Download Ca Signaling11

Synapse-to Nucleus Calcium
Signalling
Why Calcium?
• Na+ and Cl- are sea water
– Excluded to maintain low osmotic pressure
– [K+]i kept high for electrical neutrality
• [Ca2+]i maintained very low
– Prevents precipitation of organic anions
• Mg2+ helps solubilize organic anions
Calcium has been ‘selected’ by evolution as an
intracellular messenger in preference to other
monoatomic ions in the cell
• Divalency - stronger protein binding than
monovalent ions.
• More flexible that smaller divalent Mg2+ ions  more
effective coordinate with protein-binding sites.
• Energetically favourable to use Ca2+ as 2nd messenger
(large [Ca2+] gradient) (10-7 vs. 10-3 M) – rel small amt
needed to enter cell to incr signaling  relatively
little energy needed to pump it back out of the cell.
• Higher [Ca2+] would ppt with PO43- ions  lethal.
How cells keep [Ca]i low
• All eukaryotic cells have PM Ca2+-ATPase
– Excitable cells also have Na+/Ca2+ exchanger (NCX)
• ER Ca2+-ATPase (against a high grad)
• Mitochondrial high capacity (low affinity) pump
– When [Ca]i very high (dangerous) levels (>10-5 M)
– Inner mitochondrial membrane
– Uses the electrochemical gradient generated during
electron-transfer of oxidative-phosphorylation
Calcium Concentrations
• [Ca2+]o / [Ca2+]i >104
– [Ca2+]o ~10-3 M
– [Ca2+]ER ~10-3 M
– [Ca2+]i <10-7 M at rest
Ca2+ - a versatile signal
Target Tissue
Signaling Molecule
Major Responses
Liver
Vasopressin
Glycogen breakdown
Pancreas
ACh
Amylase secretion
Smooth muscle
ACh
Contraction
Mast cells
Antigen
Histamine secretion
Blood platelets
Thrombin
aggregation
Ca2+ - a versatile signal
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Synaptic vesicle release (ms)
Excitation-contraction coupling (ms)
Smooth muscle relaxation (ms-sec)
Excitation-transcription coupling (min-h)
Gene transcription (h)
Fertilization (h)
Ways the Cell (neuron) uses to Partition Ca2+
Fig 5.3, Purves et
al., 2001
How cells ↑ [Ca]i
• Voltage-gated Ca2+ Channels
– Membrane potential drives Ca2+ down its chemical
gradient
– Different channels in different cells
• Different properties for different purposes
Ca2+ shut-off pathways
• Voltage-gated Ca2+ channels inactivate
• IP3 rapidly dephosphorylated
• Ca2+ rapidly pumped out
Ca2+ as a 2nd Messenger
Ca2+ as a 2nd Messenger (cont’d)
Ca2+ as a 2nd Messenger (cont’d)
Ca2+ as a 2nd Messenger (cont’d)
Gq signaling pathways and Calcium
Fertilization of an egg by a sperm triggering an increase
in cytosolic Ca2+
3 major types of Ca2+ channels:
1. Voltage dependent Ca2+ channels on plasma
membrane
2. IP3-gated Ca2+ release channels on ER membrane
3. Ryanodine receptor on ER membrane
Calcium uptake and deprivation
1. Na/Ca exchanger on plasma membrane, 2. Ca pump on ER membrane, 3. Ca binding
molecules, 4. Ca pump on Mitochondia
Ca2+ as a 2nd Messenger (cont’d)
Ca2+ as a 2nd Messenger (cont’d)
Ca2+ as a 2nd Messenger (cont’d)
Ca2+ as a 2nd Messenger (cont’d)
Ca2+ as a 2nd Messenger (cont’d)
Synaptotagmin and neurotransmitter
release
Ca2+ as a 2nd Messenger (cont’d)
Ca2+-Activated Signalling of Glu Receptor in the Postsynaptic Neuron
Synaptic Plasticity in the Nervous System
• Activity-dependent plasticity is mediated by
electrochemical activity of the synapse.
• Activity-dependent plasticity is a change in
neural connections and synaptic strength that
are the hallmarks of learning and memory.
Ca2+ in Synaptic Plasticity
Targeting molecules for Calcium
Calcium binding protein Calmodulin
Ca2+/calmodulin dependent protein kinase (CaM-kinase)
Memory function: 1. calmodulin dissociate after 10 sec of low calcium level; 2. remain
active after calmodulin dissociation
Ca2+/calmodulin dependent protein kinase (CaM-kinase)
Frequency decoder of Calcium oscillation
High frequence, CaM-kinase does not return to basal level before the second wave of
activation starts
Synaptic plasticity in the Nervous
System
• Nervous system adapts to environmental
changes.
• Such stimulation  activity-dependent
plasticity or alterations in the number of
synapses and/or in the strength of existing
synapses.
