Transcript Calcium Channels - FSU Program in Neuroscience

Voltage-Gated
Calcium Channels
“Excitable cells translate their electricity into action by Ca2+ fluxes
modulated by Voltage-Sensitive, Ca2+ permeable channels.”
Brad Groveman
Membrane Biophysics
Brief History
• Discovered accidentally by Paul Fatt and
Bernard Katz in neuromuscular
transmissions in crab legs
• Carbone and Lux termed LVA and HVA in
in mammalian sensory neurons
• Kurt Beam identified voltage-gated
calcium channels as the voltage sensors
in skeletal muscle
The Ion… Ca2+
Found in all Excitable Cells
Shapes the Regenerative A.P.
[Ca2+]i Three Best Studied Roles:
1. Contraction of Muscle
2. Secretion
3. Gating
Structure/Function
• Positively charged lysine and arginine residues
in the S4 transmembrane segment thought to
form the voltage sensor
• Key negatively charged glutamate residues in
each pore loop contributes to selectivity
• Inactivation mechanism still unclear
– [Ca2+]i elevation
– Mode switching
Classes of VGCC
http://calcium.ion.ucl.ac.uk/a1-nomenclature.html
Classes, Location, Blockers
http://en.wikipedia.org/wiki/Voltage_gated_calcium_channel
Example Currents
A. C. Dolphin 2006
Alpha-1 Subunit Structure
http://calcium.ion.ucl.ac.uk/calcium-channels.html
Ribbon Structure of Alpha-1
http://calcium.ion.ucl.ac.uk/calcium-channels.html
Accessory Subunits
http://calcium.ion.ucl.ac.uk/calcium-channels.html
http://www.sigmaaldrich.com
Accessory Subunits
• β - Contains Guanylate Kinase domain and
SH3 domain
• GK domain binds α1I-II intracellular loop
– Stabilizes α1 and helps to traffic to membrane
– Allows more current (higher amplitudes) for
smaller depolarizations (HVA)
• Shifts towards negative membrane potentials
Accessory Subunits
• α2δ- co-expressed, linked by disulfide bond.
– α2 extracellularly glycosylated
– δ has a single transmembrane region
– Co-expression enhances α1 expression
• causes increased current amplitude, faster kinetics, and
a hyperpolarizing shift in the voltage dependence of
inactivation
• Associates with all HVA calcium channels
– Binding site for some anticonvulsant drugs
Accessory Subunits
• γ- 4 transmembrane
helices
– Found in skeletal
muscles
– May have an
inhibitory effect on
calcium currents
– Interact with AMPA
and Glutamate
receptors
Modulation
• Upregulation of cardiac L-type channels by
cyclic AMP-dependent protein kinase
• Inhibitory modulation occurs via the activation of
heterotrimeric G-proteins by G-protein-coupled
receptors (GPCRs)
• Calcium and Ca2+/CaM
• Intracellular effector proteins (RyR, SNARE)
Synaptic Transmission
• P/Q-types channels mainly responsible for
transmitter release at central terminals
• N-type channels prevalent in peripheral nerve
terminals, responsible for synaptic transmission
in autonomic and sensory terminals
• L-type channels of the CaV1.3 and 1.4 class
support synaptic transmission at specialized
terminals
– Continuous transmitter release in the retina and
auditory hair cells with low depolarizations.
Pathologies
• Neuropathic pain
• Epilepsy
• Congestive heart failure
• Familial hemiplegic migraine
• Several cerebellar ataxias
Important Domains
EF Hand Motif
•Alloserically couples
Ca2+ sensing apparatus
with inactivation gate
Pre-IQ / IQ
•Bind Calmudulin
(Primary Ca2+ sensor)
Peptide A
•Unknown Importance
ICDI
•Inactivator of Calcium
Dependent Inactivation
•CaM1234
•CaM cant bind Ca2+
Inactivation
• Typical fast channel
inactivation conferred by
voltage, but enhanced by
Ca2+ feedback mechanism
– Cav1.2
• Photoreceptors generate
graded electrical response
 requires sustained Ca2+
influx
– Seem to be devoid of CDI
– Cav1.4
• major channel mediating
Ca2+ influx in
photoreceptors
Cav1.4 shows no CDI
Ba2+ blocks CDI,
focusing inactivation on
voltage dependence
f = Difference in
normalized IBa and ICa
remaining after 300ms
of depolarization
Cav1.2 shows typical
“U” f curve
Cav1.4 shows no
difference
Black – IBa
Red – ICa
CaM binding in C-Terminal
Proximal
CaM Binding
In presence of Ca2+
Distal
No CaM Binding
*Co-IP
CaM1234 binding shows CaM binds Cav1.2 and 1.4 at basal Ca2+ conditions
 Loss of Calcium Sensor CaM NOT responsible for CDI insensitivity
CDI masked by inhibitory domain?
