Transport Across Membranes

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Transport Across Membranes
Endocytosis and Exocytosis are bulk processes--
one vesicle at a time Most transport across
membranes is small molecules and ions, ie.
solutes Cells must be able to bring in solutes
against a concentration gradient 20 of the E
coli genome are involved in transport
processes some molecules pass freely through a
membrane-- usually small, nonpolar molecules
that are soluble in both water and lipids ie.
ethanol, oxygen, carbon dioxide, DMSO, some
hormones many of the molecules require specific
transport proteins
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Transport Across Membranes
transport proteins proteins which recognize a
substrate and catalyze its movement across a
membrane for facilitated diffusion, solutes move
down their concentration gradient active
transport energy requiring reactions moving them
against their free energy gradients ions are
frequent targets of transporters membrane
potential relative net charge on opposite sides
of a membrane typical cell membrane potential,
or resting potential, is -60 mV electrochemical
gradient combined electrical and chemical free
energies for a given ion ie. just because
potassium is positive doesn't mean it moves into
a cell
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Simple Diffusion Across Membranes
solutes move from a higher concentration to a
lower concentration ie. most disordered state
from the second law of thermodynamics ie. oxygen
diffuses across a red blood cell membrane and
binds hemoglobin in the lungs the blood
circulates, and oxygen is released in the
tissues where oxygen is at a lower
concentration carbon dioxide diffuses across the
membrane in the tissues and is released in the
lungs if water can cross a membrane and ions
can't, water will move to reduce solute
concentration osmosis movement of water across
a differentially permeable membrane in
response to a difference in solute concentration
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Simple Diffusion Across Membranes
liposomes small vesicles 100 nm in diameter
with aqueous centers made from cell membranes
that do not contain protiens (extracted) if
liposomes are made in a high concentration of a
solute, centrifuged, and resuspended in pure
water, it is possible to measure the diffusion
of the solute through the liposome
membrane potassium and sodium didn't change for
days, oxygen and carbon dioxide went to
equilibrium almost immediately-- definitely
semi-permeable membrane bilayers are 1) more
permeable to small molecules than large ones
membranes have about 0.01 of the diffusion
of water 2) more permeable to non-polar than
polar solutes 3) very impermeable to charged
ions, both positive and negative
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Simple Diffusion Across Membranes
simple diffusion is always energetically
favorable-- no cellular energy required
because it is always a decrease in DG diffusion
rate is directly proportional to the difference
in concentration for a cell, v pDS (NOT
entropy!) p permeability constant gives a
linear graph of reaction velocity vs. S (not
Michaelis-Menton)
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Facilitated Diffusion
Facilitated diffusion protein catalyzed
transport across a membrane in the direction
of the concentration gradient (ie. DGlt0 still!)
cell doesn't have to put in any metabolic
energy 2 major types of proteins facilitiate
diffusion across membranes 1) carrier proteins
(transporters or permeases) specifically bind
to individual molecules on one side of a
membrane and release them on the other
substrate binding induces a conformational change
in the protein to move the 'active site'
from one side of the membrane to the other
now that it is on the lower concentration side,
the substrate is released and the
transporter switches back to the binding site
being on the membrane side with a higher
solute concentration
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Facilitated Diffusion
Carrier proteins are highly specific their
'active site' recognizes only a small group of
chemically related molecules Like other enzymes,
follows Michaelis-Menton kinetics and shows
saturation in high S ie. a transporter can
only function so fast Also undergoes competitive
