Biochemistry of membrane transport

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Cell Structure and Function 2003 - 2004
Transport of small molecules across membranes
Part II Structure and Physiological Function of
Membrane Transporters
Svetlana Lutsenko MRB 625, phone
494-6953 lutsenko_at_ohsu.edu
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Learning objectives

• Know the function and structural organization of
  channels
• Be able to define channel gating
• Know the differences in transport characteristics
  of channels, primary and secondary active
  transporters
• Be able to define and describe a primary active
  transport system or pump.
• 2. Know the role of P-type ATPases (or ion pumps)
  including the Na, K-ATPase, Ca-ATPase,
  H,K-ATPase, and Cu-ATPases.
• 3. Know the energetic basis of secondary active
  transport and the role of secondary active
  transport systems in cellular systems.

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Channels
Essential for rapid propagation of various
signals and for muscle contraction Malfunction
of channels lead to myotonia, periodic paralysis,
cardiac arrhythmia, certain forms of epilepsy,
and others
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Gated channels
The conductance of the ion channel is typically
regulated by the conformational switching of the
protein structure between open and close
states
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- - - -

• Depending on the type of the channel, this gating
  process may be driven by
• ligand binding (ligand-gated channels)
• changes in electrical potential across cell
  membrane (voltage-gated channels)
• mechanical forces acting on cellular
  components (mechanosensitive channels)

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Voltage -gated channels

• Play a crucial role in neurotransmission and
  muscle contraction
• Highly selective for the transported ion, unlike
  ligand-gated channels ( for example,
  acetylcholine receptor is equally permeable for
  Na and K , while potassium channel is 100
  times more permeable for K than for Na )
• Many channels have distinct pharmacology and
  represent targets for various toxins
  (tetrodotoxin, saxitoxin, veratridin)
• Stay open for a very short time, than proceed to
  inactive form

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• In a cell unequal distribution of ion generates
  resting potential
• Ion mM Intracellular
  Extracellular
• Na 5-15
  145
• K 140
  5
• Mg 0.5
  1-2
• Ca 10-7
  1-2
• H 7 x 10-5 (pH 7.2) 4 x 10-5
  (pH 7.4)
• Cl 5-15
  110
• DY - RT ln(SPiMi out SPX-jin) -60
  mV
• ZF SPiMi in
  SPX-jout
• At this potential K is much closer to
  equilibrium than is Na (DYK -75 mV, while DYNa
  55 mV ). Therefore, if the membrane were to
  become fully permeable for ions, the major
  effects would be a massive influx of Na

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Action potential is generated when the membrane
is locally depolarized by 20 mV
Na goes in, K goes out
For this process to occur, the voltage-gated
channels should be (a) highly selective c)
voltage sensitive (b) very fast d) have a
mechanism for rapid
inactivation
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K channel is perfect for its job

• highly selective (permeability for K is at
  least 10,000 times higher than for Na )
• ion conductance is highly efficient (close
  to free diffusion limit, 108 ions/sec)
• has voltage-sensor
• inactivates rapidly

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Molecular Architecture of the Channel
Tetramer K-channel is a tetramer of 4 identical
subunits, Na-channel and Ca-channel have 4
repetitive subdomains in a single protein)
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Ion Conduction Pathway

• Both the intracellular and extracellular
  entryways are negatively charged
• The 45A pathway consists of the 18A internal
  pore and wide 10A cavity in the middle of the
  membrane, which are lined by hydrophobic
  aminoacid residues and through which ion moves in
  a hydrated form
• Selectivity filter separates the central cavity
  from the extracellular medium and is very polar

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A typical voltage-gated ion channel
S4 - voltage sensor P segment - selectivity
filter of the pore
- S4-segment, a voltage sensor, contains
motif X-A-A, where X is a charged residue, and A
is a hydrophobic residue
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Ligand-gated ion channels

• Mediate rapid action of neurotransmitters at
  synapse by changing the potential of the membrane
  in response to neurotransmitter (ligand) binding
• selectively activated by specific ligand
• discriminate between negatively and positively
  charged ions, but otherwise are not strongly
  selective

Cation-conducting channels - acetylcholine-,
serotonin- and glutamate receptors Anion-conductin
g channels - glycine and g-aminobutiric (GABA)
acid -gated receptors
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Structural Organization
Oligomers of five different subunits which are
30-50 homologous The pore is formed primarily by
M2 transmembrane segment of each monomer
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Acetylcholine Receptor
Five subunits, 4 homologous polypeptides ?
subunits contains AcCh binding sites AcCh
binding opens gate -activates channel
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Molecular Basis of the ATP-driven Transport
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P-type ATPases

• Large family of more than 150 different
  transporters
• Transport various cations against the
  electrochemical potential gradient
• Use energy of ATP hydrolysis and have similar
  catalytic cycle
• Form stable acylphosphate intermediate by
  transferring g-phosphate of ATP to invariant Asp
  residue in the ATP-binding domain
• Characterized by three conserved motifs DKTG,
  TGES/A and GDGxxG