C1884STOP
*Modified
Removal of last 100aa of Cav1.4 restored CDI but not Ba2+ inactivation
Restored typical
“U” shape voltage
dependence and
fmax nearly identical
to Cav1.2
ICDI Domain
C1884Stop co-expressed
with CaM1234 Mutant to
demonstrate that CDI is
CaM dependent
C1884Stop co-expressed
with peptide of last 100aa
to demonstrate presence of
an inhibitory domain (ICDI)
which is sufficient to block
CDI effects
* Red Box shows
importance of sequence
between aa1930 and
aa1953 in CDI inhibition
Does ICDI interact with the Ca2+
sensing apparatus of Cav1.4?
• Co-IP C-terminal fragments for interaction
with ICDI
– C-terminal fragments myc-tagged (IP)
– IDCI Flag Tagged (IB)
• ICDI IP with proximal C-terminal
• IP abolished with deletion of EF hand motif
• No interaction seen with peptides A or C
from distal C-terminal
EF Hand target sequence for ICDI
• GST-tagged IP of EF hand or EF hand
with N-terminal Pre-IQ sequence
• Both bound ICDI  Target sequence
• EF Hand motif and ICDI Domain both
helical
– Form paired helix which uncouples Ca2+
sensing apparatus from inactivation gate
Is inactivation of Cav1.2 rendered
insensitive by Cav1.4 CT?
Generated Cav1.2/1.4 Chimeras
Cav1.2/1.4 Chimeras demonstrate CDI inhibition
Inhibit CDI
Complete C-terminal replacement
C + ICDI replacement
A + ICDI replacement
Do No Block CDI
Addition of ICDI
Fusion of ICDI to IQ
Replacement of A
•Peptide A and ICDI sufficient to abolish
CDI
•Peptide A does not bind ICDI  Indirect
Proposed Model
Gate opens Ca2+ interacts with
CaM pre-bound to IQ motif
causing conformational change in
EF hand promoting interaction
with channel conferring CDI
ICDI constitutively binds EF hand
impairing Ca2+/CaM induced
conformation change.
 Inactivation strictly voltagedependent with kinetics intrinsic to
channel core
Pathophysiological Relevance
• Loss of function mutation in Cav1.4 cause
Congenital Stationary Night Blindness
• Two mutations discovered in CSNB2
patients  truncations in distal C-termial
• Frameshift mutation identified in first 10aa
of ICDI
– All cause loss of ICDI function, allowing for
CDI of photoreceptor Ca2+ channels
Amyloid Precursor Protein
Chronic Hypoxia
• Chronic Obstructive Pulmonary Disease
• Arrhythmia
• Stroke
Reduction of Oxygen in brain
Previous Studies
• APP expression increased following
cerebral hypoxia or ischemia
• Prolonged hypoxia enhances Ca2+ influx in
PC12 cells apparently dependent on Aβ
enhanced expression
– Suggested Aβ composed Ca2+ pores as well
as up-regulation of L-Type Ca2+ channels
• THIS CANNOT BE EXTRAPOLATED TO
CENTRAL NEURONS!!!
Mean Current Density vs Voltage Relationships
Currents based on VGCC
Current density in chronic
hypoxic cells enhanced
from normoxic conditions
•Significantly at -10mV
and 0mV
•Inset shows no change in
kinetics
Cd2+ non-selectively blocks
VGCC
•Abolished whole-cell
Ca2+ current in both
normoxic and hypoxic
 Augmentation of current do
to up-regulation of
endogenous VGCC
Mean Current Density vs Voltage Relationships
L-Type VGCC Responsible
No difference seen in
current under normoxic
or hypoxic conditions in
presence of L-Type
Channel blocker
Nimodipine
Exaggerated difference
seen in current under
hypoxic conditions in
presence of N-Type
Channel blocker ω-CgTx
What does this have to do with APP?
• Current augmentation caused by upregulation in L-Type Ca2+ Channels
• Immunohistochemical studies show
increase in Aβ in hypoxic cells
– This increase is abolished to normoxic
conditions in presence of either γ or βSecretase inhibitors
To beat a dead horse…
• Hypoxia up-regulates L-Type Ca2+
Channels
• Hypoxia increases Aβ production
But are they related?