inhibition like other enzymes
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Facilitated Diffusion
Transporters can move either 1 or 2 types of
solutes at a time uniport transports 1
specific solute cotransport transports 2
different solutes at the same time (coupled)
functionally, it requires both solutes so if 1 is
absent, transport fails 2 types of
cotransporters symport two solutes moved in the
same direction antiport two solutes moved in the
opposite direction note cotransporters can be
either facilitated or active transport molecules
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Facilitated Diffusion
Glucose transporter uniport, speeds diffusion
through membrane by about 50,000x compared to
free diffusion through a lipid bilayer like all
carrier proteins, it functions in both
directions-- whichever way a concentration
gradient goes, so goes the transport ie. won't
change the point of the equilibrium, only when it
gets there several types of glucose
transporters, usually optimized by tissue
type ie. in liver, most transport is OUT of the
cell after glycogen breakdown also true for
cells in the digestive system that put glucose
into the blood most cells will transport
glucose from the blood into themselves inside
the cell, glucose is rapidly reduced by
phosphorylating it via energy provided by ATP
it now cannot bind to the normal transporter
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Facilitated Diffusion
Erythrocyte anion exchange protein antiport
protein facilitates the exchange of bicarbonate
ions HCO3- for chloride Cl- very selective and
specific 1 chloride, 1 bicarbonate, no other
ions, both must be present to
transport because it is an antiport, either
chloride goes in and bicarbonate goes out or
bicarbonate goes in and chloride goes out
antiport is required to overcome the electical
work of transporting a single ion across
the membrane erythrocytes have the enzyme
carbonic anhydrase to convert CO2 into
bicarbonate HCO3- goes from a membrane permeable
molecule to an impermeable form required to
get CO2 from tissues to lungs in lungs, the
process is reversed
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Channel Proteins
unlike transporters, channels form a hydrophilic
corridor through a membrane to allow ions to
move across a membrane directly ie. no
individual ion binding is necessary in a
channel channels are usually ion selective
allow movement of 1 or few ions ie. sodium
channels allow little potassium flow and vice
versa mechanism involves several steps of
anion/cation selectivity, followed by size
selection based on the sphere of hydration ion
channels are usually gated closed until
specifically opened usually only opened for a
period of time before closing again 3 broad
types of channels 1) ligand gated channels
open in response to a chemical signal 2) voltage
gated channels open after changes in membrane
potential 3) mechanosensitive mechanical forces
ie. touch and hearing (sound)
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Channel Proteins
porins large, poorly selective channels with a
water filled pore in center formed by b sheets
with hydrophobic residues facing the membrane
and hydrophilic residues lining the channel
the width of the b barrel determines the maximum
size of solute which can pass through
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Channel Proteins
aquaporins channels that facilitate the rapid
movement of water across a membrane in specific
tissues ie. kidney tubules, red blood cells,
vacuoles NOT part of the porin family! 6
transmembrane regions with b sheets lining the
pore, functions as a tetramer
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Active Transport
active transport energy requiring process to
move a solute up a concentration gradient
must not only move the solute but couple it to
an energy yielding reaction 3 primary fuctions
of active transport 1) concentrate essential
nutrients inside a cell 2) remove secretory or
waste products from a cell 3) maintains a fairly
constant concentration of intracellular ions
temporary changes (ie. action potentials in
nerve cells) later must be reversed so the
neuron can continue functioning unlike diffusion
mediated events, active transport is in one
direction ie. the cell does not need to use
energy for diffusion with a gradient!
