PSIO 404 Exam 5 Flashcards
Dynorphin
is an “endogenous opiate”
Calcium
can cross the membrane and into the cytoplasm of a cell can bypass a lot of integration and processes right to the generation of cellular responses like muscle contractions or changes in cell shape
Principles of Signal transduction by ion channels
Ion channels exist in allosteric equilibrium between active (open) and inactive (closed) states.
– Inputs for the ion-channel switch favor either the active or the inactive state of the relevant ion channel.
* The resting membrane potential powers ion-channel switches.
*the gate region becomes open and closed
how input signals make a receptor on or off
input -> on = agonist, that is highly specific and non covalent interaction will bind to discriminator domain and the effector domain will have a conformational change to active state to put out output signals
input -> off an antagonist will bind to a different receptor the discriminator domain which will change the effector domain to in inactive conformation leading to no output signals
All ion channels are controlled by
agonists and antagonists
False
on channels can be gated (opened) by:
1. Membrane Potential Δs (for voltage-gated ion channels)*
2. Molecules (for ligand-gated ion channels)**
3. Mechanical force (for mechanosensitive ion channels)
4. Temperature (for temperature-gated ion channels)
*The majority of human ion channels (of the 500 we have).
**Nevertheless, these channels are still the 3rd most
common receptor type used in cell-cell communication.
the three most common types of membrane receptors and the energy supply
- protein kinase coupled receptors = phosphorylation switch
- GPCR = GTPase switch
- ion channel receptors = membrane potential discharge
Resting Membrane potential and ion in signal transduction
outside : K = 100 mV
Na - 20 mV
Cl- = 5 mV
Ca2+ = 0.01 mV
Inside: K+ = 5 mV
Na+ = 140 mV
Cl- = 120 mV
Ca2+ = 1 mV
Describe the common structural features of ion channels.
Cations are Na, K, and Ca
- they have at least 4 subunits that form the central cavity
and contribute to the selectivity filter (K = 2 subunits)
Describe the common structural features of ion channels.
The common structural features of cation channels are
1. selectivity filter, these are formed by p-loops in cation channels and they remove water from ions and permit passage of one or more specific ions
2. central cavity, these are formed by multiple subunits that rehydrate ions that pass into it
3. gate, these are formed by inner helices for many cations channels and are input dirven for opening or closing
Discuss the general structure of prototype 1 and prototype 2 cation channels, and name an example for each type of channel.
prototype 1:
– 6 transmembrane
domains (S1-S6)
– One P-loop between
S5 and S6
-need 4 P-loops to create a selectivity filter
– Where applicable,
S4 is a voltage sensor
Example: (not voltage sensitive InsP3R
prototype 2:
– 2 transmembrane
domains (S5 and S6)
– One P-loop between
S5 and S6
-need 4 P-loops to create a selectivity filter
– For K2P channels,
only need two S5-S6 subunits
are covalently linked for a complete channel
Example:
Kir
Describe voltage gating’s basic theme (basic mechanism).
-Helices 3b and 4 (the “S3-S4 paddle”) are voltage-
sensitive in that they change position, moving
further into the membrane upon depolarization.
-As helices 3b and 4 “swing” upward, the channel is
opened
Briefly describe the structure of voltage-gated Na+ channels.
-propagate action potentials
-have four influx openings and four efflux openings
-are composed of one alpha subunit and three beta subunits
-the alpha subunit forms the channel
-they are gated by the s subunit S4 voltage sensor
-prototype 1
-are inactivated by a separate inactivation domain on the alpha subunit
Briefly describe the function of voltage-gated Na+ channels.
the function of voltage gated sodium channels is to propagate action potentials by traveling the length of the neurons
-voltage gating = > inactivation for a time “(the refractory period) for unidirectional movements of action potentials, once the refractory period passes it is ready for the next AP
Targets:
-local anesthetics such as lidocaine
-neurotoxins like tetrodotoxin (puffer fish), histrionicotoxin, saxitoxin (frog)
Name and very briefly describe the action of two of the four anesthetics or toxins which target the voltage-gated sodium channel.
lidocaine= a local anesthetic
tetrodotoxin= leads to insufficient activation of skeletal muscles which leads to inhibition of contracting muscle to the diagram, paralysis of receptor muscle leading to respiratory arrest and death
histrionicotoxin = used by native tribes on poison arrow
saxitoxin = produced by Gonyaulax catenella is disastrous red tides
they all inhibit the function of of voltage gated sodium channels
Briefly describe the functions of ligand-gated Na+ channels (epithelial Na+ channels, or ENaCs). How do ENaCs act in concert with ion pumps and K+ channels to accomplish their function?
-ENaCs are ligand gated
-some regulate electrolytes balance across epithelia (vasopressin and aldosterone vs. ANP action in the kidney)
-all function in concert with ion pumps and K channels
-some ENaCs are involved in sensation of salty and sweet taste
-Na enters from the environment to the body by entering through an increase in ENaC, sodium will enter in to the cell from the tubular fluid along the collecting ducts, this is accompanied with an increased amount of sodium potassium ATPase and potassium channels so that you can take the sodium that is moving into the cell and cause it to be pumping into the interstitial fluid to be removed
Briefly explain the role of ENaCs in the sensation of taste: in which one or two types of taste do ENaCs play an initiating role?
ENacs are the primary channel involved in salty and sour taste. The signal to the brain with the regulation of electrolytes and water balance if the food is salty or sour
Briefly discuss the structure of voltage-gated K+ channels.