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Na/ K ATPase
Greatest consumer cellular energy Sets up
concentration electrical gradients
Hydrolysis of 1 ATP moves 2K in and 3Na out
against their concentration gradients
Na,K-ATPase is a receptor of digitalis and
related cardiac glycosides used to strengthen
the heartbeat
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In a cell Na,K-ATPase generates unequal
distribution of Na and K across the membrane

• Composed of two subunits the catalytic
  a-subunit and b-subunit which is required for
  proper folding of the a-subunit and for
  modulation of K binding and occlusion
• a-subunit contains two major domains the
  ATP-binding domain and the membrane portion that
  forms the cation-translocation pathway
• the ATP-binding domain is directly connected to
  transmembrane segments providing direct
  structural link between two functional domains
  and coupling transport and ATP-hydrolysis

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Na,K -ATPase

• maintains uneven distribution of Na and K
  ions across cell membrane by transporting 3Na
  and 2K per each ATP hydrolyzed
• during the transport cycle the ions become
  occluded
• cycles between two major conformational states
  E1 which has high affinity for Na and ATP and
  E2, which has high affinity for K -ions
• can be specifically inactivated by ouabain

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Therapeutic action of cardiotonic steroids like
digitalis (ouabain derivatives)

• Inhibition of Na,K-ATPase by ouabain-like
  cardiotonic steroids leads to decrease in Na
  -gradient and decrease in the activity of
  Na/Ca2 exchanger
• This in turn leads to increases in intracellular
  Ca2 concentration and better cardiac muscle
  contraction

ouabain
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Ca2-ATPase of Sarcoplasmic Reticulum

• Plays a major role in muscle relaxation by
  transporting released Ca back into SR
• A single subunit protein with 10 transmembrane
  fragments
• Is highly homologous to Na,K-ATPase

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Four major domains M - Membrane-bound domain,
which is composed of 10 transmembrane segments N
- Nucleotide-binding domain, where adenine moiety
of ATP and ADP binds P Phosphatase domain,
which contains invariant Asp residue, which
became phosphorylated during the ATP
hydrolysis A domain essential for
conformational transitions between E1 and E2
states
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H,K-ATPase Mediates acid secretion in gastric
mucosa by exporting protons in exchange for
extra-cellular potassium ions Structurally is
very similar to Na,K-ATPase Gastric and duodenal
ulcer depend on acid secretion, therefore
H,K-ATPase is an important pharmacological
target
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Human Copper-transporting ATPases
Lumen
Cytosol
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Menkes Disease

• A progressive neurodegenerative X-linked disorder
  caused by mutations or deletions in the gene
  ATP7A
• Basis dietary copper is trapped in intestinal
  cells, leading to overall copper deficiency in
  tissues
• Symptoms developmental delays, mental
  retardation, connective tissue and vascular
  abnormalities, kinky hair

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Autosomal recessive disorder, lethal if
untreated. Caused by various mutations in the
ATP7B gene (chromosome 13)First described in
1860 as a neurological disease and thought to be
complication from syphilisCopper accumulates to
very high levels in the liver, brain, and kidneys
leading to liver malfunction, neurological and
psychiatric abnormalities
Wilsons Disease copper overload
normal
ATP7B-/-
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Secondary active transport

• DG RTln(C2/C1)
• If C2occurs spontaneously down the concentration
  gradient - facilitators, ionophores, pores, gated
  channels
• If C2C1 then DG is positive and then the
  energy source, such as ATP, is required to
  transport molecules against their concentration
  gradient - P-type ATPases, F1FO-ATPases,
  multidrug transporters, V-type ATPases

• If two molecules A and B are unequally
  distributed across cell membrane and DG for A is
  negative and gradient transport of A can be utilized to
  transport B against its concentration gradient -
  Na/Ca2-exchanger, Na,glucose transporter,
  lactose-permease

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General characteristics of secondary active
transport

• Widely used for uptake of glucose, amino
  acids, neurotransmitters, and other nutrients
• Rate of transport is fairly slow. Compare
• Ion channel - 107-108 ions/sec
• ATP-driven transport - 100-103 ions/sec
• Transporter - 102-104 ions/sec

• Cotransporters selectively bind two or more
  transported molecules simultaneous binding is
  essential to initiate transport
• These transporters do not have a pore or
  channel structure, and their transport mechanism
  is based on a series of conformational
  transitions - that is why the rate of transport
  is so slow

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Transport of intestinal glucose to blood
Basolateral Na/ K ATPase generates Na gradient
that drives the Symporter
Na-glucose symporter
couples transport of 2 Na -and 1 glucose
Energetics of transport Entry of 1 sodium
contributes about 2.2-3 kcal/mol For uncharged
glucose DGRTln(C2/C1) therefore co-transport
with 2 Na allows to generate about 1000 fold
higher concentration of glucose inside the cell