Download Why Ca2+ is chosen by nature for diverse regulations?

10 th PBL in calcium- and phospholipid signaling
May 3-14, 2010
Md. Shahidul Islam, M.D., Ph.D.
Associate Professor
Department of Clinical Sciences and Education,
Södersjukhuset
Karolinska Institutet
Forskningscentrum, Södersjukhuset
118 83 Stockholm, Sweden
[email protected]
Sydney Ringer (1883)
Survival of Fish
Muscle contraction
Fertilization of eggs
Development of tadpole
Locke and Overton (1894)
Impulse transmission
Why Ca2+ is chosen by nature for
diverse regulations?
Why Ca2+ is chosen by nature for
diverse regulations?
• Specific and tight binding to effector
proteins
• Suitable coordination chemistry
• Larger diameter and flexible coordination
number
• Can bind in to irregularly shaped protein
cavities
Evolutionary history of calcium
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Ancient sea water was free of calcium
Water contaminated by rocks
Protection against calcium toxicity evolved
Cells chose calcium as a signaling ion
Total
2+
Ca
and free
2+
Ca
• Total cell calcium: 2.5 mmol/Kg wet
weight. Higher total calcium in tumors
• 60-80% of it is bound to the extracellular
coat
• Large amount of calcium in secretory
granules and ER
• Free Ca2+ concentration is important for
regulatory purposes
What are the functions of
2+
Ca ?
• Structural.
– Bone
– Membrane fluidity and integrity (Ca2+
phospholipid)
– Protein structure and function
– Chromatin structure
What are the functions of
• Co-factor for enzymes
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–
–
–
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Protein Kinase C
Phospholipase A2
Prothrombin
Calpain
DNAse 1
• Electrical
– Ca2+ current during action potential
2+
Ca ?
Intracellular Regulation
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Second messenger
Muscle contraction
Secretion
Metabolism
Gene expression
2+
Ca
is a two-edged sword
• Excessive rise of [Ca2+]i
– Activation of
• Proteases
• Phosphatases
• Endonucleases
Excessive rise of [Ca2+]i
• Impaired mitochondrial function
• Perturbation of cytoskeletal organization
• Apoptosis, cell death
Cellular components that
2+
2+
determine Ca fluxes and Ca
homeostasis: Plasma membrane
• Ca2+ channels
– Voltage-gated
– Receptor operated
– TRPs
• Plasma membrane Ca2+ ATPase
• Na+/Ca2+ Exchanger
Na+/Ca2+ exchanger vs PM Ca2+
ATPase
• Na+/Ca2+ exchanger: Low affinity, high
capacity. Takes care of large Ca2+ loads
• PM Ca2+ ATPase. High affinity low
capacity
• Three Na+/Ca2+ exchanger genes.
• Gene products NCX1, NCX2, NCX3;
several splice variants
PM Ca2+ ATPase
• P-type family of transport ATPases
• Four genes PMCA1, PMCA2, PMCA3,
PMCA4
• Four splice variants
• Calmodulin-dependent
Cellular components that
2+
2+
determine Ca fluxes and Ca
homeostasis: ER 2+
• Serco-Endoplasmic Reticulum Ca ATPase
(SERCA)
• IP3 Recepotors
• Ryanodine receptors
• TRIC (trimeric intracellular cation) channel
(Yazawa M, Nature 2007)
• Luminal Ca2+-binding proteins
Cellular components that
2+
2+
determine Ca fluxes and Ca
homeostasis:
• Mitochondria
– Ca2+ Uniporter
– Na+/Ca2+ exchanger
• Cytoplasm
– Calmodulins
– Parvalbumin
– Other Ca2+-binding proteins
Calcium Channels
• Voltage Dependent
– Slow, fast
• Intracellular Ca2+ channel
Receptor-operated Ca2+ channels
2+
Ca Channels
• Receptor-operated Ca2+ channels
– Nicotinic cholinergic receptor
– NMDA receptor ion channel
– Purinergic receptor P2X
Voltage-gated Ca2+ channels
Gene Superfamily of VoltageGated Ion Channels
• Voltage-gated Na+ channels
• Voltage-gated K+ channels
• Voltage-gated Ca2+ channels
Distinct classes of
2+
Ca
curents
• L-type
–
–
–
–
High activation voltage
Large conductance
Long lasting
Blocked by dihydropyridine, phenylalkylamine,
benzothiazepine
Other high-voltage-activated
Ca2+ channels
• N-type
– Neuronal
• P/Q-type
• R-type
Not blocked by DHPs, blocked by
polypeptide toxins
Low-voltage activated Ca2+
current
• T-type
– Tiny conductance
– Transient current
–
Nomenclature
• Ion conducted
• Main regularor
• Alpha-1 subunit gene family
Cav1.1
EA Ertel Neuron 25:533, 2000.
Molecules and curents
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Cav1.1
Cav1.2
Cav1.3
Cav1.4
Cav2.1
Cav2.2
Cav2.3
Cav3.1
Cav3.2
Cav3.3
• L-type
•
•
•
•
P/Q-type
N-type
R-type
T-type
Blockers
•
•
•
•
•
•
•
•
•
•
Cav1.1
Cav1.2
Cav1.3
Cav1.4
Cav2.1
Cav2.2
Cav2.3
Cav3.1
Cav3.2
Cav3.3
•
•
•
•

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DHP
DHP
DHP
Not known
w-Agatoxin IVA
Conotoxin
None
None
None
None
Fluorescence
Jablonski Diagram
Fluorescence Resonance Energy Transfer
What is FRET
It is a distance-dependent interaction
between the electronic excited states
of two dye molecules in which
excitation is transferred from a donor
molecule to an acceptor molecule
without emission of a photon.
Conditions for FRET
• Proximity (10-100Å)
• Absorption spectrum of acceptor overlaps
emission spectrum of donor
• Donor and acceptor transition dipole
orientation is parallel
Excitation and emission spectra of different ”GFP”
Cameleons
Miyawaki A. 1997, Nature 388:6645