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Active Transport
direct active transport accumulation of solute
is coupled directly to an exergonic reaction,
usually hydrolysis of ATP direct active
transporters are often referred to as pumps 4
distinct types of ATPase pumps P-type ATPases
pumps themselves become transiently
phosphorylated on a specific aspartic acid--
hydrolysis of the phosphate provides -DG 8-10
transmembrane a helices always cation
transporters (), all blocked by vanadate ions
VO43- best known example Na/K pump moving
Na out and K in common in eukaryotes, less
common (still present) in prokaryotes
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Active Transport
V-type ATPases 'vesicle' pumps force protons
into organelles such as vacuoles, endosomes,
and golgi complex transport subunit is a
transmembrane protein peripheral protein
component is the ATPase allosteric changes in
the peripheral protein are coupled to changes in
the transport subunit that causes the actual
movement of protons F-type ATPases 'factor'
multicomponent pumps superficially like V-type
moves protons across a membrane, and composed of
a transmembrane component and a peripheral
ATPase component found in mitochondria,
chloroplasts, and bacteria is reversible--
proton gradients can force the synthesis of ATP
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Active Transport
ABC-type ATPases 'ATP binding cassette' large
family of pumps from mostly bacteria, but
eukaryotes as well contains 4 domains, 2
integral membrane domains, 2 peripheral
generally different polypeptides assoicated in a
complex, some 1 protein ATP is hydrolyzed in
the peripheral subunits, coupled to transport in
the integral membrane subunits each
integral membrane subunit has 6 transmembrane
domains ABC type transporters can transport
ions, sugars, amino acids, peptides also can
pump some drugs out of a cell multi-drug
resistance protein (MDR) ABC-type pump
hydrophobic drugs out of the cell,
particularly hydrophobic chemotherapy
drugs cystic fibrosis is a disease of a chloride
specific ABC-type pump (CFTR)
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Active Transport
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Active Transport
Indirect active transport transport driven by
ion gradients often associated with the
simultaneous movement of other ions,
usually Na or H down their concentration
gradient ie. often symport mechanisms animal
cells use sodium ion gradients to power uptake of
many sugars bacterial cells typically use proton
(H) gradients cells also use indirect active
transport to remove Ca2 as an antiporter ie.
Na ions come in while Ca2 leave
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Active Transport
Na/K pump is a direct active transporter
cells keep sodium ions out and potassium ions in
Naout/Nain 221, while Kout/Kin
0.03 P-type ATPase that is inherently
directional ie. Na ions on the inside along
with ATP binding, with K ions on the
outside usually 3 Na ions are moved out as 2 K
ions are pumped in per ATP composed of a
(affinity subunit) and b (???) subunit (external
side) b subunit is glycosylated and important
but with an unknown function
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Active Transport
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Active Transport
Sodium/ glucose symporter is an indirect active
transporter found in epithelial cells lining the
intestine-- need to absorb glucose against a
concentration gradient Na ions provide the
driving force (-DG) to make the symporter
work similar transporters absorb amino acids in
intestinal epithelial cells related proton
symporters drive absorption in bacteria, plant,
fungi
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Active Transport
bacteriorhodopsin is a proton pump capturing
light energy to move H proton pump can be used
to synthesize ATP via F-type ATPases retinal
light absorbing pigment in bacteriorhodopsin (and
your eyes) and is a derivative of Vitamin A (b
carotene), 7 transmembrane a helices and was
the first membrane protein structure protein is
covalently bound to the retinal, a prosthetic
group exact mechanism of proton transport is
still unknown ie. knowing the protein structure
isn't always enough to solve mechanism believed
to involve the photoconversion of all-trans
retinal to 11-cis H pump by light is an
absolute requirement for efficient ATP synthesis
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Transport Energetics
just like every other chemical reaction, there is
an overall -DG in every transport reaction
(even in light driven ones, some energy is
wasted) 2 different factors play a role in the
energetics concentration and charge for
uncharged molecules, DG is determined only by
concentration for the reaction Sout
Sin DG RTlnSin/Sout ie. if Sin
ltSout, then -DG and spontaneous if Soutlt
Sin, energy is required to drive the solute
into the cell
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Transport Energetics
for charged solutes, DG depends upon the
electrochemcial gradient membrane potential of
most animal cells is usually around -60mV ie.
electrically, positive ions want inside and
negative ions outside for the reaction
Scout Scin DG RTlnScin/Scout
zFVp F Faraday constant (23062 cal/mol) z
charge on the ion Vp membrane potential in
volts ie. negative charge with a negative
membrane potential, DG goes up positive charge
with a negative membrane potential, DG goes down
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Transport Energetics
What is the DG of Na ions moving into a cell at
25 oC if the resting Vp is -60 mV, the
internal Na 12mM and the external Na150
mM? the chemical 'reaction' for this transport
is Naout Nain DG
RTlnNain/Naout zFVp substitute into
the equation... DG 1.987298ln(0.012/0.150)
(1)23062(-.060) DG 592ln(0.08)
(-1383.72) DG -1495 - 1384 DG -2879 cal/mol