Blocking Aβ production by γ-Secretase inhibitor
abolishes hypoxia effect
Normoxic
γ-Secretase inhibitor shows no
effect on Ca2+ currents under
normoxic conditions
Hypoxic
γ-Secretase inhibitor fully
prevents Ca2+ currents
augmentation by hypoxic
conditions
In presence of N-Type channel blockers
Blocking Aβ production by β -Secretase
inhibitor abolishes hypoxia effect
Normoxic
β -Secretase inhibitor shows
no effect on Ca2+ currents
under normoxic conditions
Hypoxic
β -Secretase inhibitor fully
prevents Ca2+ currents
augmentation by hypoxic
conditions
In presence of N-Type channel blockers
Conclusions
• Hypoxia increases formation of Aβ in
primary culture neurons
• Functional expression of L-Type Channels
increased
– Dependent on Aβ
• Aβ do not form Ca2+ permeable pores
Status Epilepticus
• Single episode can be evoked using
chemical or electrical stimulation to mesial
temporal lobe. <Pilocarpine>
• Latent period of up to several weeks after
first episode of normal behavior
– Electrophysical changes including acquisition
of low-threshold bursting behavior and high
frequency clusters of 3-5 spikes
Bursting
• Somatic bursting generated when spike
after-depolarization (ADP) is large enough
to attain spike threshold and trigger
additional spikes
• INaP currents drive bursting in ordinary cells
• Intrinsic bursting in SE-experienced cells
suppressed by Ni2+  Ca2+ driven
• T-type Ca2+ channels (ICaT) implicated
Purpose
• Contribution of ICaT vs ICaR
• Subcellular localization of ICaT
• Contribution of INaP
Bursting in early epileptogenesis driven by
Ni2+ Sensitive Ca2+ Current
“Jitters” seen in later spikes
indicating a subthreshold
ψR
Small subthreshold hump
Ni2+ suppresses bursting
into single spike
T-Type Ca2+ channels are
blocked by Ni2+
ICaT vs ICaR
• Ni2+ blocks both ICaT and ICaR
• Previous studies show ICaT up-regulated
after SE, but not ICaR
• Cav3.2 T-type Ca2+ channel is 20-fold more
sensitive to Ni2+ than other 2 splice variants
– CaV3.2 provide critical depolarization for bursting
Amiloride suppresses bursting
Blocks ICaT preferentially
over HVA ICaR
Also bock Na2+ exchangers
Induces bursting by
blocking KCNQ K+
Channels
Bursting in normal cell not
suppressed by Amiloride
 non-specific channel
block not responsible for
burst suppression
SNX-482 does not suppress bursting
Blocks ICaR
SNX-482 did not
suppress bursting,
however subsequent
treatment with Ni2+ did
ICaR not critical, but
is possibly auxiliary
to bursting
Ni2+ and Amiloride
block bursting in SE
cells, but SNX-482
does not
 ICaT Critical Bursting
INaP Contribution
• PDB and Riluzole block INaP completely in
pyramidal neurons without reducing
transient Na+ currents
• Subthreshold depolarizing potentials
(SDP) also monitored
– SDP blocked by TTX and INaP blockers, but not
Ca2+ blockers  INaP driven
SDP Reduced by PDB
INaP blockage by
PDB does not effect
bursting, but
reduces SDP to
passive membrane
response
Subsequent
addition of Ni2+
suppressed
bursting
INaP activation not mandatory for bursting
Same effects seen
as with PDB
Localized effects
• ICaT localized predominantly in distal apical
dendrites in ordinary cells
– ICaT driven bursting may also be localized to
distal apical dendrites
• Ni2+ focally applied to axo-soma or apical
dendrites
Axo-Soma application had no effect on bursting
Apical Dendrite application suppressed bursting
SDP was unaffected by Ni2+ application
Burst generation requires activation of ICaT in
distal apical dendrites
Subsequent Ni2+ application and recovery in different regions shows
burst suppression only in apical dendrites
Backpropagation
• Proximal axon spikes backpropagate to
apical dendrites
– Results in recruitment of Ca2+ Channels to
apical dendrites
• Blocking backpropagation should block
bursting from apical dendritic Ca2+
currents
Somatic spike backpropagation into apical
dendrites is critical step in burst electrogenesis
TTX on dendrites stopped bursting, but did not effect SDP
TTX on axo-soma stopped burtsing, and greatly reduced SDP
Primary spike is unchanged in all  TTX Blocks bursting by acting at distal portion
Retigabine Studies
• M-Type K+ channel agonist  enhances IM
– Shifts activation curve to more negative potential
• Retigabine applied to apical dendrites of
normal cells locally suppresses Ca2+ spikes
and bursting without affecting spike
generation in axo-soma
Bursting requires interplay between apical
dendrites and axo-soma conductances
Application to apical dendrites suppressed bursting but did not affect SDP
Application to axo-soma suppressed bursting and SDP
Increased IM conductance in apical dendrites suppresses bursting
Intradendritic
Recordings
Truncated Dendrites
High-threshold busting
Breif stimuli evoked single spike
Recap
• Bursting is present during second week
after stimulation, before symptoms present
• ICaT has predominant and critical role in
bursting
• Bursts are product of interplay between
backpropagating Na+ spikes in the axosoma and ICaT driven depolarizations in
apical dendrites
“Ping Pong”
3) Spike
ADP boost
triggered
fast spikes
2 & 4) ICaT driven depolarization
1) Somatic spike backpropagation
End) opposing slow K+ currents
repolarize neuron
Epileptogenesis
• Persistent increases in excitatory synaptic
transmission further lowers threshold
– Increased seizure generation
• Bursting neurons drive network into population
bursting
– Drives epileptogenesis
• T-type Ca2+ important pharmacological targets
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