– are highly variable
– have prototype 1 structure w/ four
un-joined, pore-forming a subunits
(homo- and hetero-tetramers)
– are gated by depolarization
– composed of pore-forming a
subunits encoded by >50 genes w/
extensive tissue-specific expression
-each alpha subunit is associated with
a beta subunit which reversibly
plugs pore
Briefly discuss the function of voltage-gated K+ channels.
– function primarily to terminate the
action potential
-important for repolarization
Name and very briefly describe one of the two KV channel blockers mention in lecture.
– KV channels at NMJ synapses are
blocked by dendrotoxins (such as
the venom of the mamba snake)
– Other KV channel blockers
(derivatives of 4-aminopyridine)
are used to treat multiple
sclerosis (MS)
Compare and contrast the function of BK and SK channels.`
-BK channels (Big conductance K+ channels)
– voltage controlled (depolarization), but require an increase in [Ca2+] as well to act (logical AND gates).
– aid in relaxation of smooth muscle
* SK channels (Small conductance K+ channels)
– Gated by Ca2+ alone (not voltage controlled)
– responsible for after-hyperpolarization phase of action potential
Name and very briefly describe one of the two poisons/venoms mentioned in lecture which target BK and SK channels.
- both BK and SK are targets of apamin (component of bee venom) and charybdotoxin (scorpion poison)
-if you block these channels you block repolarization and continued activity of neuron and muscle
Discuss the basic structure and important physiological function of inwardly-rectifying K+ (Kir) channels.
- have prototype 2 structure and open at
negative membrane potentials and close
upon depolarization - set resting membrane potentials and
shape action potentials - Controlled by the ATP/ADP ratio in insulin-secreting cells
1. G-protein-gated inwardly rectifying K+ (GIRK) channels
– are even more signal-regulated
than other types of K+ channels
– gated by Gbg subunits of Gi-proteins
– are the way inhibitory neurotransmitters effect
hyperpolarization in the postsynaptic cell
*how they set the resting membrane potential
Describe the activation (gating) and function of GIRK channels (also known as Kir3 channels).
- G-protein-gated inwardly rectifying K+ (GIRK) channels
– are even more signal-regulated
than other types of K+ channels because they are gated by G proteins (inhibitory G protein)
– gated by Gbg subunits of Gi-proteins
– are the way inhibitory neurotransmitters effect
hyperpolarization in the postsynaptic cell by the beta gamma subunits that interact with these channels leading to a more hyperpolarized cell which means the neuron is less likely to be activated by an action potential and all other cells are suppressed in their polarization status
Briefly describe one of the two examples of the operation of GIRK channels mentioned in class (Hint: both examples are effects of the autonomic nervous system, i.e. sympathetic or parasympathetic effects).
– We’ve seen GIRK effects:
-1.parasympathetic actions on heart,
-with pacemaker cells, activation of the M2 receptor by acetylcholine will lead to activation of the inhibitory G protein that it is coupled too. The alpha subunit that inhibit adenylate cyclase means less cyclic AMP which means less opening of the HCN sodium channel so less sodium can enter the cell. this will reduce the steepness of the pacemaker cell potential. Then the beta gamma subunits bind to the GIRK channels, which the beta/gamma subunit activates the channel and causes it to open, opening of the channel causes potassium to leave, but not enter therefore resulting in more polarized of the cell (hyperpolarization)
-this means the threshold if reach less often in a period of time slowing the heart rate
-2. a2-adrenergic feedback inhibition of NE
release at sympathetic synapses.
– Inhibitory signaling takes two directions:
1. reducing membrane potential via Gbg
and 2. reducing cAMP concentration via Ga
Discuss the structure, function, and critical physiological importance of KATP channels. Specifically, be able to explain how KATP channels enable beta cells of the pancreas to depolarize and thus release insulin when blood glucose levels are high.
– For the KATP channel of pancreatic b-cells, the
ATP/ADP-binding transmembrane protein SUR
(SulfonylUrea Receptor) gates the activity.
– Channel closes when ATP/ADP ratio rises
– SURs are targets of sulfonylureas (anti-diabetes
drugs!
-SUR is activated by the presences of high ATP levels (causing the cell to be more polarized) and is closed with low ATP levels (causing the cell to become more depolarized)
-closed SUR channels - more depolarization of beta cells = release of more insulin
-good diabetes drug
Describe the basic structure and function of K2P channels, and give a couple of examples of how K2P channels are used by our cells.
NOT voltage controlled therefore they are always active and they do not open or close (like background channels)
- Cell sensation of internal mechanical pressure
(osmotic pressure in, ex: a hypotonic solution.)
– To prevent rupture, the cell opens K+ and Cl- channels to move solutes out in an electroneutral way so that cell function is not impaired and osmotic pressure is relieved! Section 14.7.1 in your textbook covers more of this. - Cell sensation of temperature in the hypothalamus.
– These K2P channels close as temperature drops. This leads to depolarization and generation of action potentials. Prostaglandin
E2 & pyrogens also block these channels to increase body temperature. Changed neuron activity mediates proper response. - Cell sensation of disturbance in blood supply to cells.
– Arachidonic acid and lysophospholipids respond to brain oxygen deprivation and prevent fatal accumulation of Ca2+ in brain cells.
Explain how K2P channels are employed by a cell 1) to regulate cell osmolarity, 2) to determine and respond to ↑ body temperature, and 3) to protect against neuron damage in the event of oxygen deprivation in the brain.
