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Transcript of Synapse-to Nucleus Calcium Signalling. Why Calcium? Na + and Cl - are sea water – Excluded to...
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Synapse-to Nucleus Calcium Signalling
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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
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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.
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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
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Calcium Concentrations
• [Ca2+]o / [Ca2+]i >104 – [Ca2+]o ~10-3 M
– [Ca2+]ER ~10-3 M
– [Ca2+]i <10-7 M at rest
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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
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Ca2+ - a versatile signal
• Synaptic vesicle release (ms)• Excitation-contraction coupling (ms)• Smooth muscle relaxation (ms-sec)• Excitation-transcription coupling (min-h)• Gene transcription (h)• Fertilization (h)
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Ways the Cell (neuron) uses to Partition Ca2+
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Fig 5.3, Purves et al., 2001
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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
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Ca2+ shut-off pathways
• Voltage-gated Ca2+ channels inactivate• IP3 rapidly dephosphorylated• Ca2+ rapidly pumped out
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Ca2+ as a 2nd Messenger
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Ca2+ as a 2nd Messenger (cont’d)
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Ca2+ as a 2nd Messenger (cont’d)
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Ca2+ as a 2nd Messenger (cont’d)
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Gq signaling pathways and Calcium
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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
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Calcium uptake and deprivation1. Na/Ca exchanger on plasma membrane, 2. Ca pump on ER membrane, 3. Ca binding molecules, 4. Ca pump on Mitochondia
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Ca2+ as a 2nd Messenger (cont’d)
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Ca2+ as a 2nd Messenger (cont’d)
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Ca2+ as a 2nd Messenger (cont’d)
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Ca2+ as a 2nd Messenger (cont’d)
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Ca2+ as a 2nd Messenger (cont’d)
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Synaptotagmin and neurotransmitter release
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Ca2+ as a 2nd Messenger (cont’d)Ca2+-Activated Signalling of Glu Receptor in the Postsynaptic Neuron
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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.
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Ca2+ in Synaptic Plasticity
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Targeting molecules for Calcium
Calcium binding protein Calmodulin
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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
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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
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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.
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The 3 Phases of Synaptic Plasticity1. 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.
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• 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.
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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 high-
frequency 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+.
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• 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.
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LTP in the Hippocampus (cont’d)
LTP-inducing stimuli Ca2+ IEGs
zif268c-fosc-jun
NMDA receptors
Secondary wave of txn, leading to the struct/funcchanges required formaintenance of LTP
e.g.,tissue plasminogenactivator; activity-regulatedcytoskeletal-assoc protein
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Synaptic plasticity in the Nervous System – Control of Gene Expression
• Pre-initiation complex.• Histone acetylase activity.• RNA pol.• Transcription factors.• Promoter, enhancers, silencers.• REST/NRSF binding NRSE.• Signal-inducible transcription factors.
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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
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TranscriptionFactors
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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 receptor-dependent 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 L-type 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.
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Pre-initiationcomplex
Core PromotorElement
RNA Pol II Basal txn
Recall: Activating txn factorsbind here, upstream, enhance the rate of PIC formation by contactingand recruiting the basal txn factorsvia adaptors or co-activatorsTxn factors can also acetylate histones,disrupting/modifying chromatin structure
Stimulus
A wide variety ofintracellular signaling
pathways can influencethe rate if txn initiation
by many txn factors
Phosphorylation
Reactions
There are several well-characterized DNA
elements that act as bindingsites for txn factors that are
regulated by Ca-activated signalingpathways
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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.
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Ca-Responsive DNA Regulatory Elements and their Transcription Factors
• Serum Response Element (SRE) – binding site for serum-response factor (SRF)
SRE
SRF
Ternary complexfactor (TCF)
5’
SAP-1 Elk-1
SAP-2
Ca signaling pathways – dependent synaptic activation
Rsk 2 ERK 1/2 Ras
TCF recognizes and binds SREonly with SRF bound
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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
Calcineurin
Ca2+
NFAT-P
ExtracellularIntracellular
CytoplasmicNuclear
NFATP
NFAT
GSK-3β ATP
Ca2+
Calcineurin(decr activity)
NFAT-P
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Ca2+ as a 2nd Messenger (cont’d)
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Terminology: CRE(cyclic AMP response element); CREB: CRE binding protein; CBP: CREB binding protein
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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.
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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.
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Mechanism of CREB ActivationCREB Activation Requires a Crucial Phosphorylation EventCa2+-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) .
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MAPK Cascade
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Parallel Activation of CaMK and MAPK pathways by Synaptic Activity
CREB – end-point of several signaling pathways
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The Role of CREB Binding Protein in CREB-Mediated Transcription
Phosphorylated CREB activates transcription by recruiting its coactivator, CREB binding protein, CBP
CREB has an inducible domain, the kinase-inducible 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).
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Ca-Responsive DNA Regulatory Elements and their Transcription Factors
• cAMP Response Element (CRE)
CRE
RNAPol II
(CBP has intrinsic histoneacetyl transferase (HAT) activity)
5’
CBP TFIIB
TATA BP
Ca signaling pathways – dependent synaptic activation
Rsk 2 ERK 1/2 Ras
HAT p/CAF HAT
SRC1
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Cytoplasmic Ca2+
Ras
ERK 1/2
Rsk 2
Nuclear Ca2+
CBP
CREB
CRE
Ser133
P
CaM Kinase IV
P – Ser301
Slow activation, long-lasting
Fast activation,Short -lasting
Physiological Importance of CREB:A Model for Nuclear Ca2+-Regulated Txn:
Regulation of CBP
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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).
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In contrast, ERK pathway, located just adjacent to the PM inside the cell and tethered to PM-bound 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.
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NEXT SLIDE:Successive recruitment of signalling molecules
by 3 distinct spatially distinct Ca2+ pools may underline differential gene expression by synaptic activity.
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Weak
Stronger
Strongest
MAP KinaseTCF/SRECREB (weak)
MAP Kinase TCF, CREB (weak)Calcineurin NFAT
MAP Kinase TCF, CREB (weak)
Calcineurin NFATCaM Kinase IV CREB (strong)
c-Jun
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Ways the Cell (neuron) uses to Partition Ca2+ reveals different buffering capacities or Ca2+ clearance
mechanisms in different areas of the cell