Membrane Transport: To Accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham

Biochemistry 2/e - Garrett & Grisham
Chapter 10
Membrane Transport
to accompany
Biochemistry, 2/e
by
Reginald Garrett and Charles Grisham
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Outline
• 10.1 Passive Diffusion
• 10.2 Facilitated Diffusion
• 10.3 Active Transport
• 10.4 - 10.6 Transport Driven by ATP, light, etc.
• 10.7 Group Translocation
• 10.8 Specialized Membrane Pores
• 10.9 Ionophore Antibiotics

Passive Diffusion
No special proteins needed
• Transported species simply moves
down its concentration gradient - from
high [c] to low [c]
• Be able to use Eq. 10.1 and 10.2
• High permeability coefficients usually
mean that passive diffusion is not the
whole story



Facilitated Diffusion
G negative, but proteins assist
• Solutes only move in the
thermodynamically favored direction
• But proteins may "facilitate" transport,
increasing the rates of transport
• Understand plots in Figure 10.3
• Two important distinguising features:
– solute flows only in the favored direction
– transport displays saturation kinetics





Active Transport Systems

Energy input drives transport
• Some transport must occur such that
solutes flow against thermodynamic
potential
• Energy input drives transport
• Energy source and transport machinery
are "coupled"
• Energy source may be ATP, light or a
concentration gradient

The Sodium Pump

aka Na,K-ATPase
• Large protein - 120 kD and 35 kD subunits
• Maintains intracellular Na low and K high
• Crucial for all organs, but especially for
neural tissue and the brain
• ATP hydrolysis drives Na out and K in
• Alpha subunit has ten transmembrane
helices with large cytoplasmic domain



Na,K Transport
• ATP hydrolysis occurs via an E-P
intermediate
• Mechanism involves two enzyme
conformations known as E1 and E2
• Cardiac glycosides inhibit by binding to
outside




Na,K Transport
• Hypertension involves apparent
inhibition of sodium pump. Inhibition in
cells lining blood
• Vessel walls results in Na,Ca
accumulation
• Studies show this inhibitor to be
ouabain!

Calcium Transport in Muscle

A process akin to Na,K transport
• Calcium levels in resting muscle cytoplasm are
maintained low by Ca-ATPase - a Ca pump
• Calcium is pumped into the sarcoplasmic
reticulum (SR) by a 110 kD protein that is very
similar to the alpha subunit of Na,K-ATPase
• Aspartyl phosphate E-P intermediate is at Asp-
351 and Ca-pump also fits the E1-E2 model




The Gastric H,K-ATPase

The enzyme that keeps the stomach at pH 0.8
• The parietal cells of the gastric mucosa
(lining of the stomach) have an internal
pH of 7.4
• H,K-ATPase pumps protons from these
cells into the stomach to maintain a pH
difference across a single plasma
membrane of 6.6!


The Gastric H,K-ATPase

• This is the largest concentration
gradient across a membrane in
eukaryotic organisms!
• H,K-ATPase is similar in many respects
to Na,K-ATPase and Ca-ATPase

Osteoclast Proton Pumps

How your body takes your bones apart!
• Bone material undergoes ongoing remodeling
•
– osteoclasts tear down bone tissue
– osteoblasts build it back up
• Osteoclasts function by secreting acid into the
space between the osteoclast membrane and
the bone surface - acid dissolves the Ca-
phosphate matrix of the bone
• An ATP-driven proton pump in the membrane
does this!


The MDR ATPase

aka the P-glycoprotein
• Animal cells have a transport system
that is designed to recognize foreign
organic molecules
• This organic molecule pump recognizes
a broad variety of molecules and
transports them out of the cell using the
hydrolytic energy of ATP


The MDR ATPase

• MDR ATPase is a member of a
"superfamily" of genes/proteins that
appear to have arisen as a "tandem
repeat"
• MDR ATPase defeats efforts of
chemotherapy


Light-Driven H + Transport
The Bacteriorhodopsin story
• Halobacterium halobium, the salt-loving
bacterium, carries out normal respiration if
O2 and substrates are plentiful
• But when substrates are lacking, it can
survive by using bacteriorhodopsin and
halorhodopsin to capture light energy
• Purple patches of H. halobium are 75% bR
and 25% lipid - a "2D crystal" of bR - ideal
for structural studies




Bacteriorhodopsin
Protein opsin and retinal chromophore
• Retinal is bound to opsin via a Schiff
base link
• The Schiff base (at Lys-216) can be
protonated, and this site is one of the
sites that participate in H+ transport


Bacteriorhodopsin
• Lys-216 is buried in the middle of the 7-
TMS structure of bR, and retinal lies
mostly parallel to the membrane and
between the helices
• Light absorption converts all-trans
retinal to 13-cis configuration - see
Figure 10.22

Bacteriorhodopsin
The protons visit the aspartates....
• Asp-85 and Asp-96 lie on opposite
sides of a membrane-spanning helix
• These remarkable aspartates have pKa
values around 11! (Why?)
• Protons are driven from Asp-96 to the
Schiff base at Lys-216 to Asp-85 and
out of the cell

Halorhodopsin
• Halorhodopsin transports Cl - instead of H +
• Halorhodopsin has Lys-242 Schiff base but
no aspartates and no deprotonation of
Schiff base during the transport cycle



Secondary Active Transport

Transport processes driven by ion
gradients
• Many amino acids and sugars are
accumulated by cells in transport
processes driven by ion gradients

Secondary Active Transport

• Symport - ion and the amino acid or
sugar are transported in the same
direction across the membrane
• Antiport - ion and transported species
move in opposite directions
• Several examples are described in
Table 10.2

