key phosphorylated intermediate
• Phosphoryl group linked to a specific aspartate
  residue in ATPase
• Aspartate residue conserved
• Conformation change
• ATP hydrolysis
• E1 and E2
• Six conformational states E1, E1-(Ca2)2, E1-
  (Ca2)2 (ATP), E1- (Ca2)2 (ADP), E2-P, and E2

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Na-K Pump

• Same mechanism
• Three Na ions transported out of cells as result
  of phosphorylation and transition to E2 state
• E2 binds two K ions and transport into the cells
  by hydrolysis of phosphoaspartate.

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Ion Gradient

• Important energy storage forms
• Animal cells intracelllular concentrations
• High concentration of K
• Low concentration of Na
• Na-K ATPase / Na-K Pump
• Specific transport system
• Enzyme
• Energy provided from ATP hydrolysis

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Na-K Gradient

• Controls cell volume
• Renders neurons and muscle cells electrically
  excitable
• Drives the active transport of sugars and amino
  acids

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Active Transport

• ATP-Driven Pump
• P-type ATPase
• ATP-Binding Cassette
• Secondary Transporters
• E. coli lactose transporter

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ATP-Driven Pumps

• ATP-Binding Cassette
• Conformation change upon ATP binding and
  hydrolysis
• Bound ion transported across membrane

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ATP Binding Cassette

• Undergo conformational changes on ATP binding and
  hydrolysis
• Conformational change coupled with each dimeric
  transporter unit
• Drive uptake or efflux of specific compounds or
  act as gates for open membrane channels

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Multidrug Resistance

• MDR or P-glycoprotein
• Membrane protein 170 kd
• ATP-dependent pump
• Extrude wide range of small molecules from cells
  that express it
• Cystic Fibrosis
• CFTR-cystic fibrosis transmembrane conductance
  regulator
• ATP-regulated chloride channel in plasma
  membranes of epithelial cells

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Active Transport

• ATP-Driven Pump
• P-type ATPase
• ATP-Binding Cassette
• Secondary Transporters
• E. coli lactose transporter

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Secondary Transporters

• Energy from concentration gradient downhill
  flow of one gradient power formation of another
  gradient
• Antiporters
• Drive uphill flow in opposite direction
• Symporters
• Drive uphill flow in same direction
• Sodium-Calcium Exchanger

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Sodium-Calcium Exchanger

• Lower affinity for Ca2 than Ca2 ATPase
• Exchanger lowers cytosolic Ca2 level to several
  micromolar
• Pump lowers cytosolic Ca2 level to submicromolar
  range
• Greater capacity to extrude Ca2 than Ca2 ATPase
• Exchanger can transport 2000 Ca2 /second
• Pump can transport 30 Ca2 /second

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Passive Transport/Ion Channels

• Highly selective for particular ions
• Open and Closed States
• Regulated transitions between open and closed
  states
• Spontaneous conversion of open state to an
  inactivate state

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Passive Transport/Ion Channels

• Ligand-Gated Channel
• Open and close in response to binding of specific
  chemicals
• Voltage-Gated Channel
• Open and close in response to electrical
  potential across membrane

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Ion Channels Propagation of Nerve Impulses

• Ligand-Gated Channel
• Acetylcholine Receptor Channel
• communicates nerve impulse between certain
  neurons
• Votage-Gated Channels
• Na Channel
• K Channel
• Conduct the nerve impulse down the axon of a
  neuron

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Axon

• The part of a nerve cell or neuron that transfers
  a nerve impulse from the nerve cell body to a
  synapse with another cell.

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Nerve Cell
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Nerve Cell
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Synaptic Cleft

• The minute space between the cell membrane
• of an axon terminal and that of the target cell
• with which it synapses.
• 50 nm

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Ligand-Gated ChannelAcetylcholine Receptor

• Integral membrane protein that responds to the
  binding of the neurotransmitter acetylcholine
• Communication of nerve impulse
• Neurotransmitter acetylcholine
• Binding of acetylcholine to receptor changes the
  ionic permeability
• Large influx of Na and smaller outflux of K

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Action Potential

• A momentary change in electrical potential on
  the surface of a nerve or muscle cell that takes
  place when it is stimulated, especially by the
  transmission of a nerve impulse
• Stimulating a nerve fiber causes an action
  potential to spread across the nerve cell, making
  it contract.

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Acetylcholine Receptor

• Purification
• Nonionic detergent to postsynaptic membrane
  prepartion
• Affinity chromatography using immobilized
  cobratoxin
• Structure
• 268 kd
• Pentamer ?2, ?, ?, ?
• Arranged in a ring that creates a pore through
  the membrane
• Acetylcholine binds at the ??-? and ?-? interfaces

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Mechanism of Channel Opening???

• Alternating ridges of small polar or neutral
  residues and large nonpolar residues
• Closed State
• Large residues may occlude channel by forming
  tight hydrophobic ring
• Open State
• Acetylcholine binding could allow allosteric
  rotation of membrane-spanning helices to allow
  small polar residues to line the pore and allow
  passage of Na and K

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Ion Channels Propagation of Nerve Impulses

• Ligand-Gated Channel
• Acetylcholine Receptor Channel
• communicates nerve impulse between certain
  neurons
• Votage-Gated Channels
• Na Channel
• K Channel
• Conduct the nerve impulse down the axon of a
  neuron

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Nerve Cell
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Voltage-Gated Channel

• Na Channel
• K Channel
• Propagation of Nerve Impulses

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Membrane Polarization

• A typical neuron
• -60 mV at rest (polarized)
• 30 mV at peak of action potential (depolarized)
• Amplified depolarization propagated along nerve
  terminal
• Polarized
• Membrane of resting neuron
• Inside of membrane negatively charged relatively
  to the outside of the membrane
• Depolarized
• Nerve impulse causes membrane to undergo a large
  and short-lived increase in ion permeability
  across the membrane
• First Na influx is so great that for a msec
  the inside becomes positively charged relative to
  the outside.
• Second Permeability of Na returns to normal
  and the permeability of K outflux increases and
  restore the inside of the cell to its original
  negative state within few msec.
• Efficient signaling over large distances only
  1/one million of sodium and potassium ions in
  nerve cells flow across plasma membrane during
  action potential

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Rapid Rates of Transport

• Tight Binding
• How can rapid flow of ions be achieved with tight
  binding of K ions to the carbonyl oxygen
  (channel)??????

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Ca2 Channel Selectivity

• Glutamate residue in region between segments S5
  and S6 plays a major role in determining ion
  selectivity

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Gap Junctions

• Cell - to - cell channels
• Connect interiors of contiguous groups of cells
• Polar molecules with mass of less than 1 kd can
  pass through gap junctions
• Important for intercellular communication

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Gap Junctions

• Traverse two membranes
• Connect cytosol to cytosol
• Connexons forming channel synthesized by
  different cells
• Form readily when cells brought together
• Open for seconds minutes
• Closed by Ca2 and H
• Also controlled by membrane potential and
  hormone-induced phosphorylation

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Connexins

• Several inherited disorders of humans such as
  certain congenital heart defects and certain
  cases of congenital deafness have been found to
  be caused by mutant genes encoding connexins.