Download BIOL 201: Cell Biology and Metabolism

BIOL 201: Cell Biology and Metabolism
WEEK 12
Receptor Protein Tyrosine Kinases:
GalphaO, Galpha Q for DAG
Always want low levels of calcium in the cell
 They are proteins that are at the cell membrane, exist as monomers in an
unbound form
 When they bind to there ligands, this induces a change in the monomers that they
will come together, forming a dimmer
o Many of the growth factors work through RTKs
 The monomeric receptors have a very low level of tyrosine kinase activity. It will
phosphorylate tyrosines. However unliganded, very low intrinsic kinase activity
 Once they become dimmers, the low level kinase activity is enough to
phosphorylate the tyrosine of the Activation Lip on the
other monomer. Causes an major conformation change
o Activation lip opens up and it becomes a better kinase
 Get a number of the phospho-tyrosine residues
 These residues are recognized by intracellular molecules
with specific domains that interact with theses residues
o The domains are the SH2 domains and PTB Domain
(for insulin)
 Proteins with these domains come to the surface an interact with the RTKs
 For IRS-1, the PTB domain brings it near the receptor, the it is phosphorylated.
These residues that are phosphorylated are then recognized by other SH2 domain
containing proteins
Receptor Tyrosine Kinase Signaling Activity:
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JRB2 is an adapter molecule with SH2 domain that binds to the
receptor. It serves as a platform to bring in SOS
SOS serves a GEF (Kick out GDP and replace with GTP).
This activates the GTP binding proteins. SOS interacts with RAS
o RAS is one of the major targets involved in cancer
When RAS has GTP, it is active. When it is active, turns on a
whole cascade of kinase
RAS to MAP Kinase Cascade:
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When RAS is activated, it goes on an effects a key protein kinase:
RAF (also oncogenetic)
RAF is then activated, it then interacts with other kinases, to
eventually phosphorylate MAP Kinases
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Once MAP kinases are activated, they dimerize and go into the
nucleus and phosphorylate key transcription factors (mostly turn on growth genes,
but can turn off genes involved in growth)
MAP Kinase Induction of Transcription:
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Once they dimerize, enter nucleus and phosphorylate at least two
transcription factors:
o TCF, SRF: Factors for c-fos gene
Scaffold Proteins Limit MAP Kinase Cascades in Yeast:
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Cascade is conservative all the way to yeast (from organization
to order) that requires small GTPase proteins
Except, in yeast, whole thing gets activated by a GPCR
Mating factors interact with GPCR, activating the small
GTPase pathway. They do this by brining all the things close to the membrane by
the Scaffolding proteins (Important for recruiting all the
components together so they can interact)
Receptor Cross-Talk:
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RTKs can also work through a Phospholipase C pathway
They are in a pathways that effects the levels of IP3 and DAG
Take-Hope: Work through 3 different branches
Nervous System – Neurons, Ion Pumps/Channels and Resting Membrane Potential:
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Human brain ~1011 neurons
Brain is ~2% of body mass, but counts for ~20% of resting energy consumption,
Why so much energy: The brain's cells have many elongated processes, so there
is an enormous amount of cell membrane. There is an electrical potential across
the membrane generated and maintained by an energy-dependent process - ion
pumping
Neurons have elongated processes
Why Neurons have Elongated Processes:
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Neurons communicate electrochemically with each other by
direct contact (synapse), often over great distances (Spinal motor reflexes)
Individual neurons can receive input from > 1,000 other neurons. A complex
branching pattern of processes (dendrites) is needed to provide enough surface for
all those other neurons to connect to
Romon y Cajal’s Law of Dynamic Polarization:
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Communication comes in through the dendrites and out through the axon
o Dendrites are input synapses
o Axon terminus is output synapse
Long distance communication along axons is by action potentials, originating in
cell body
Action Potentials:
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It is the output of a neuron that travels down the axon
Transient disturbance of the cell resting membrane potential
Resting membrane potential is -60 mV (inside is negative)
Depolarization: Caused by the AP, cell gets more positive
Repolarization/Hyperpolarization: Cell gets more negative
APs move
Basis of the Resting Membrane Potential:
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Phospholipid Bilayer: Phospholipid bilayer membranes are
intrinsically impermeable to many molecules and ions
Sodium/Potassium pump
Potassium Channels
Sodium/Potassium Pump:
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The sodium/potassium pump, (Na/K ATPase) is the main
energy-requiring component of the resting membrane potential
The pump generates electrically neutral Na+ and K+ gradients
across the membrane by hydrolyzing ATP. Pumps 3 Na out and 2 K in
In a pure phospholipid bilayer these Na+ and K+ gradients would have no
electrical consequences
Potassium:
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But cellular membranes are not pure phospholipid, they contain K+ channels,
transmembrane proteins that allow K+, and only K+, cross the membrane
The K+ is allowed to pass, it is not pushed. No energy is needed
K+ flows down its concentration gradient, so + charges accumulate outside the
cell. We now have an electrical potential (inside negative) across the
membrane
Membrane Impermeable to Na, K, Cl:
Membrane Permeable Only to K:
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Eventually, net K+ efflux will stop because once there is an
electrical gradient, a K+ ion in the channel will be attracted by
the negative charge behind it on the cytosolic side and repelled by the positive
charge ahead of it on the extracellular side
At some point the K+ concentration gradient is exactly balanced by the
electrical potential. This is the Equilibrium Potential (-59 mV)
The Nernst Equation:
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A physical-chemical formula that gives the equilibrium potential for a membrane
permeable to an ion as a function of the initial concentration difference across the
membrane
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R = gas constant, T = temperature (oKelvin), Z = charge on ion, F = Faraday
constant
o For Z = 1, and T = 293 oKelvin (20 o Celsius), this becomes:
o EK = 0.059 log10 ([K outside] / [K inside]) in volts
So, for a [K outside] / [K inside] ratio of 10, we get EK = 0.059 V, or 59 mV
And for a [K outside] / [K inside] ratio of 0.1, we get EK = -0.059 V, or -59 mV
This is close to real cellular resting potentials. In fact, K+ channels establish the
resting potential
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Membrane Permeable Only to Na:
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Turns out that the Membrane resting potential,
because Na goes the other way, of + 59 mV
Molecular Basis of Ion Channel Selectivity:
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K+ Channel from Stretomyces:
o 2 Helical (S5, S6) domains that traverse the membrane.