Group Translocation
The phosphotransferase system (PTS)
• Discovered by Saul Roseman in 1964
• Sugars are phosphorylated from PEP
during transport into E. coli cells
• Four proteins required: EI, HPr, EII, and
EIII


Group Translocation
• EI and HPr are universal and work for
all sugars
• EII and EIII are specific for each sugar
• Mechanism involves transfer of P from
PEP to EI and then to HPr and then to 2
sites on EIII and then finally
phosphorylation of sugar


Porins
Found both in Gram-negative bacteria and in
mitochondrial outer membrane
• Porins are pore-forming proteins - 30-50 kD
• General or specific - exclusion limits 600-6000
• Most arrange in membrane as trimers
• High homology between various porins
• Porin from Rhodobacter capsulatus has 16-
stranded beta barrel that traverses the
membrane to form the pore (with eyelet!)



Why Beta Sheets?

for membrane proteins??
• Genetic economy
• Alpha helix requires 21-25 residues per
transmembrane strand
• Beta-strand requires only 9-11 residues
per transmembrane strand
• Thus, with beta strands , a given amount
of genetic material can make a larger
number of trans-membrane segments

The Pore-Forming Toxins

• Lethal molecules produced by many
organisms
• They insert themselves into the host cell
plasma membrane
• They kill by collapsing ion gradients,
facilitating entry by toxic agents, or
introducing a harmful catalytic activity

Colicins
• Produced by E. coli
• Inhibit growth of other bacteria (even
other strains of E. coli)
• Single colicin molecule can kill a host!
• Three domains: translocation (T),
receptor-binding (R), and channel-
forming (C)


Clues to Channel Formation

• C-domain: 10-helix bundle, with H8 and
H9 forming a hydrophobic hairpin
• Other helices amphipathic (Fig. 10.30)
• H8 and H9 insert, with others splayed
on the membrane surface
• A transmembrane potential causes the
amphipathic helices to insert!


Other Pore-Forming Toxins

• Delta endotoxin also possesses a helix-bundle
and may work the same way
• There are other mechanisms at work in other
toxins
• Hemolysin from Staphylococcus aureus forms
a symmetrical pore
• Aerolysin may form a heptameric pore - with
each monomer providing 3 beta strands to a
membrane-spanning barrel



Amphiphilic Helices
form Transmembrane Ion Channels
• Many natural peptides form oligomeric
transmembrane channels
• The peptides form amphiphilic -helices
• Aggregates of these helices form channels
that have a hydrophobic surface and a polar
center
• Melittin (bee venom), magainins (frogs) and
cecropin (from cecropia moths) are examples

Amphipathic Helices
• Melittin - bee venom toxin - 26 residues
• Cecropin A - cecropia moths - 37
residues
• Magainin 2 amide - frogs - 23 residues
• See Figure 10.35 to appreciate helical
wheel presentation of the amphipathic
helix

The Magainin Peptides

• Discovered by Michael Zasloff
• He noticed that incisions on Xenopus
laevis (African clawed frog) healed
without infection, even in bacteria-filled
aquarium water
• He deduced that the frogs produced a
substance that protected them from
infection!


The Cecropins
• Produced by Hyalophora cecropia (the
cecropia moth - see Figure 10.36)
• Induced when the moth is challenged by
bacterial infections
• These peptides are thought to form -
helical aggregates in membranes,
creating an ion channel in the center of
the aggregate


Gap Junctions
Vital connections for animal cells
• Provide metabolic connections
• Provide a means of chemical transfer
• Provide a means of communication
• Permit large number of cells to act in
synchrony

Gap Junctions
• Hexameric arrays of a single 32 kD
protein
• Subunits are tilted with respect to
central axis
• Pore in center can be opened or closed
by the tilting of the subunits, e.g. as
response to stress



Ionophore Antibiotics
Mobile carrier or pore (channel)
• How to distinguish? Temperature!
• Pores will not be greatly affected by
temperature, so transport rates are
approximately constant over large
temperature ranges
• Carriers depend on the fluidity of the
membrane, so transport rates are highly
sensitive to temperature, especially near the
phase transition of the membrane lipids


Valinomycin
A classic mobile carrier
• A depsipeptide - a molecule with both
peptide and ester bonds
• Valinomycin is a dodecadepsipeptide
• The structure places several carbonyl
oxygens in the center of the ring structure
• Potassium and other ions coordinate the
oxygens
• Valinomycin-potassium complex diffuses
freely and rapid across membranes

Selectivity of Valinomycin
Why?
• K + and Rb + bind tightly, but affinities for
Na + and Li + are about a thousand-fold lower
• Radius of the ions is one consideration
• Hydration is another - see page 324 for
solvation energies
• It "costs more" energetically to desolvate
Na+ and Li+ than K+

Gramicidin
A classic channel ionophore
• Linear 15-residue peptide - alternating D & L
• Structure in organic solvents is double helical
• Structure in water is end-to-end helical dimer
• Unusual helix - 6.3 residues per turn with a
central hole - 0.4 nm or 4 A diameter
• Ions migrate through the central pore


Membrane Transport: To Accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham

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Membrane Transport: To Accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham

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Biochemistry 2/e - Garrett & Grisham

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Active Transport Systems

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The Sodium Pump

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Calcium Transport in Muscle

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The Gastric H,K-ATPase

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The Gastric H,K-ATPase

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Osteoclast Proton Pumps

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The MDR ATPase

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The MDR ATPase

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Secondary Active Transport

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Why Beta Sheets?