Membrane Transport

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Membrane Transport

• The question
• How does a cell Membrane serves as both
  barrier and gate for communication between
  the outside and inside of the cell (or among
  organelles.
• Lipid bilayerbarrier, transport proteinsgates.

2. Permeability of lipid bilayer and a cell
membrane Biologically important molecules
bilayer membrane Polar small
water, glycerol, oxygen
Polar large amino acids, sugars,
nucleotides -- Ions K,
Na, Ca2, Cl-, NO3- etc
-- Non-polar small/large
phenolics, lipids, steroids
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• 3. Membrane Transport
• Carriers and channels carriers function like
  enzymes that
• bind small molecules and release to the other
  side of the membrane (mechanical hands)
  channel is a aqueous pore formed by membrane
  proteins (tunnel).
• 2) Passive and active transport transport down
  the electrochemical gradient across the membrane
  without the need of immediate energy requirement
  (ATP) (passive) or requires ATP and transport
  against he electrochemical gradient (active). For
  uncharged molecules just mean concentration
  gradient.

Out in
Out in
Out in
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4. ATP-driven carriers or pumps The carrier
protein is an enzyme called ATPase that
hydrolyzes ATP to get needed energy for
transport. 1) Example/model
2 K
P
P
3 Na
ATP
ADP
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• Summary ATP-dependent conformational change
  powered by reversible phosphorylation (at
  aspartate residue forming a high-energy
  intermediate) conformational changes generate
  binding sites for Na/K and movement associated
  with the translocation of the ions.
• This example Na/K pump is only found in animals
  but not in fungi and plants!
• 2) Plant pumps
• H-ATPase the most important active transporter
  that produce a proton gradient and maintain the
  membrane potential for other secondary transport
  (across PM).

Amino acids Other organic acids
pH gradient between inside and outside of the
cell. Membrane potential charge Difference
across the membrane For plant cells typically
around 120 mV Meaning more negative inside the
cell
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A lot of secondary transport is dependent on
H-gradient
Amino acids(symport) And Anions Cations (channels)
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b. Vacuolar H-ATPase A complex
molecular Machine that consists of 13 distinct
subunits. It pumps H into the Vacuole from
cytoplasm. The two H-ATPases in PM or
tonoplast take Care of the H in the Cytoplasm
and pump them into the inactive Space and keep
the pH Neutral in the cytosol.
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• c. Other pumps Ca2-ATPases in the PM, ER,
  vacuole
• These are the pumps that like the H-ATPases keep
  the Ca2 concentration in the cytoplasm low by
  pumping Ca2 into the cell wall, ER, or vacuole.
  Very important because Ca2 serves as a signal
  for many processesdiscuss in later sessions.
• 5. Ion channels structure and function
• Ion channels conduct ion flows down the
  electrochemical gradient and is considered as
  passive transport.
• General properties
• a) selectivity
• b) gating open / close (like a door)
• 2) Voltage- or ligand-gated channels the
  gate/door is operated by voltage or ligand
  (chemical binding to the channel protein)
• 3) Voltage-gated channels from plants K
  channels
• a) Identification of the first plant ion channel
  KAT1 and AKT1the yeast and oocyte model system

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Yeast mutant that cannot survive on low K
medium---transform this mutant with a cDNA
library that represents all possible
genes----select the mutant cells that can survive
the low K medium---isolate the plant cDNA
inside the yeast cells---likely represent the
gene coding for K transporter.
One of such clones was KAT1standing for K
transporter of Arabidopsis thaliana. KAT1
expression in the yeast rescued the mutant on low
K medium. Interestingly, it turns out to be a
voltage-gated K channel like those found in our
nerves!
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b) Characteristics of voltage-gated K
channels Tetrameric complex and each subunit
has ---6 transmembrane domains (one
subunit) ---voltage sensor (voltage
sensing) ---pore domain (for K selectivity) Orig
inally discovered in Animals And KAT1 has all
these Elements! The structure is solved at atomic
level
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c) Functional Analysis Oocyte expression And
patch-clamp Involves --microinjection of
mRNA --recording of electrical Current across the
membrane ---analysis of the current
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This shows that the channel conduct both inward
and outward currentthe ions can flow both ways
depending on the membrane potential (voltage)
given by the machine.
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Results The K current conducted by
KAT1 rectifying inward current---unidirectional
influx of K when the membrane voltage is
negative enough (more negative than 100 mV)this
is consistent with the voltage gating
theory. K-selective not permeable to other
monovalent cations such as Na. This is
consistent with the Selectivity property. D)
Functions Transport and signaling
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The journey of K Soil---root epidermis (inward
channels)---root cortex---endodermis---xylem
cells (outward channels)---xylem vessels
--transpiration stream/mass flow---leaf
xylem---mesophyll cells (inward/outward
channels)---back to phloem and recycling
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6. Water channels---new concept on water
permeability Earlier idea lipid bilayer is
somewhat permeable to water New idea water
transport across Membrane is facilitated by
channels Discovery of water channels In plants
The most abundant protein in vacuole
membrane Turns out to be an ion-channel -like
molecule that conduct water And glycerol in
oocyte system. Make the oocytes burst due
to Excessive water uptake! Not only vacuole but
plasma membrane also has them./
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Atomic structure of a water channel from red
blood cells
It is formed by 4 subunits (like the K-channel)