Transcript How does a cell Membrane serves as both “barrier” and “gate”

Membrane Transport
1. 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 bilayer—barrier, transport proteins—gates.
2. Permeability of “lipid bilayer” and a “cell membrane”
Biologically important molecules:
bilayer
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 +
membrane
+++
+++
+++
+++
3. Membrane Transport
1) 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”).
Out
in
Out
in
Out
in
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.
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
3 Na+
ATP
ADP
P
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
a.
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
A lot of secondary transport is dependent on H+-gradient
Amino acids(symport)
And Anions
Cations (channels)
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.
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
processes—discuss in later sessions.
5. Ion channels: structure and function
Ion channels conduct ion flows down the electrochemical gradient and is
considered as “passive” transport.
1) 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 AKT1—the yeast
and oocyte model system
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 KAT1—standing for K+ transporter of Arabidopsis
thaliana. KAT1 expression in the yeast rescued the mutant on low [K+]
Interestingly, it turns out to be a voltage-gated K+
channel like those found in our nerves!
medium.
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)
Originally discovered in Animals
And KAT1 has all these
Elements!
The structure is solved
at atomic level
c) Functional Analysis
Oocyte expression
And patch-clamp
Involves:
--microinjection of mRNA
--recording of electrical
Current across the membrane
---analysis of the current
This shows that the channel
conduct both inward and
outward current—the ions
can flow both ways
depending on the
membrane potential
(voltage) given by the
machine.
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
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
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./
Atomic structure of a water channel from red blood cells
It is formed by 4 subunits
(like the K-channel)