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

• 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 deprivation1. 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 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.

• 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 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+.

• 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

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

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.

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

TranscriptionFactors

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.

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

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)

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

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

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 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) .

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 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).

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

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

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 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.

NEXT SLIDE:Successive recruitment of signalling molecules

by 3 distinct spatially distinct Ca2+ pools may underline differential gene expression by synaptic activity.

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

Ways the Cell (neuron) uses to Partition Ca2+ reveals different buffering capacities or Ca2+ clearance

mechanisms in different areas of the cell