Calcium Signaling II Flashcards
what are the two Ca binding domains
C2 domain and EF-hand domain
what is the structure of EF-hand domain
“helix-loop-helix” protein motif, with the loop specialized to
bind a metal ion
what part of the EF hand domain bind to Ca ion?
helix-loop
what are the two classes of EF-hand domain
Ca buffers and Ca sensors
__________ types of EF-hand domain does not alter protein and _____________ change protein conformation
Ca buffers
Ca Sensors
Give 3 examples of Ca buffer and 5 examples of Ca sensors
Ca2+ buffers
• Calbindin D-28k (6 EF-hands)
• Parvalbumin (3 EF-hands)
• Calretinin (6 EF-hands)
Ca2+ sensors • Calmodulin (4 EF-hands) • Troponin C (4 EF-hands) • Calcineurin B (4 EF-hands) • Calpain (5 ER-hands) • S100 proteins (a large family)
where does Ca interact in the EF-hand domain ?
ca interacts with with O2 atom mainly of glutamate and aspartate side-chain
groups) in the loop
what does s100% mean
Soluble in 100% ammonium sulfate
what is calmodulin
A ubiquitous Ca2+-binding protein
what and how many Ca does one calmodulin bind to be activated
Each molecule of CaM cooperatively binds 4 Ca2+(4 EF-hand domains); all 4
sites must be occupied by Ca2+ for CaM function
does CaM have enzymatic activity?
CaM has no enzymatic activity, even when bound to 4 Ca2+ ions!!!
how does CaM function?
interaction with Ca2+/CaM à change
conformation of target protein (often
by relieving autoinhibition) à altered
target protein function
What is CaM-Kinase
A family of serine/threonine kinases that mediate many of the effects of Ca2+/CaM
what are the types of CaM-kinases
- Substrate-restricted: Responsible for phosphorylation of a specific substrate
- Multifunctional
Broad substrate specificity
CaM-kinase II (CaMKII) is the most prominent example
what are CaM-kinase II substrates
tyrosine hydroxylase
ion channels
CaM-kinase II
what factors increase Ca
Receptor complexes
that generate IP3
Membrane Ca2+
channels
what factors decrease Ca
Ca2+ pumps and
exchangers
Ca2+ buffers
what are membrane Ca channels
§ Located in the plasma membrane and in membranes delimiting organelles
Channel opening allows _____________movement of Ca2+ down______________
passive
electrochemical gradient.
what are the membrane potentials for plasma membrane and mitochondrial membrane
Plasma membrane is inside-negative (-70 mV)
Mitochondrial membrane is matrix-negative (-150 mV)
what is the Ca concentration for resting state for extracellular fluid, cytosol and ER?
EF: 2mM
cytosol: 100nM
ER: 300-800uM
what is the Ca concentration for stimulated state for extracellular fluid, cytosol and ER?
EF: 2mM
cytosol: 1000nM
ER: 100uM
How does Plasma membrane involved in Ca channels
Provide an avenue for sustained Ca2+ entry into the cell, allowing prolonged elevations
of Ca2+
Multiple types of Ca2+ channels
what are types of Ca channels
ligand-gated
voltage-gated
store-operated
Voltage gated ion channel has _______ pore forming ___________ subunit
4
pore-forming (a1) subunit
how many subunits does Voltage gated Ca Channel
4 accessory subunits
discuss structure of Voltage gated Ca channel a1 subunit
4 pore-forming domains (I-IV), each with 6 transmembrane
segments (S1-S6) connected by intracellular or extracellular loops
Nifedipine
specifically blocks Cav1 (L-type)
important antihypertensive agent
EGTA
avidly binds extracellular Ca2+ (calcium chelator)
blocks all responses dependent upon Ca2+ influx
regardless of the type of Ca2+ channel
a1 subunit pore loop confer to ______
Ca selectivity
a1 subunit Regulatory domain confer to _____________
- EF-hand (Ca2+)
- Ca2+/CaM
- PKC
How does ER/SR membrane involved in Ca channels
Allows rapid Ca2+ mobilization from intracellular “stores”
ER/SR Ca channels are ________________ gated
what are the two types
ligand
two types
IP3 receptor
Ryanodine receptor
what are IP3 receptors
A ubiquitous ligand-gated ion channel in the ER or SR
membrane
homo- or hetero-tetramer
IP3 regulatory domain has
Ca binding
ATP binding
phosphorylation site
how many domains does IP3 has
Regulatory domain
IP3 binding domain
Channel domain
Ca2+ release via IP3Rs is a
primary signal to activate
processes in ______________
cells.
give examples
non-excitable cells
• Fertilization (oocytes) • Proliferation (lymphocytes) • Metabolism (hepatocytes) • Secretion (salivary gland; pancreas)
IP3R activation usually triggers brief ______________
transients of Ca2+ release
_________ responses generally involve repeated spikes/oscillations.
longer duration
Oscillation frequency characteristically depends on the level of _____________
stimulus intensity.
what are the three IP3R isoforms
IP3R1, IP3R2, IP3R3
what is the point of the three IP3R isoforms
similar primary structures but different
physiological properties
allows cells to create IP3-dependent
[Ca2+]i signals with distinct spatial and
temporal characteristics to control many
cellular functions
_____________ in the cytosol are required for channel opening (primary regulators)
IP3 and Ca2+
Channel activation
↑[IP3]i à 4 IP3 molecules bind to the IP3R tetramer à conformation change
sensitizes Ca2+ binding site à Ca2+ binds à conformation change opens the pore à Ca2+ release into
cytosol.