- 2P-Domain K+ Channels
– are of prototype 2 structure and are not
voltage-controlled
– also control the resting potential; do not fully
close upon depolarization (they are called
background channels, adjusting resting
membrane potential at different levels)
– are gated by a variety of physical and chemical stimuli
– mechanically-gated for osmotic pressure relief to prevent rupture
– temperature-gated for control of body temperature (these 2P-
channels are in sensory nerves and in the hypothalamus)
– ligand-gated for transmission of pain sensations, protection of
neurons from overstimulation (as in stroke)
– are opened by gaseous narcotics (chloroform, halothane, etc.)
Voltage-operated Ca2+ channels (VOCCs)
IIC. Voltage-operated Ca2+ channels (VOCCs)
* Voltage-operated Ca2+ channels (VOCCs) are
– gated by depolarization and are found in many tissues, in all eukaryotes
* VOCCs connect changes of membrane potential with Ca2+-
dependent cellular processes like secretion & movements.
– VOCCs are critical for neurotransmitter release at synapses, cellular
motility and muscle contraction. The neuron AP’s job is to gate VOCCs
Signal Transduction by Anions
- Primary ion transported is chloride (Cl–), but other ions include
bromide, iodide and nitrate (very low abundance for all of these) - Gating of a Cl– channel can either hyperpolarize (skeletal m. and
neurons) or depolarize (smooth m.) depending upon the
steepness of the concentration gradient of chloride (a
determining factor for the membrane potential value).
Briefly describe the general function of anion channels in the body.
- Primary ion transported is chloride (Cl–), but other ions include bromide, iodide and nitrate (very low abundance for all of these)
- Gating of a Cl– channel can either hyperpolarize (skeletal m. and neurons) or depolarize (smooth m.) depending upon the steepness of the concentration gradient of chloride (a determining factor for the membrane potential value)
Cell sensation of internal mechanical pressure
(osmotic pressure in, ex: a hypotonic solution.)
– To prevent rupture, the cell opens K+ and Cl- channels
to move solutes out in an electroneutral way so that cell function
is not impaired and osmotic pressure is relieved! Section 14.7.1 in
your textbook covers more of this
Cell sensation of temperature in the hypothalamus.
– These K2P channels close as temperature drops. This leads to
depolarization and generation of action potentials. Prostaglandin
E2 & pyrogens also block these channels to increase body
temperature. Changed neuron activity mediates proper response
Cell sensation of disturbance in blood supply to cells.
– Arachidonic acid and lysophospholipids respond to brain oxygen
deprivation and prevent fatal accumulation of Ca2+ in brain cells.
List the 3 families of chloride channels discussed in lecture.
- There are 3 families of Chloride Channels:
1. ClC (Cl– Channel) family
2. CFTR (Cystic Fibrosis Transmembrane conductance
Regulator) family
3. GABA (Gamma-AminoButyric Acid) & Gly receptor anion
channels
- Anion Channels: CFTR (Cystic Fibrosis
Transmembrane conductance Regulator) family
- CFTR governs the
movement of salt
& water across
epithelia - Various toxins
operate by hyper-
phosphorylation of
the regulatory
region
Briefly discuss the structure and function of ClC channels, and be able to give 2 of the 4 specific physiological roles/examples for ClC channels mentioned in lecture.
- composed of 2 subunits to make a single 2-pore channel
- major functions include: control of cell volume and
osmolarity, trans-epithelial electrolyte transport, pH
regulation, acidification for bone resorption, setting the
resting membrane potential, and (mostly-inhibitory)
influences on neurotransmission - a special isoform called
ClC-K is involved with
chloride reabsorption in
the thick ascending limb
of the loop of henle - ClC-K is also important to
endolymph production in
the inner ear - ClC7 is used by
osteoclasts to secrete HCl - other ClCs work with K2P
channels for cellular
osmotic pressure relief
Briefly discuss the structure and function of GABA and glycine receptor anion channels.
- are ligand-gated (GABA and glycine)
– Ligand binding opens the channel and
causes chloride influx (thanks to low
cytosolic Cl− levels) to cause
hyperpolarization - composed of 5 subunits (see righ
List and briefly explain the effects of, two of the five types of agonists for GABA and glycine receptor anion channels given in lecture.
Agonists: alcohol, halothane (also
targets K2P channels), sleep-inducer
barbiturates, benzodiazepines used as
fear reliever
List one of the two industrial chemicals mentioned in lecture which are also antagonists for GABA & glycine receptor anion channels.
- are ligand-gated (GABA and glycine)
– Ligand binding opens the channel and
causes chloride influx (thanks to low
cytosolic Cl− levels) to cause
hyperpolarization - composed of 5 subunits (see right)
- Agonists: alcohol, halothane (also
targets K2P channels), sleep-inducer
barbiturates, benzodiazepines used as
fear relievers - Antagonists: analeptics such as
avermectin (insecticide), picrotoxin
(plant poison) and clinical treatment
for alcohol and barbiturate intoxication
Briefly explain the use of analeptics for the treatment of alcohol or barbiturate intoxication (not addiction, just intoxication).
