Calcium Signalling - Ca2+ as second

messenger: Essay Prep

Intracellular Ca has effects on several cellular processes… many hormones and other extracellular
stimuli (neurotransmitters) evoke increases in cytosolic Ca leading to several cellular responses:

- Exocytosis/Secretion: e.g. neurotransmitter release

- Muscle contraction.
- Control of metabolism.
- Regulation of Ca2+-dependent ion channels: membrane excitability.
- Control of cell-cycle/survival/differentiation.
- Reg. of gene expression + protein synthesis
- Reg. of Ca2+-dependent kinases and phosphatases
- “” proteases and nucleases
- Reg. of fertilisation

The concentration of cytosolic Ca2+ is controlled by its uptake and release from membrane enclosed
Ca2+ storage compartments, and inward/outward flux across the plasma membrane between
cytosol & intracellular space.

- [Ca2+] outside cell usually much higher than inside cell.

- [Ca2+] outside = (1-2mM)
- [Ca2+] cytosol resting = (100nM)
- [Ca2+] cytosol active = uM
- [Ca2+] inside mitochondria/ER = (10-100uM)

2 Main Ca2+ stores: Endoplasmic reticulum (Sarcoplasmic reticulum in muscle cells), and
mitochondria.

- Ca2+ crossing membrane via: Ca2+ channels (to cytosol), and Ca2+ ATPase (from cytosol to
ER/mit./extracellular space)
- Ca2+ ATPase = type of ATPase pumps, use energy from ATP hydrolysis to pump calcium either
out of the cell, or into calcium storage organelles (ER/SR/mit.)
There are 2 Mechanisms for increasing cytosolic Ca2+ concentration:

1. Influx of Ca2+ ions from EXTRACELLULAR environment via ion channels.

2. Release of [Ca2+] from INTRACELLULAR stores: ER/SR in most cells

1: Influx of Ca2+ ions from EXTRACELLULAR environment via ion channels:

a) Voltage-Operated Ca2+ Channels (VOCCs):
Open in response to membrane depolarisation (voltage/charge separation across
membrane). E.g. dihydropyridine receptor
a.k.a “Voltage-dependent Ca2+ channels”.

b) Receptor-Operated Ca2+ Channels (ROCCs):

Are directly coupled to receptors at the cell surface.
e.g. ionotropic glutamate receptors (can conduct calcium ions)

c) Second Messenger-Operated Ca2+ Channels (SMOCCs):

Open in response to increased cytosolic concentration of second messengers such as IP4
receptors, cGMP-gated ion channels.

d) Mechanically-Operated Ca2+ Channels (MOCCs):

Gated in response to mechanical stimulation of the cell, i.e from shear stress.

2: Release of [Ca2+] from INTRACELLULAR stores: ER/SR in most cells:

Two different families of channel protein involved in Ca2+ release:

a) IP3 Receptors (IP3Rs)

- Located on the ER
- Tetrameric complexes: 4 subunits of ~310kDa
- Expressed in all/most cells with greatest abundance in neuronal tissue/ brain (NT release)
- Activated by IP3 which binds this IP3 Receptor, causing it to open, allowing Ca2+ to flow from
the ER lumen, to the cytosol. Thus, cytosolic [Ca2+] increases.
- Targets Protein Kinase C… Ca2+ and DAG (diacylglycerol) will activate PKC.

b) Ryanodine Receptors (RyRs)

- Located on the SR of heart muscle, skeletal muscle, and the nervous system.
- Sensitive to the plant alkaloid ryanodine
- Tetrameric structure (~560kDa)
- At least 3 RyR isoforms in mammals.

Opening of IP3R and RyR channels depends on cytosolic Ca2+ concentration:

= Calcium Induced Calcium Release (CICR).
There is a threshold concentration of cytosolic calcium required for complete opening of the
channels. This acts as an amplification mechanism in such a way that it will exaggerate the effect
of IP3. Once cytosolic [Ca2+] has increased beyond a certain level, this inhibits further channel
opening. ( = negative feedback). Different IP3R and RyR isoforms have differing, distinct
dependencies on cytosolic Ca2+.
 (e.g. Type 1 RyR: max activity at 10uM Ca2+ / Type 2 RyR max at 1uM Ca2+)
IP3Rs require IP3 to be activated. Ca2+ acts as a co-activator (CICR: calcium-induced calcium
release).
RyRs can be directly activated by Ca2+ (CICR), or by physical/mechanical interaction with
dihydropyridine receptors (a type of voltage-dependent Ca2+ channel), which act as voltage
sensors in the PM and respond to membrane depolarisation.

Removal of Calcium from the cytosol:

1. Ca2+ ATPases:
- A type of pump found in the PM (plasma membrane), on the ER/SR, and mitochondria.
- Can bind ATP, then use energy from ATP hydrolysis to pump calcium out of the cell or into
calcium storage organelles (ER/SR/mit.)
2. Ca2+ Binding Proteins:
- e.g. Calmodulin, Calsequestrin, Calreticulin.
- => act to lower the concentration of cytosolic Ca2+.
3. Ca2+ may also bind to negatively charged phospholipids (lesser extent: uM range)

Measurement of intracellular calcium:

1. Calcium binding fluorescent dyes (fluo-3, fluo-2)… i.e. Fura-2 changes to yellow as [Ca2+]
increases (it fluoresces at different wavelength due to Ca2+ binding).
2. Calcium sensing proteins e.g. Aequorin (Ca2+-activated photoprotein) (isolated from
Aequorea Victoria, the crystal jellyfish).
3. Radiolabelled Ca2+: 45Ca2+ (used less nowadays)

Proteins act as effectors for Ca2+ signals directly or indirectly:

A) Directly: Ca2+ binding proteins whose structure/function changes on binding calcium:

Calmodulin, Calreticulin

B) Indirectly: effectors whose activity is modulated indirectly by calcium, via Ca2+ binding
proteins.