what are the secondary regulation of IP3R activity
Factors that tend to ↑IP3R activity
cytoplasmic nucleotides (ATP)
PKA-mediated phosphorylation
reactive oxygen species tumor suppressors
Factors that tend to ↓IP3R activity
PKG-mediated phosphorylation Protooncogenes
Very high [Ca2+]i (> 300 nM), likely through Ca/CaM
Xestospongin C
membrane-permeable
sterically blocks Ca2+ pore
Heparin
membrane impermeable
competes for IP3 binding
Ryanodine receptor (RyR)
A ligand-gated ion channel in the ER/SR
membrane, structurally related to the IP3R
The functional channel of Ryanodine receptor (RyR)
is a
homotetramer
what are the isoforms Ryanodine receptor (RyR)
3 isoforms:
RyR1 (skeletal muscle)
RyR2 (cardiac muscle)
RyR3 (more ubiquitous)
what is calsequestrin
a Ca2+-binding
protein
________________ draw calsequestrin into a complex with the RyR
Junctin & triadin
___________ maintains proper spacing between SR
and plasma membran
Junctophilin
Ryanodine
a plant alkaloid
μM concentrations inhibit channel opening
Caffeine
mM concentrations activate the RyR
Ca2+ release via RyRs and influx via VGCCs are primary signals for activating processes in \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_
give example
- Contraction (muscle)
- Insulin secretion (b cells)
- Excitability (neurons)
______________ is the primary activator of RyRs
Cytosolic Ca2+
Source of the an increase in Ca is
Ca2+ channels in the
plasma membrane or in the ER/SR, even nearby
RyRs (resulting in a wave of RyR activation and
Ca2+ release from the ER/SR)
In excitable cells, plasma membrane depolarization sensed by _______________ opening of RyRs via _____________ or _____________ mechanisms
voltage-gated Ca2+ channels
(VGCCs)
direct or indirect mechanisms
direct mechanism of RyR is ____________ muscle and indirect mechanism is _______ muscle
skeletal
cardiac
direct mechanism of RyR is through ________________ of plasma membrane VGCCs with
RyRs in SR membrane
Physical coupling
Indirect mechanism of RyR is through _____________ positions RyRs in SR membrane close to
plasma membrane VGCCs
Junctophilin
what is the role of Ca2+ pumps and exchangers
to remove Ca2+ from the cytosol
Keep [Ca2+]i low under resting conditions
Restore low [Ca2+]i after a Ca2+-dependent stimulus
Either extrude Ca2+ from the cell or sequester it into organelles
what are the two types of Ca pumps
Plasma membrane Ca2+-ATPase (PMCA)
Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)
what are the two types of Ca exchange
Na+/Ca2+ exchanger (NCX)
Na+/Ca2+/K+ exchanger (NCKX)
PMCA Hydrolyzes __________ molecule to extrude _____________ Ca2+ from the cell against its electrochemical
gradient
1 ATP
1 Ca2
PMCA has ____________ Ca affinity but _______ capacity
High affinity low capacity
_____________ Provides the major route of Ca2+ extrusion at resting [Ca2+]i (~100 nM)
PMCA
PMCA is Regulated primarily by________
Ca2+/CaM
What is the similarity between PMCA and SERCA
A primary active transporter
High affinity & low capacity
SERCA Hydrolyzes ___________ molecule to pump _________ into the SR/ER against its
concentration gradient
1 ATP
2Ca2+
Thapsigargin
specific & potent
forms a dead-end complex
Cyclopiazonic acid
competitive at the ATP
binding site
how do we regulate SERCA
↑ [Ca2+]i à ↑ SERCA activity
Protein kinase A-dependent phosphorylation of
phospholamban (PLB) à ↑ SERCA activity
Inhibited by high [Ca2+] in the ER or SR lumen
Ca2+-binding proteins in the lumen keep [Ca2+]ER/SR in the ________range
uM
______________(in ER) and ___________(in SR) are high capacity, low affinity Ca2+ buffers
that can bind up to 50 Ca2+ per molecule
Calreticulin (in ER) and Calsequestrin (in SR)
_______________ is a secondary active transport
NCX
NCX relies on the inwardly-directed __________ to drive uphill extrusion
of _____________ from the cell.
3 Na+ electrochemical gradient t
2 Ca2+ from the cell
why is NCX important when cytosolic Ca rise
Low Ca2+ affinity, but very high capacity
NCX processes ______ cycles/sec Ca during influx
5000 cells/sec
how is NCX regulated?
by Ca
[Ca2+]ià ↑ NCX activity
NCX has ________ Ca binding domains?
2 Ca
Refilling the Ca2+ store is called _______________
Capacitative Ca2+ influx
↓ [Ca2+]SR/ER à activation of “X ________________ in the plasma
membrane à Ca2+ influx into a sub-plasmalemmal space
“Store-Operated Channels” (SOCs)
EGTA
Ca2+ chelators that bind free Ca2+, thus preventing Ca2+-
dependent effects
A23187, others
Ca2+ ionophores that let Ca2+ flow freely across cell
membranes
Thapsigargin, Cyclopiazonic Acid
SERCA inhibitors that prevent Ca2+ reuptake into ER/SR
Xestospongin
current best IP3R blocker
Nifedipine (a dihydropyridine), others
blocks Ca2+ influx through
voltage-gated Ca2+ channels (DHPRs)
Ryanodine (µM concentrations)
blocks RyRs
Caffeine (mM concentrations)
activates RyR
PMA (phorbol ester)
activates conventional & novel PKC isoforms
Calphostin C
inhibits PKC