Agonists: alcohol, halothane (also
targets K2P channels), sleep-inducer
barbiturates, benzodiazepines used as
fear relievers
* Antagonists: analeptics such as
avermectin (insecticide), picrotoxin
(plant poison) and clinical treatment
for alcohol and barbiturate intoxication
output signal can depend on
-pH dependent
-Calcium dependent
-Voltage dependent
signal transduction by cations for sodium channels
- voltage gated Na+ channels
- ENaCas (epithelial Na+ channels)
signal transduction by cations for potassium channels
- Kv voltage gated potassium channels
- Kir inward rectifying potassium channels
- K2p channels
signal transduction by cations for calcium channels
- VOCC voltage operated calcium channels
signal transduction by anions for chloride channels
- CIC (CL- channel) family
- CFTR (cystic fibrosis transmembrane conductance regulator ) family
- GABA (gamma aminoButyric Acid) and Gly receptor and channel
Prototype 1 cation channels can be “gated” by multiple things including voltage, cyclic nucleotides, or InsP3.
True
Which of the following anion channels is used by mucosal epithelia to maintain hydration of mucous membranes?
CFTR channels
Which of the following channels can be assembled on demand based upon the action of G-proteins?
inwardly-rectifying potassium channels
Which of the following is NOT a typical part of cation channels?
a selectivity filter
a pressure relief valve
a gate
a carbohydrate linker for additional specificity
a central cavity
a ligand binding site
-a ligand binding site
a carbohydrate linker for additional specificity
a pressure relief valve
Which of the following is not true regarding the axonal sodium channel?
It is regulated by calcium ions as part of negative-feedback mechanisms.
It assembles in the membrane with a total of 24 transmembrane domains.
It contains a voltage sensor in its S4 transmembrane domain(s).
It has four influx and four efflux openings.
It is regulated by calcium ions as part of negative-feedback mechanisms.
Signal Transduction by Anions
- Primary ion transported is chloride (Cl–), but other ions include
bromide, iodide and nitrate (very low abundance for all of these) - Gating of a Cl– channel can either hyperpolarize (skeletal m. and
neurons) or depolarize (smooth m.) depending upon the
steepness of the concentration gradient of chloride (a
determining factor for the membrane potential value).
To have a noticeable affect on downstream effectors,
the cytosolic [Ca2+] must be between 1-10mM.
How is cytoplasmic Ca generated and maintained
by Ca2+ pumps which operate against steep concentration gradients (with direct
or indirect powering by ATP hydrolysis).
Describe how [Ca2+] differs throughout the cytoplasm vs. within organelles vs. the extracellular space for a typical non-stimulated eukaryotic cell?
outside the cell: typically very high at a concentration of 1 mM
extracellular space (cytoplasm) : typically very low at a concentration of 0.0001 mM
within the organelle : ER = 0.1 - 1 mM
mitochondria = 10 mM
In the cytoplasm of non-stimulated cells, the [Ca2+] is maintained at about 0.1uM. This is a [Ca2+] which is 4 orders of magnitude lower than extracellular [Ca2+] or ER lumenal [Ca2+], ormitochondrial [Ca2+]
- Cytoplasmic [Ca2+] is also 2-4 orders of magnitude lower than [K+], [Na+] and [Cl-], and thus Ca2+ influx rarely changes the membrane potential significantly.
Explain how [Ca2+] in the cytosol is maintained by a cell.
- Persistent cytoplasmic [Ca2+] of roughly 1mM or more will trigger apoptosis, so how can Ca2+ signaling be effective without killing the cell?
Answer: signal transitoriness by means of Ca2+ waves.
Briefly discuss the function of ligand-gated calcium channels of the plasma membrane involved in calcium signaling
- Ligand-controlled cation channels for Ca2+ are gated by intercellular signaling molecules (like arachidonic acid) or as a result of capacitative Ca2+ influx mechanisms.
- Ligand-controlled cation channels for Ca2+ are effectors of signals such as hormones, neurotransmitters, cytokines, and environmental
stimuli which activate phospholipase A2 (PLA2).
Discuss the role of PLA2 in calcium signaling across the plasma membrane
- PLA2 is a signal-activated intracellular enzyme
- PLA2 forms 2 products from phospholipids:
1. Lysophospholipids
2. Arachidonic acid - Lysophospholipids influence cell proliferation, apoptosis, vesicle transport, cytoskeletal dynamics, etc.
- Arachidonic acid is the substrate for eicosanoid synthesis but it is itself a genuine second messenger.
B. Ca2+ Channels:
ARCCs
* Arachidonic acid is formed by PLA2 in response to a
wide variety of extracellular signals.
* Arachidonic acid gates arachidonic acid regulated
calcium channels (ARCCs)
Briefly discuss the function of voltage-gated calcium channels of the plasma membrane involved in calcium signaling.
- Voltage-operated Ca2+ channels (VOCCs) are
– gated by depolarization and are found in all eukaryotes in many tissues.
1. L-type (L for “long -lasting) in the heart
2. N/P/Q/R-type (N for neural) induced NT release
3. TRPP (T for transient) for heart pacemaker threshold channels - VOCCs connect changes of membrane potential with Ca2+- dependent cellular processes like secretion & movements.
– VOCCs are critical for neurotransmitter release at synapses and in cellular motility & muscle contraction. In essence, purpose of APs is to gate VOCCs
-AP travels down the presynaptic end bulb to get to the VOCC to open them and allow an calcium to rush in and get a neurotransmitter at that part of the cell
Briefly discuss the function of PMCA and EXNa in the regulation of cytoplasmic [Ca2+] in calcium signaling
- The 0.0001mM concentration of Ca2+ is maintained in the cytoplasm by several ion channels and pumps. PMCA and EXNa are calcium pumps located in the plasma membrane.
- Plasma membrane Ca2+-dependent ATPase (PMCA) pumps Ca2+ out of the cell while the electrogenic Na+-Ca2+ exchanger (EXNa) moves Ca2+ out in exchange for three Na+ ions. EXNa therefore has a depolarizing effect as well
Name and give the ligands for the calcium channels of the ER.