Between them is the P (pore) segment that includes
the selectivity filter
o There are 4 of these subunits that form the channel (tetramer)
o Selectivity filter is the narrowest part of the pore
Molecular Basis of K+ Channel Ion Specificity:
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Carbonyl oxygens within the selectivity filter precisely replace the water of
hydration of K+, but do not fit Na+. So Na+ "prefers" to stay associated with its
water of hydration, and that hydrated complex is too large to fit through the
channel pore
The length of the selectivity filter pore is considerable,
there is space for 4 K+ ions
At any time the filter has two K+ ions and two
"empty spaces"
K+ can go either way, depends on [] gradient
Pumps:
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The Na+ /K+ pump is not the only pump
o There are other pumps, including Ca2+ pumps (pumps Ca2+
out of cell)
+
The K channels are not the only channels
o There are many others, including Na+, Ca2+, ClThe combined action of the pumps and channels generates the
differences between extracellular and intracellular ionic makeup
Electrophysiology Measurements:
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Membrane potential measured using intracellular and extracellular
electrodes. Tells us the voltage across the cell
Requires penetration into the cell, a good seal is formed
Patch Electrodes – Another Recording:
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Glass can make an extremely tight seal with cell membrane
Measure ion channel activity as current carried by the ions (while membrane
potential held constant)
Very small membrane patches can be studied: Single channel molecule
recording
Patch has small # of channels, therefore, can study the activity of a single
channels. Measured 0.5 pA ~ 104 Na+ ions per millisecond
Nervous System – Action Potentials:
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Action potentials are initiated at threshold level of depolarization: -50 mV
Threshold provides an integrating mechanism: Input from a single synapse may
give a subthreshold depolarization, whereas simultaneous input from multiple
synapses may exceed the action potential threshold potential
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Experiment with a stimulation and recording electrode
Records the resting membrane potentials:
o Hyperpolarization: Push level more negative
o Depolarization: Push level more positive
If it does not pass the threshold, it gives a passive response
When it passes the threshold, allows the initiation of an action potential
Move fast along axon up to 100 m/s
All-or-none: Get an action potential or you done
All action potentials from any given cell are identical
o Basically similar in all neurons (e.g. sensory and motor)
Signaling information resides in the frequency, not the amplitude
o Amplitude is always the same
As you increase a stimulus, increase frequency of AP generation
Action potential based on voltage-gated channels
Depolarization phase: Voltage-gated Na+ channels
Repolarization phase: Voltage-gated K+ channels
Voltage-Gated Channels:
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Channels that are closed at the resting membrane potential, but are opened when
depolarized to a certain threshold
The action potential depolarization threshold is the opening threshold of the Na+
channel
The opening of the voltage-gated Na+ channels leads to Na+ influx, which
depolarizes the cell up to a maximum depolarization (or rather opposite
polarization) equal to the Na+ equilibrium potential ~=+59mV
Two things happen that reverse the depolarization due to opening of the Na+
channels
o The Na+ channels inactivate, or shut themselves off within ~ 1ms
o Voltage-gated K+ channels are opened by the strong depolarization
With the Na+ channels inactivated, and the voltage-gated K+ channels
open, K+ flows out of the cell, which restores the resting membrane
potential
Upon exposure to the resting membrane potential, self-inactivated Na+
channels become reactivated (though closed)
Na Channel – Voltage Gated, Self-Inactivating Channel:
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In a resting cell, the channels are closed by a gate
Voltage-Sensing Alpha Helix can exist in two
conformations: High & Low. It moves by the
membrane potential
When the cell gets depolarized, it moves High,
changing the conformation of the molecule,
opening the gate, allowing for Na to flow through
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However, the Channel-Inactivating segment
will flow into the pore, inactivating the channel
When the cell repolarizes, the alpha helixes move down, and kick out the
Inactivating segment
Inactive Na Channel leads to a refractory period
Voltage-Gated K and Na Channels:
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K Channel: Voltage-Gated K Channel is in the form of tetramers
o Evolutionary relationship to Na Channel
o Each helix 4 of the tetramer is the voltage sensing helix
o Each tetramer has an inactivating segment
Na Channel: It is one polypeptide, but with 4 units
o Helix 4 still the voltage sensing helix
o However, has only 1 inactivating segment
As you increase membrane potential, increase the % of time
that the channel is open. Patch Experiment:
Na Channel Inactivation:
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Means that action potentials always move forward, however, the spread of the
charge moves in both direction
o In axonal regions near the region that has open Na+ channels, the
membrane is partly depolarized due to passive spread of charge
o In immediate neighboring regions, this partial depolarization exceeds the
Na+ channel opening threshold, so the action potential is self-propagating
o However, it can only self-propagate forward because the Na+ channels in
the axon segment behind the currently active region are still inactivated,
and they cannot respond to even suprathreshold depolarization, Only the
channels ahead of the active region are able to open
o Inactivation/reactivation also limits the maximal firing rate