Download 03_Membrane rest potential. Generation and radiation action

Membrane rest potential.
Generation and radiation action
potential.
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).
Valinomycin
O
O
+
O
K
O
Hydrophobic
O
O
Valinomycin
O
O
+
O
K
O
O
O
Hydrophobic
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).
Val-K+
Val-K+
K+
K+
Val
Val
membrane
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.
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.
conformation
change
conformation
change
Carrier-mediated solute transport
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.
conformation
change
conformation
change
Carrier-mediated solute transport
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.
Classes of
carrier
proteins
Uniport
A
Symport
A
Antiport
B
A
B
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.
Symport (cotransport) carriers bind two dissimilar
solutes (substrates) & transport them together across a
membrane.
Transport of the two solutes is obligatorily coupled.
Uniport
A
Symport
A
B
Antiport
A
Lactose permease catalyzes uptake of the disaccharide
lactose into
E. coli bacteria, along with H+, driven by
a proton electrochemical gradient.
Uniport
A
Symport
A
B
Antipor
A
The substrate binding site is at the apex of an aqueous
cavity between two domains, each consisting of six
trans-membrane a-helices.
TDG
substrate
analog
Lactose Permease
PDB 1PV7
conformation
change
conformation
change
Carrier-mediated solute transport
As in simple models of
carrier transport based on
functional assays, the tilt
of transmembrane ahelices is assumed to
change, shifting access of
lactose & H+ binding sites
to the other side of the
membrane during the
transport cycle.
TDG
substrate
analog
Lactose Permease
PDB 1PV7
Uniport
Symport
Antiport (exchange diffusion)
carriers
A
A
B
exchange one solute for another across a
membrane.
 Usually antiporters exhibit "ping pong"
kinetics.
Antiport
A
B
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.
mitochondrial
matrix
ATP 4
ADP
3
adenine nucleotide translocase
Example of an antiport carrier:
Adenine nucleotide translocase (ADP/ATP
exchanger) catalyzes 1:1 exchange of ADP for ATP
across the inner mitochondrial membrane.
Active
Transport
ADP + Pi
S2
S1
ATP
Side 1
Side 2
Active transport enzymes couple net solute
movement across a membrane to ATP hydrolysis.
An active transport pump may be a uniporter or
antiporter.
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.
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.
The reaction mechanism for a P-class ion pump
involves transient covalent modification of the
enzyme.
O
Enzyme-C
OH
ATP
Pi
ADP
H2O
O
Enzyme- C
O
O
P
O-
P-Class Pumps
O-
The ER Ca++ pump
is called SERCA:
Sarco(Endo)plasmic ADP
Reticulum
ATP
++
Ca -ATPase.
E~P-Ca++2
E~P-Ca++2
2Ca++
E-Ca++2
2Ca++
E
Pi
cytosol
membrane
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.
ER
lumen
Reaction cycle:
E~P-Ca++2
ADP
E~P-Ca++2
1. 2 Ca++ bind tightly
ATP E-Ca++
from the cytosolic
2
side, stabilizing the
++
2Ca
conformation that
E
allows ATP to react
Pi
with an active site
cytosol
membrane
aspartate residue.
2Ca++
ER
lumen
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++.
E~P-Ca++2
E~P-Ca++2
ADP
ATP
2Ca++
E-Ca++2
2Ca++
E
Pi
cytosol
membrane
ER
lumen
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.
This X-ray structure
of muscle SERCA (Ca++ATPase) shows
2 Ca++
ions (colored magenta)
bound between
transmembrane a-helices in
the membrane domain.
Asp351
cytosolic
domain
2 Ca++
PDB 1EUL
membrane
domain
Muscle SERCA
Active site Asp351, which is transiently phosphorylated
during catalysis, is located in a cytosolic domain, far
from the Ca++ binding sites.
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.
++
Ca
SERCA Conformational Cycle
enzyme
phosphorylation
phosphate
hydrolysis
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.
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).
Ion Channels
conformation
change
closed
open
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 unusual
peptide, with alternating D
& L amino acids.
In lipid bilayer membranes,
gramicidin dimerizes &
folds as a right-handed bhelix.
The dimer just spans the
bilayer.
Gramicidin dimer
(PDB file 1MAG)
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.
Gramicidin dimer
(PDB file 1MAG)
current
ion flow
through one
channel
time
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).
Gating (opening & closing) of a gramicidin channel
is thought to involve reversible dimerization.
Proposed mechanism of
gramicidin gating
open
closed
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)
Patch Clamping
A
electrode
glass pipet
B
electrode
electrode
The technique of patch clamping is used to
study ion channel activity.
amplifier with
voltage control
Patch
Clamping
reference
electrode
electrode in
patch pipet
salt solution
membrane
patch
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.
current
open
closed
time
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.
20
pA
102 msec per trace
Patch clamp recording at 60 mV. Consecutive
traces are shown. Note that at a negative voltage,
increased current is a downward deflection.
Current Amplitude
Histogram
Occupancy of current levels
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.
-60
-55
-50
Amplitude in pAmp
-45
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