- Ca2+ channels (in the ER) are gated by ligands:
1. InsP3 and sphingosine 1-phosphate for InsP3 receptors (InsP3Rs)
2. cADPR (and possibly NAADP) for ryanodine receptors (RyRs)
3. Ca2+ itself for 1. & 2. (Ca2+-induced Ca2+ release).
What is cADPR and how does its formation link cell redox state of a cell to calcium signaling?
- Formed from NAD+ by ADP-ribose cyclase
-removes nicotinamide and creates a covalent bond between ribose and the adenine - Cyclic ADP ribose (cADPR) likely gates RyRs,
but probably not directly. - Since it’s formed from NAD+, cADPR
formation is linked to the NADP+/NAD+ ratio,
which is a measure of cell redox state
-this redox state means that it is tied to how much NAD+ and NADH which means it is tied to how much electrons are around therefore tied to the energy status of the signaling
Discuss in detail calcium-induced calcium release, and explain how the release of calcium is stopped
- ER Ca2+ channels (InsP3R and RyR) release Ca2+ upon ligand binding which gates other ER Ca2+ channels
- ER Ca2+ channels (InsP3R and RyR) are regulated by Ca2+ in a biphasic mode: positive feedback below
300nM [Ca2+] and negative feedback above
-when you ramp up the concentration of the other side of the smooth ER then you shut off the movement of calcium you get a aredion of increaseconcntrion of calcium that can move like a wave
-which ever signal (1. InsP3 and sphingosine 1-phosphate for InsP3 receptors (InsP3Rs) 2. cADPR (and possibly NAADP) for ryanodine receptors (RyRs) 3. Ca2+ itself for 1. & 2. (Ca2+-induced Ca2+ release).) this will lead to an induce of calcium release which induces more calcium release leading to a flood out of Ca into the smooth ER
Describe SERCA, and explain it’s contribution to Ca2+ wave formation
- Sarco/endoplasmic reticulum calcium-dependent ATPase (SERCA) enables the ER to take up Ca2+ from the cytoplasm. Along with pumps in the PM and mitochondria, this rapidly returns cytoplasmic
[Ca2+] to 0.0001mM.
Discuss intracellular calcium waves: formation, frequency, and how constant signals lead to depletion of Ca2+ from the ER
Due to the biphasic mode of ER Ca2+ channels
and to Ca2+ pumps, [Ca2+] will spike and then be rapidly lowered. This continues as long as the stimulus is applied.
- Weak signals lead to a lower Ca2+ wave frequency than strong signals.
- A cell can interpret Ca2+ wave frequency.
- Ca2+ signals can be “sparks” (1-μm and <1sec) or longer-lasting “puffs” and “spikes.”
- Wavelengths of oscillating Ca2+ signals can vary from milliseconds to several minutes.
- Said another way, Ca2+ oscillations vary between tens of Hz (waves/sec) in neurons to tens of mHz in non-excitable cells
Control of Ca2+ Wave Frequency
- The arachidonic acid- regulated Ca2+ channel
(ARCC) controls the frequency of Ca2+ waves. - Arachidonic acid is formed by the enzyme phospholipase A2 (PLA2) in response to a wide variety of extracellular signals
by using Ca to start the Ca wave because the more calcium that is flooding in the more frequent the calcium waves will become
how can a long-term signal be maintained by the cell
- At low [Ca2+], STIM proteins aggregate in the ER membrane next to clusters of CRAC (Ca2+-release-activated Ca2+) channels.
- Association between STIM & CRAC subunits (Orai) leads to interaction between the ER and PM so Ca2+ entering the cell is immediately pumped into the ER by SERCA!
- SOCCs control amplitude
of Ca2+ waves by controlling
the level of Ca2+ “charge” in the ER.
Discuss in detail how Ca2+ stores are replenished during prolonged Ca2+ signaling
- A transient signal creates a wave, but a persistent signal will create a stationary pattern throughout the cell.
- This is of paramount importance because practically all cellular processes are modulated directly or indirectly by Ca2+
However, in a long-term Ca2+ signal,
Ca2+ is slowly lost by the cell (more is
pumped out than can be
recovered by the ER).
What is a hot spot in Ca wave
large numbers of calcium waves are being generated at a particular location in the cell
Briefly discuss calmodulin’s (CaM’s) role in Ca2+ signaling
- EF-hand domains
* Ca2+ binding changes protein structure (like exposing a lipid anchor
– the Ca2+-myristoyl switch).
* Examples: Calmodulin (CaM) and many others
Calmodulin binds four calcium ions (built-in filtering) via EF-hand
domains. It changes conformation, and then forms a myriad of
interactions with signaling proteins
How is CaMKII able to sense calcium wave frequency in a cell
CaMKII responds to Ca2+ wave frequencies between 100 and 3000mHz.
- Activated by Ca2+/CaM-dependent
transautophosphorylation in a stepwise manner - Activated by Ca2+/CaM-dependent transautophosphorylation in a stepwise manner
- The stronger the signal, the more frequent the Ca2+ waves and more active CaMKII.
- CaMKII has molecular memory in that it’s activity remains long after Ca2+ waves.
- As mentioned earlier, CaMKII targets many proteins involved in neurotransmission.
- CaMKII phosphorylates transcription factors such as CREB and SRF.