Calmodulin:
A protein that binds calcium and in turn regulates the structure and function of several other
proteins in response to the binding of calcium.
 EF-hand and C2 domains of calmodulin are capable of binding calcium.
- 17kDa
- Ubiquitously expressed: meaning present in most cells (in eukaryotes).
- 4 calcium binding sites: EF-hands, arranged in pairs at either end of the protein. Up to 4 Ca2+
ions can bind the 4 EF-hands (one each)
- On binding Ca2+, calmodulin undergoes a large conformational change from a dumbbell
structure to a more compact, spherical form, wrapped around a target protein.
- Calmodulin transmits the Ca2+ signal to another protein, the target protein. (one of several..)

Calmodulin’s Target Proteins:

1. Protein Kinases: Calmodulin-dependent protein kinase II, myosin light chain kinase.
2. Protein Phosphatases: Calcineurin
3. Enzymes: glycogen phosphorylase kinase
4. Transporters: Ca2+ ATPase
5. Ion channels: RyR, IP3R

Calmodulin-dependent protein kinase II (CaMKII):

= a serine/threonine kinase
- Key cellular effector of Calcium. Activity depends on Calmodulin and Ca2+.
- Ubiquitous tissue distribution.
- Multifunctional kinase
- Soluble cytosolic protein
- 5 isoforms…Mw 50-60 kDa… Active CaMKII: 600kDa
CaMKII is a multifunctional kinase which phosphorylates numerous effector proteins and hence
influences a number of cellular processes including:
1. Stimulation of NT release: phosphorylates synapsin 1
2. Has effects on membrane trafficking machinery: SNAREs, Rabs, ARF proteins.
3. Activation of neurotransmitter and hormone synthesis: CaMKII phosphorylates and
stimulates tyrosine and tryptophan hydroxylases, involved in synthesis of the
catecholamines (dopamine, epinephrine, norepinephrine) and serotonin (5-
hydroxytryptamine), respectively.
4. Stimulation of calcium sequestration in heart muscle: phosphorylation of phospholamban
in heart muscle, a protein which regulates the activity of Ca2+ ATPase pumps involved in the
uptake of this ion into the SR.
5. Regulation of transcription through phosphorylation of several transcription factors.
6. Glycogen synthase: suppression of glycogen synthesis
 Misregulation of CaMKII is linked to Alzheimer’s, Angelman’s syndrome, heart arrythmia.
 => Targets of CaMKII: RyRs, IP3R’s, PM Ca2+ ATPase… etc.

Calcineurin:
 - Ubiquitous serine/threonine protein kinase
- Activated by DAG and Ca2+ (Ca dependent)
- 6 isoenzyme forms in mammals
- Calmodulin acts as a cofactor for activity. (calcineurin is structurally similar to calmodulin)
- Substrate specific: Target = Protein Kinase A
- Thus, involved in cross-talk between cAMP-coupled receptors and Ca2+ second messenger
systems.
- 2 subunits:
o A subunit = catalytic (61kDa)
o B subunit = regulatory (19kDa)

Protein Kinase C (revision):

= serine/threonine protein kinase… activated by DAG and Ca2+
Mammals: at least 6 isoenzymes. It is ubiquitous but enriched in brain/neuronal tissue.
It is cytosolic, but can translocate to the plasma membrane (PM) in presence of DAG. Once
activated by DAG, PKC can phosphorylate membrane-associated proteins.
- Functions as a 70kDa monomer in cell proliferation, differentiation, angiogenesis (formation
of new blood vessels from existing ones), and apoptosis. (So incorrect function can promote
oncogenesis)
Contains a C-Terminal catalytic domain, and also an N-Terminal regulatory domain (this part binds
DAG, calcium.)
 PKC can be abnormally activated by phorbol esters, e.g. phorbol myristyl acetate (PMA) that
mimic DAG, but act as tumour promoters. Phorbol esters are able to cross the PM, then
mimic DAG (which regulates PKC activity), hence causing misregulation to take place… can
cause tumours and cancer.

Case Study: Malignant Hyperthermia (MH):

Malignant hyperthermia is an inherited disorder of skeletal muscle that affects humans, horses and
pigs. It usually arises from problems with the Ryanodine Receptors (RyRs). It results in a
hypermetabolic response to the inhalation of anaesthetics including halothane and succinylcholine. If
given anaesthetic during, for example, surgery: cytosolic [Ca2+] (conc.) increases dramatically, leading
to complications such as hyperthermia (increase in body temperature), tachycardia (elevated heart
rate), skeletal muscle rigidity (paralysis) and muscle spasms, and eventually organ failure and possibly
death. Increased O2 consumption and CO2 consumption are also observed. The onset of this
condition can occur in minutes or hours.
- MH gene: RyR1 gene on chromosome 19 (humans), however MH has also been linked to
other loci such as chromosomes 1, 3, 7, and 17.
- Many different mutations (up to 18) may occur, leading to uncontrolled release of Ca2+ from
sarcoplasmic reticulum in muscle cells (this occurs in 50-70% of cases)
- Diagnosis: Treatment of muscle biopsy with caffeine/halothane and measure of muscle
contraction.
- Treatment: Dantrolene (muscle relaxant).