The 3 Phases of Synaptic Plasticity
1. Early (sec-min) after electrical activity:
changes in neural connections via
modifications (phosphorylation) of existing
proteins (ion channels) or delivery of proteins
to postsynaptic membrane.
2. Intermediate (min-hr): synthesis of new
proteins by existing levels of genes.
3. Late (days - longer ): changes in gene
expression: txn and tln => long-lasting
changes.
All of these phases triggered by Ca2+ influx.
• Hippocampus – site of much plasticity and LTP
studies.
• Patients with hipp lesions  anterograde and
retrograde amnesia.
• LTP – induced into postsynaptic neuron by
high-freq. train of electrical impulses into
presynaptic afferents.
- model for learning and memory.
- activity-dependent incr in synaptic efficacy
that can last days-weeks in vivo.
LTP in the Hippocampus.
• A model for plasticity - learning and memory.
• Is an activity-dependent increase in synaptic efficiency
that can last for days – weeks.
• Induced in the postsynaptic neuron by repeated highfrequency stimulation of presynaptic afferents.
• Characterized by an early, protein synthesis
independent phase and late phases, which can be
blocked by protein synthesis inhibitors.
• During the longest phase, there is a critical period of
transcription after the LTP-inducing stimuli has been
applied.
• Induction of LTP is critically dependent on an elevation
of postsynaptic Ca2+.
• IEGs – genes whose txn can be triggered
without de novo protein synthesis (e.g., txn
factors)  2ary wave of txn for other proteins
required for LTP.
LTP in the Hippocampus (cont’d)
LTP-inducing stimuli
Ca2+
IEGs
e.g.,tissue plasminogen
activator; activity-regulated
cytoskeletal-assoc protein
zif268
c-fos
c-jun
Secondary wave of txn,
leading to the struct/func
changes required for
maintenance of LTP
Synaptic plasticity in the Nervous System –
Control of Gene Expression
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Pre-initiation complex.
Histone acetylase activity.
RNA pol.
Transcription factors.
Promoter, enhancers, silencers.
REST/NRSF binding  NRSE.
Signal-inducible transcription factors.
Control of Gene Expression
• Control of gene expression can occur at any
stage in the process.
• By far, the most common point of regulation is
at transcription initiation (RNA Pol II).
• Transcription factors
Transcription
Factors
Synaptic plasticity in the Nervous System – Ca2+Responsive DNA Regulatory Elements and their
Txn Factors
• Cyclic-AMP response element (CRE).
- Incr of synaptic activity  synaptic NMDA receptordependent transient Ca2+ currents and long-lasting LTP in
hipp (CA1 region) activate (phosphorylate) CREB txn factor
 CaMKII and MAPK (ERK) signalling pathways
• Serum response element (SRE).
- Induce expression of c-fos promotor  activation of Ltype Ca2+ channels.
• Nuclear Factor of Activated T cells (NFAT) response
element.
- NFAT activity regulated by Ca2+-activated calcineurin.
- Calcineurin dephos cyto NFAT  transport into nucleus.
- W/o Ca2+-activated calcineurin activity, NFAT becomes
rephos by GSK and re-exported to cytoplasm.
Recall: Activating txn factors
bind here, upstream, enhance the
rate of PIC formation by contacting
and recruiting the basal txn factors
via adaptors or co-activators
Txn factors can also acetylate histones,
disrupting/modifying chromatin structure
Pre-initiation
complex
RNA
Pol II
Core Promotor
Element
There are several wellcharacterized DNA
elements that act as binding
sites for txn factors that are
regulated by Ca-activated signaling
pathways
Basal txn
A wide variety of
intracellular signaling
pathways can influence
the rate if txn initiation
by many txn factors
Stimulus
Ca-Responsive DNA Regulatory Elements
and their Transcription Factors
• cAMP-response element (CRE) – bound by
CRE binding protein (CREB).
- Ca activation of CREB is mediated by CaM
KII and Ras-ERK1/2 signaling pathways.
Ca-Responsive DNA Regulatory Elements
and their Transcription Factors
• Serum Response Element (SRE) – binding site
for serum-response factor (SRF)
Ternary complex
factor (TCF)
Elk-1
SAP-1
SAP-2
5’
TCF recognizes and binds SRE
only with SRF bound
SRF
SRE
Rsk 2
ERK 1/2
Ras
Ca signaling pathways –
dependent synaptic activation
Ca2+ as a 2nd Messenger (cont’d)
Ca-Responsive DNA Regulatory Elements
and their Transcription Factors
• Nuclear Factor of Activated T cells (NFAT)
Response Element
Extracellular
Intracellular
Calcineurin
Ca2+
Ca2+
Calcineurin
(decr activity)
P
NFAT
GSK-3β
NFAT-P
NFAT
Cytoplasmic
Nuclear
NFAT-P
ATP
Ca2+ as a 2nd Messenger (cont’d)
Terminology: CRE(cyclic AMP response element);
CREB: CRE binding protein; CBP: CREB binding protein
Physiological Importance of CREB
• LTM
• Information storage (Aplysia).