- Calcium also has manifold indirect effects on transcription through CaM,
cPKC, and other proteins
Discuss the unique structure of CaMKII and explain how it is able to sense calcium wave frequency in a cell
- Calcium has direct effects on calcium-sensitive proteins by
means of calmodulin (CaM) and other similar proteins. - How could calcium wave frequency possibly be detected by
something like a protein? An answer is shown in the
activation of Ca2+/CaM-dependent kinase II (CaMKII) - CaMKII phosphorylates many proteins involved in
neurotransmission (RyRs, neurotransmitter receptors, and
transcription factors) linked to memory and learning
the transcription factor Ca selective Channel family
- Several SOCCs are in the TRP ion channel family
- TRP (Transient Receptor Potential) channels are
– classified into 6 subfamilies and encoded by >28 genes
– structured like KV channels; most are not voltage-sensitive
– used (usually via PLC activation) to
1. process sensory stimuli like sweet/umami/bitter, sour,
heat, pressure, pain, and even pheromone chemicals
2. mediate capacitative Ca2+ influx
3. carry out transepithelial transport of Ca2+, the acrosomal
reaction of sperm, vasoconstriction, osmosensation, and
synthesis of NO and prostacyclins by epithelial cells
What are the TRP (Transient Receptor Potential) channels that are classified into 6 subfamilies and encoded by >28 gene
- TRPC (C for canonical)
- TRPV (V for vanilloid receptor)
- TRPM (M for tumor suppressor protein melastatin)
- TRPP (P for related to polycystins)
- TRPML (ML for mucolipin)
- TRPA (A for ankyrin-like protein)
Calcineurin
is a phosphate the one and only controlled bu Ca 2+ / CaM
Discuss how calcium controls the transcription factor NFAT.
- At correct frequencies, Ca2+ waves trigger
activation of NFAT. - NFAT responds to Ca2+ wave
frequencies between 3 and 90mHz - NFAT is critical for development of the CNS,
integument, CV system and skeletal muscle. - NFAT is tissue-specifically expressed, and is
important for learning & memory fixation.
Describe NFAT signaling in Down Syndrome patients.
- Regulatory protein DSCR-1 is overexpressed
in individuals with Down syndrome.
DSCR-2 is an inhibitor of Calcineurin which is a promotor of NFAT - “Dosage issues” for expressed proteins are
a consequence of trisomies in general.
Trisomy 21 leads to overproduction of
DCSR-1 and underexpression of proteins
critical for organ development & function.
Describe how calcium controls DREAM, and discuss how calcium plays a role in the transmission of pain signals
- Ca2+ also directly interacts with one, and only one, transcription factor: DREAM.
by enacting it
-Pain transmission to the brain from the periphery
-Pain transmission decreased by spinal interneuron thanks to inactivation of DREAM by calcium
Explain how and why Calpains are activated, and summarize their action.
- At high frequency, Ca2+ waves eventually become a persistent,
elevated cytoplasmic Ca2+ concentration, activating Calpains - Calpains
– are Ca2+-dependent cysteine proteases
– are inactive proenzymes (activated by autocatalytic cleavage triggered by Ca2+)
– are found in ubiquitous and tissue-specific isoforms
– have variable Ca2+ affinity (from μM for μ-calpains to mM for m-calpains)
– have direct impact on execution of apoptosis (they cleave key elements in the
apoptotic machinery like members of the Bcl-2 family, Bid, caspase-12, etc.)
Which of the following proteins does not respond to calcium wave frequency?
troponin
PKC
CaMKII
NFAT
troponin
The action of which of the following proteins is absolutely critical for cells that need to reload their ER with calcium?
STIM
Ligand-controlled Ca2+ channels initiate calcium signaling in cells in response to signals such as hormones, neurotransmitters, cytokines and environmental stimuli. This is done by activating which enzyme in response to the signals?
PLA2
Which of the following active transporters is located in the membrane of the endoplasmic reticulum?
SERCA
Calcium channels in the ER of our cells are gated by a select number of ligands. Which of the following is not one of those ligands?
arachidonic acid
Which of the following calcium wave frequency detector proteins is activated by dephosphorylation within a particular range of calcium wave frequencies?
NFAT
How many signal proteins are missing between Ras and MAPK
At least one is missing: a MAP2K.
How many signal proteins are missing between MAPK and Ets/TCF?
None. The second protein is a substrate of the first.
How many signal proteins are missing between cPKC and AP-1?
At least three are missing: a MAP3K, MAP2K, and MAPK.
How many signal proteins are missing between cPKC and AP-1?
At least three are missing: a MAP3K, MAP2K, and MAPK.
In chapter 14, we looked at the CaM kinases. What is another name for CaM kinase III?
eEF2 kinase
Name the five families of small G-proteins
I. The Ras Superfamily of Small G-Proteins: Central
Switching Devices of Eukaryotic Cell Signal Processing
II. The Rho Family of Small G-Proteins: Controllers of Cell Shape and Cell Motility
III. The Arf & Rab Families of Small G-Proteins: Control of Vesicle Transport
IV. The Ran Family of Small G-Proteins: Control of Nuclear Transport
Briefly discuss each of the following for the Ras family of small G-proteins: localization, types of signaling it transduces, and activity regulation by GEFs and GAPs.
localization:Ras is a small G-protein which upon activation anchors to the inner side of the membrane to create signaling complexes
types of signaling it transduces: that transduce mitogenic, differentiation-controlling, and pro- apoptotic signal
activity regulation by GEFs and GAP:
– Ras is activated by Ras-guanine nucleotide exchange factors (Ras-GEFs) activated by all kinds of input signals.
