Membrane rest potential. Generation and radiation action potential.

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• Membrane rest potential. Generation and radiation
  action potential.

2

• This discussion will focus on selected examples
  of transport catalysts for which
  structure/function relationships are relatively
  well understood.
• Transporters are of two general classes
• carriers and channels.
• These are exemplified by two ionophores (ion
  carriers produced by microorganisms)
• valinomycin (a carrier)
• gramicidin (a channel).

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Puckering of the ring, stabilized by H-bonds,
allows valinomycin to closely surround a single
unhydrated K ion. Six oxygen atoms of the
ionophore interact with the bound K, replacing O
atoms of waters of hydration.

• Valinomycin is highly selective for K relative
  to Na.
• The smaller Na ion cannot simultaneously
  interact with all 6 oxygen atoms within
  valinomycin.
• Thus it is energetically less favorable for Na
  to shed its waters of hydration to form a complex
  with valinomycin.

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• Whereas the interior of the valinomycin-K
  complex is polar, the surface of the complex is
  hydrophobic.
• This allows valinomycin to enter the lipid core
  of the bilayer, to solubilize K within this
  hydrophobic milieu.
• Crystal structure (at Virtual Museum of Minerals
  Molecules).

5

• Valinomycin is a passive carrier for K. It can
  bind or release K when it encounters the
  membrane surface.
• Valinomycin can catalyze net K transport because
  it can translocate either in the complexed or
  uncomplexed state.
• The direction of net flux depends on the
  electrochemical K gradient.

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• Proteins that act as carriers are too large to
  move across the membrane.
• They are transmembrane proteins, with fixed
  topology.
• An example is the GLUT1 glucose carrier, in
  plasma membranes of various cells, including
  erythrocytes.
• GLUT1 is a large integral protein, predicted via
  hydropathy plots to include 12 transmembrane
  a-helices.

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• Carrier proteins cycle between conformations in
  which a solute binding site is accessible on one
  side of the membrane or the other.
• There may be an intermediate conformation in
  which a bound substrate is inaccessible to either
  aqueous phase.
• With carrier proteins, there is never an open
  channel all the way through the membrane.

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• The transport rate mediated by carriers is faster
  than in the absence of a catalyst, but slower
  than with channels.
• A carrier transports one or few solute molecules
  per conformational cycle, whereas a single
  channel opening event may allow flux of many
  thousands of ions.
• Carriers exhibit Michaelis-Menten kinetics.

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Classes of carrier proteins

• Uniport (facilitated diffusion) carriers mediate
  transport of a single solute.
• An example is the GLUT1 glucose carrier.
• The ionophore valinomycin is also a uniport
  carrier.

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Symport (cotransport) carriers bind two
dissimilar solutes (substrates) transport them
together across a membrane. Transport of the two
solutes is obligatorily coupled.

• A gradient of one substrate, usually an ion, may
  drive uphill (against the gradient) transport of
  a co-substrate.
• It is sometimes referred to as secondary active
  transport.
• E.g ? glucose-Na symport, in plasma membranes
• of some epithelial cells
• ? bacterial lactose permease, a H
  symport carrier.

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Lactose permease catalyzes uptake of the
disaccharide lactose into E. coli
bacteria, along with H, driven by a proton
electrochemical gradient.
It is the first carrier protein for which an
atomic resolution structure has been determined.
Lactose permease has been crystallized with
thiodigalactoside (TDG), an analog of lactose.
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The substrate binding site is at the apex of an
aqueous cavity between two domains, each
consisting of six trans-membrane a-helices.

• In the conformation observed in this crystal
  structure, the substrate analog is accessible
  only to what would be the cytosolic side of the
  intact membrane.
• Residues essential for H binding are are also
  near the middle of the membrane.

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• As in simple models of carrier transport based
  on functional assays, the tilt of transmembrane
  a-helices is assumed to change, shifting access
  of lactose H binding sites to the other side
  of the membrane during the transport cycle.

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• Antiport (exchange diffusion) carriers exchange
  one solute for another across a membrane.
• Usually antiporters exhibit "ping pong" kinetics.

• A substrate binds is transported.
• Then another substrate binds is transported in
  the other direction.
• Only exchange is catalyzed, not net transport.
• The carrier protein cannot undergo the
  conformational transition in the absence of bound
  substrate.

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• Example of an antiport carrier
• Adenine nucleotide translocase (ADP/ATP
  exchanger) catalyzes 11 exchange of ADP for ATP
  across the inner mitochondrial membrane.

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• Active transport enzymes couple net solute
  movement across a membrane to ATP hydrolysis.
• An active transport pump may be a uniporter or
  antiporter.
• ATP-dependent ion pumps are grouped into classes,
  based on transport mechanism, genetic
  structural homology.

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• P-class ion pumps are a gene family exhibiting
  sequence homology. They include
• Na,K-ATPase, in plasma membranes of most animal
  cells is an antiport pump.
• It catalyzes ATP-dependent transport of Na
  out of a cell in exchange for K entering.
• (H, K)-ATPase, involved in acid secretion in
  the stomach is an antiport pump.
• It catalyzes transport of H out of the
  gastric parietal cell (toward the stomach lumen)
  in exchange for K entering the cell.

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• P-class pumps (cont)
• Ca-ATPases, in endoplasmic reticulum (ER) and
  plasma membranes, catalyze ATP-dependent
  transport of Ca away from the cytosol, into the
  ER lumen or out of the cell.
• Some evidence indicates that these pumps may
  be antiporters, transporting protons in the
  opposite direction.
• Ca-ATPase pumps function to keep cytosolic
  Ca low, allowing Ca to serve as a signal.

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• The reaction mechanism for a P-class ion pump
  involves transient covalent modification of the
  enzyme.