• Confirmed by anti-sense oligonucleotides 
blocked LTM, but not STM formation.
• Drug addiction.
• Circadian rhythmicity.
• Neuronal survival mediated by neurotrophins
(BDNF).
• Changes in synaptic strength and efficacy.
• Besides BDNF, CREB-dependent pro-survival
genes include nNOS, bcl-2, mcl-1 and VIP.
Mechanism of CREB Activation
CREB Activation Requires a Crucial
Phosphorylation Event
• CREB binds CRE.
• Ser 133.
• Depol  incr [Ca2+]cyto  P-ser133 on CREB.
• A133S  abolished CREB-mediated gene
expression of many IEGs.
•  CREB is a Ca2+-sensitive txn factor.
Mechanism of CREB Activation
CREB Activation Requires a Crucial Phosphorylation Event
Ca2+-dependent signaling molecules capable of phoshorylating
CREB on ser133:
CaM kinases and their role in Ca2+-activated, CRE-dependent gene expression:
CaMKII, CaMKIV, and CaMKI.
-Play roles in secretion, gene expression, LTP, cell cycle regulation, tln
control.
- Activate c-fos expression:
- experiments with KN-62  decr L-type Ca2+ channel-activated c-fos
expression.
- experiments with calmodulin antagonist, calmidazolium.
- CaMKIV – the prime member for CREB-mediate gene expression by
nuclear Ca2+ signals.
- experiments with anti-sense oligonucleotide disruption of CaMKIV
expression  abolished Ca2+-acticated CREB phosphorylation in hipp
neurons.
- critical for LT plasticity.
- Knock-out mice for CaMKIV  cognition/memory deficits related to
noxious shock stimulus and related to spatial learning (hippocampus).
- both inhibition of either CREB or CaMKIV function  blocked
cerebellar LTD (late phase) .
MAPK Cascade
Parallel Activation of CaMK and MAPK pathways by Synaptic
Activity
CREB – end-point of several signaling pathways
The Role of CREB Binding Protein in CREBMediated Transcription
Phosphorylated CREB activates transcription by
recruiting its coactivator, CREB binding
protein, CBP
CREB has an inducible domain, the kinaseinducible domain (KID).
CBP and p300 (closely related protein) function
as coactivators for many signal-dependent txn
factors (e.g., c-Jun, INF-α, STAT2, ELK-1, p53,
and many nuclear hormone receptors).
Ca-Responsive DNA Regulatory Elements
and their Transcription Factors
• cAMP Response Element (CRE)
HAT
p/CAF
HAT
SRC1
TFIIB
CBP
TATA BP
5’
(CBP has intrinsic histone
acetyl transferase (HAT) activity)
RNA
Pol II
CRE
Rsk 2
ERK 1/2
Ras
Ca signaling pathways –
dependent synaptic activation
Physiological Importance of CREB:
A Model for Nuclear Ca2+-Regulated Txn:
Regulation of CBP
Nuclear Ca2+
Cytoplasmic Ca2+
Ras
CaM Kinase IV
Fast activation,
Short -lasting
ERK 1/2
Rsk 2
Slow activation,
long-lasting
P
P – Ser301
CBP
Ser133
CREB
CRE
Decoding the Ca2+ Signal
The neuron regulates Ca2+ signals on many levels:
1. Amplitude.
2. Temporal properties (oscillatory frequency).
3. Spatial properties.
4. Site of entry.
The cellular requirements for Ca2+ will determine
the extent of these 4 properties:
Subcellular localization of Ca2+ and its many
effectors involves compartmentalization (i.e.,
nuclear vs. cytosolic proteins, e.g., CaMKII is
nuclear).
In contrast, ERK pathway, located just adjacent
to the PM inside the cell and tethered to PMbound nt receptors, requires only a tiny amt of
Ca2+ to get things started.
But what about cytosolic proteins that are not
tethered to a membrane?
- Calcineurin: this membranous Ca2+ not
enough; requires global incr in [Ca2+]cyto to
trigger NFAT nuclear translocation.
NEXT SLIDE:
Successive recruitment of signalling molecules
by 3 distinct spatially distinct Ca2+ pools may
underline differential gene expression by
synaptic activity.
Weak
MAP Kinase
MAP Kinase
Stronger
Strongest
Calcineurin
MAP Kinase
Calcineurin
CaM Kinase IV
TCF/SRE
CREB (weak)
TCF, CREB (weak)
NFAT
TCF, CREB (weak)
NFAT
CREB (strong)
c-Jun
Ways the Cell (neuron) uses to Partition Ca2+ reveals
different buffering capacities or Ca2+ clearance
mechanisms in different areas of the cell