– Ras is inactivated by Ras-GTPase-activating proteins (Ras-GAPs)
Briefly describe how arrestin-coupled signaling leads to Ras activation
- mSOS (mammalian Son Of Sevenless)
– is controlled by Tyr kinase-coupled receptors (via Grb2 or Shc) and by
G protein-coupled receptors (during arrestin-coupled signaling via Src)
-heptahelical receptor, phosphorylated arrestin - coupled can recruit the tyrosine kinase Src this lead the the recruitment of adaptor proteins to tyrosine phosphorylated residues on nearby proteins like GRB2 or Shc and then these can recruit mSOS which leads to activation of Ras
Briefly describe how Tyr kinase-coupled signaling leads to Ras activation
- mSOS (mammalian Son Of Sevenless)
– is controlled by Tyr kinase-coupled receptors (via Grb2 or Shc) and by
G protein-coupled receptors (during arrestin-coupled signaling via Src)
-Tyr kinase - coupled receptors lead the the recruitment of adaptor proteins to tyrosine phosphorylated residues on nearby proteins like GRB2 or Shc and then these can recruit mSOS which leads to activation of Ras
Briefly describe how Calcium signaling leads to Ras activation.
- GRF (Guanine nucleotide Releasing Factor) and GRP (Guanine
nucleotide Releasing Protein)
– are both activated by Ca2+ (GRP is also activated by DAG).
– are both restricted to specific tissues (esp. nerve cells and T-cells).
– GRF and mSOS activate Rho as well (both have a Rho-GEF domain as
well as a Ras-GEF domain
- ion channel coupled receptor voltage dependent ion channel, calcium entry through ion channel coupled receptors of voltage gated dependent ion channels can lead to activation of GRF or GRP which leads to activation of Ras
Briefly describe how GPCR signaling leads to Ras activation.
Heptahelical receptor G -protein coupled to various types of G proteins lead to activation of PLCe which leads to activation of Ras
List 4 major GEFs for Ras.
- mSOS (mammalian Son Of Sevenless)
– is controlled by Tyr kinase-coupled receptors (via Grb2 or Shc) and by G protein-coupled receptors (during arrestin-coupled signaling via Src). - GRF (Guanine nucleotide Releasing Factor) and 3. GRP (Guanine nucleotide Releasing Protein)
– are both activated by Ca2+ (GRP is also activated by DAG).
– are both restricted to specific tissues (esp. nerve cells and T-cells).
– GRF and mSOS activate Rho as well (both have a Rho-GEF domain as well as a Ras-GEF domain). - PLCε
– is a Ras-GEF and a Ras effector protein involved in negative feedback
List 3 major GAPs for Ras.
- P120 GAP (best known)
* Activated by Ras via negative feedback. - Neurofibromin
* Known for loss-of-function mutant that causes Recklinghausen neurofibromatiosis type 1 (hereditary cancer). - CAPRI (CAlcium-Promoted Ras Inactivator)
* Activated by calcium thanks to two C2 domains which enable Ca2+-dependent association with the membrane and thus Ca2+- dependent GAP activity.
List 3 major Ras effectors.
-Phospholipase Ce
-P120 Ras -GAP
-Phosphatidylinositol 3 - kinase
-RAF kinase (Ras-Activated Factors) and they recruit MEK with the MAP kinase 2 and assemble a MAP kinase to process mitogenic signals that are mediated through Ras
Describe how RAF is involved in the mitogenesis module (MAP kinase module).
- Activation by PKC, Src, RAF, and PAK.
- Inhibition by Phosphorylation by PKA/PKB
-Human cancer mutations in the RAF → MEK → ERK MAPK module.
Which of the following GAPs for Ras are known for a herditary form of cancer named Recklinghausen neurofibromatiosis type 1?
Neurofibromin
Which members of the Ras Superfamily are involved with signaling that changes the structure of the cytoskeleton?
the Rho family
Which of the following interaction domains recognizes phosphorylated Ser residues as interaction partners?
14-3-3 domains
How do heptahelical receptors activate Ras-GEFs?
through arrestin-coupled signaling
Which of the following signaling proteins is a target of activated Ras?
RAF
Which of the following Ras isoforms is not known for predisposing individuals to cancer (i.e. for being an oncogenic protein)?
R-Ras
Which of the proteins is an activator of RAF?
PKC
What type of signaling protein is Ras?
a small G-protein
Which of the following proteins is not a GEF for Ras?
GFP
Which family of the Ras Superfamily of proteins contains the fewest number of isoforms?
Ran family
in brief, describe the overall difference between Lamellipodia, filopodia, and actomyosin (stress) fibers. Name the small G-protein which controls the development of each of the three structures.
The Rho family (Rho, Rac, and Cdc42)
* Lamellipodia (and ruffled membranes) are large
broad extensions
– formed due to the activation of Rac
* Filopodia are long conical projections
– formed due to the activation of cdc42
* actomyosin (stress) fibers are
-formed due to activation of Rho
List the three general steps of vesicular transport, and identify which step is controlled by Rab, by Arf, and by motor proteins
- Vesicle budding (controlled by Arf)
- Vesicle transport (controlled by motor proteins)
- Vesicle fusion (controlled by Rab and calcium)
Arf and Rab both have
GEFs and GAPs
But only
* Rab proteins also have GDP-dissociation inhibitor (GDI) and GDI dissociation Factor (GDF) regulator proteins
– GDIs conceal Rab lipid anchors.
* In this way, GDIs prevent Rab-GEF action
– GDFs release GDI from Rab.