At one stage of the reaction cycle, phosphate is
transferred from ATP to the carboxyl of a Glu or
Asp side-chain, forming a high energy anhydride
linkage (P). At a later stage in the reaction
cycle, the Pi is released by hydrolysis.
20
The ER Ca pump is called
SERCA Sarco(Endo)plasmic Reticulum
Ca-ATPase.
In this diagram of SERCA reaction cycle,
conformational changes altering accessibility of
Ca-binding sites to the cytosol or ER
lumen are depicted as positional changes. Keep
in mind that SERCA is a large protein that
maintains its transmembrane orientation.
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Reaction cycle 1. 2 Ca bind tightly from the
cytosolic side, stabilizing the conformation that
allows ATP to react with an active site aspartate
residue.

• 2. Phosphorylation of the active site aspartate
  induces a conformational change that
• shifts accessibility of the 2 Ca binding sites
  from one side of the membrane to the other,
• lowers the affinity of the binding sites for
  Ca.

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• 3. Ca dissociates into the ER lumen.
• 4. Ca dissociation promotes
• hydrolysis of Pi from the enzyme Asp
• conformational change (recovery) that causes Ca
  binding sites to be accessible again from the
  cytosol.

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• This X-ray structure of muscle SERCA
  (Ca-ATPase) shows 2 Ca ions (colored
  magenta) bound between transmembrane a-helices in
  the membrane domain.

Active site Asp351, which is transiently
phosphorylated during catalysis, is located in a
cytosolic domain, far from the Ca binding sites.
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• SERCA structure has been determined in the
  presence absence of Ca, with without
  inhibitors.
• Substantial differences in conformation have been
  interpreted as corresponding to different stages
  of the reaction cycle.
• Large conformational changes in the cytosolic
  domain of SERCA are accompanied by deformation
  changes in position tilt of transmembrane
  a-helices.
• The data indicate that when Ca dissociates
• water molecules enter Ca binding sites
• charge compensation is provided by protonation
  of Ca-binding residues.

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• This simplified cartoon represents the proposed
  variation in accessibility affinity of
  Ca-binding sites during the reaction cycle.
• Only 2 transmembrane a-helices are represented,
  and the cytosolic domain of SERCA is omitted.

26
More complex diagrams animations have been
created by several laboratories, based on
available structural evidence. E.g. animation
(lab of D. H. MacLennan) diagram (by C.
Toyoshima, in a website of the Society of General
Physiologists - select Poster). website of the
Toyoshima Lab (select Resources for movies).
website of the Stokes Lab (select Download
movie).
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Ion Channels

• Channels cycle between open closed
  conformations.
• When open, a channel provides a continuous
  pathway through the bilayer, allowing flux of
  many ions.
• Gramicidin is an example of a channel.

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• Gramicidin is an unusual peptide, with
  alternating D L amino acids.
• In lipid bilayer membranes, gramicidin dimerizes
  folds as a right-handed b-helix.
• The dimer just spans the bilayer.
• Primary structure of gramicidin (A)

HCO-L-Val-Gly-L-Ala-D-Leu-L-Ala-D-Val-L-Val-D-Val-
L-Trp-D-Leu-L-Trp-D-Leu-L-Trp-D-Leu-L-Trp- NHCH2CH
2OH Note The amino acids are all
hydrophobic both peptide ends are modified
(blocked).
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• The outer surface of the gramicidin dimer, which
  interacts with the core of the lipid bilayer, is
  hydrophobic.
• Ions pass through the more polar lumen of the
  helix.
• Ion flow through individual gramicidin channels
  can be observed if a small number of gramicidin
  molecules is present in a lipid bilayer
  separating 2 compartments containing salt
  solutions.

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• With voltage clamped at some value, current (ion
  flow through the membrane) fluctuates.
• Each fluctuation, attributed to opening or
  closing of one channel, is the same magnitude.
• The current increment corresponds to current flow
  through a single channel (drawing - not actual
  data).

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Gating (opening closing) of a gramicidin
channel is thought to involve reversible
dimerization.

• An open channel forms when two gramicidin
  molecules join end to end to span the membrane.
• This model is consistent with the finding that at
  high gramicidin overall transport rate depends
  on gramicidin2.

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Channels that are proteins

• Cellular channels usually consist of large
  protein complexes with multiple transmembrane
  a-helices.
• Their gating mechanisms must differ from that of
  gramicidin.
• Control of channel gating is a form of allosteric
  regulation. Conformational changes associated
  with channel opening may be regulated by
• Voltage
• Binding of a ligand (a regulatory molecule)
• Membrane stretch (e.g., via link to cytoskeleton)

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Patch Clamping

• The technique of patch clamping is used to study
  ion channel activity.
• A narrow bore micropipet may be pushed up against
  a cell or vesicle, and then pulled back,
  capturing a fragment of membrane across the pipet
  tip.

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Patch Clamping

• A voltage is imposed between an electrode inside
  the patch pipet and a reference electrode in
  contact with surrounding solution. Current is
  carried by ions flowing through the membrane.

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• If a membrane patch contains a single channel
  with 2 conformational states, the current will
  fluctuate between 2 levels as the channel opens
  and closes.
• The increment in current between open closed
  states reflects the rate of ion flux through one
  channel.
• View a video of an oscilloscope image during a
  patch clamp recording.

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• Patch clamp recording at -60 mV. Consecutive
  traces are shown. Note that at a negative
  voltage, increased current is a downward
  deflection.

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• Current Amplitude Histogram
• Occupancy of different current levels during the
  time period of a recording is plotted against
  current in picoAmperes (10-12 Amp).
• Peaks represent open closed states (note
  scale).
• Baseline current, when the channel is closed, is
  due to leakage of the patch seal and membrane
  permeability.