* GDFs therefore make subsequent Rab-GEF action possible.
In brief, discuss the critical role of coat proteins (COPs) in the process of vesicular transport
COPs can either concentrate transmembrane protein cargo directly or bind to other transmembrane proteins to concentrate soluble cargo molecules in the area
The Arf & Rab Families of Small G-Proteins
Control of Vesicle Transport
* Arf controls vesicle formation
* Rab and calcium control vesicle fusion
The Rho Family of Small G-Proteins
Controllers of Cell Shape and Cell Motility
assemble actin myosin stress fibers
involved in the assemble and disassemble of focal adhesions
The Ran Family of Small G-Proteins
Control of Nuclear Transport
in brief, discuss the role of tethering proteins and SNARE proteins in the process of vesicular fusion
tethering proteins = forbid the SNARE proteins from acting on vesicle or vesical target in the process of docking the vesicle
SNARE proteins = then calcium enters and allows SNARE to interact
Tethering proteins grab the vesicle to receive it, SNARE proteins help the membranes to fuse.
vSNARES are on vesicles,
tSNARES are on vesicle targets
guide em into place
Overview of Cell Motility Regulation by
Rho, Rac, and Cdc42
- Here is a cell at rest (anchored by focal adhesion sites)
- By actin polymerization, the resting cell develops lamelli-
and filopodia into the direction of signal. - Extensions of the cell become newly-anchored, and this
enables the rear of the cell to “contract.”
Explain the critical role of the protein synaptotagmin in vesicular fusion.
- synaptotagmin is the calcium-activated protein which enables membrane fusion
- membrane fusion doesn’t require input of energy here (energy is required to unlink the SNARE proteins).
Also it does it without using energy! Unlinking takes energy.
Action potentials gate calcium ion channels at synaptic terminals. With regard to synaptic transmission, briefly explain why it is a good design (upon calcium binding ) for SNARE proteins to not require energy input to perform vesicle fusion
once the Calcium enters this all happens as quickly as possible and this is what we want so that its effect can be as quick as possible and hydrolyzing ATP take time?
For the process of vesicular formation (budding), briefly describe the actions (and regulation by input signals) of Arf
- Input activates Arf-GEF
- Arf-GEF activates Arf
- Active Arf promotes vesicle formation
- Once vesicle buds and COPS leave, Arf-GAP inactivates Arf
In brief, describe the regulation of Rab by input signals, including the functions of both GDF and GDI.
GDI
= GDP-dissoci inhibitor
conceals Rab lipid anchors to prevent Rab-GEF action (keeping it inactive)
GDF
= GDP-dissoc factor
releases GDI from Rab, making subsequent Rab-GEF action possible and fun
In brief, discuss the process of vesicular fusion. Be sure to include the roles/actions of Rab, tethering proteins, synaptotagmin, SNARE proteins, GDI, GDF, and calcium.
- Rab starts out inactive, blocked by GDI.
- GDF comes along and frees Rab!
- Rab is now free to interact with a vesicle, activating synaptotagmin(?) and allowing for tethering of SNAREs to occur.
- Ca2+ comes in, dissolving the SNARE connection
- Rab is inactivated by its GAP
- Rab is repossessed by GDI
Very briefly, describe the control of nuclear transport by Ran. Identify the direction of protein translocation which requires the input of energy, and very briefly explain why.
When inactive, Ran-GDP comes into nucleus with NTF2.
Once in the nucleus, Ran runs into its Gef and is activated. While active, it can bind an importin, releasing its cargo. Or it can bind an exportin and accompany it outside.
Outside, it’s inactivated by its GAP
Stress fibers are composed primarily of which two proteins?
myosin actin
zone of contraction
(Rho induces the formation of
actomyosin stress fibers and enhances phosphorylation of myosin light chains to produce contraction)
towards the back of the structure by focal adhesions the fat part)
zone of transient disintegration of actin
(due to Rho inactivation) (after zone of actin polymerization, the mid front)
zone of actin polymerization
(induced by Rac and Cdc42) thowards the front of teh the structure (little and skinny)
Cdc42
Cdc stands for “cell division cycle
* Lee Hartwell identified
a number of proteins
which were required
for successful
completion of cell
division in yeast.
These were called cdc
proteins.
* This is how cdc42 was
first discovered
PKC-related kinases (PRKs)
Rho effector
– MAP4 kinases for p38 stress modules
– PRKs affect all parts of the cytoskeleton: microfilaments (actin
filaments), intermediate filaments, and microtubules
Rok
Rho effector
– Rescue contractile actomyosin system from blockade by myosin
light chains
– Promote formation of focal adhesion sites
– Promote formation of stress fibers
CRIKs
Rho effector
Involved in the contractile ring function of cytokinesis
Which of the following is a Rho effector which promotes the formation of both focal adhesion sites as well as stress fibers?
ROK
Which of the following small G-proteins uses the energy of GTP hydrolysis to move proteins out of the nucleus against their concentration gradient?
Ran
Which of the following cell structures are long, conical projections?
filopodia
To have a noticeable affect on downstream effectors,
the cytosolic [Ca2+] must be between 1-10mM.
Control of Ca2+ Wave Frequency
C. Control of Ca2+
Wave Frequency
* The arachidonic acid-
regulated Ca2+ channel
(ARCC) controls the
frequency of Ca2+ waves.
* Arachidonic acid is
formed by the enzyme
phospholipase A2 (PLA2)
in response to a
wide variety of
extracellular signals
