Voltage-Gated Calcium Channels

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Voltage-GatedCalcium Channels
Excitable cells translate their electricity into
action by Ca2 fluxes modulated by
Voltage-Sensitive, Ca2 permeable channels.

• Brad Groveman
• Membrane Biophysics

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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

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The Ion Ca2
Found in all Excitable Cells Shapes the
Regenerative A.P.
Ca2i Three Best Studied Roles 1.
Contraction of Muscle 2. Secretion 3.
Gating
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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
• Ca2i elevation
• Mode switching

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Classes of VGCC
http//calcium.ion.ucl.ac.uk/a1-nomenclature.html
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Classes, Location, Blockers
http//en.wikipedia.org/wiki/Voltage_gated_calcium
_channel
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Example Currents
A. C. Dolphin 2006
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Alpha-1 Subunit Structure
http//calcium.ion.ucl.ac.uk/calcium-channels.html
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Ribbon Structure of Alpha-1
http//calcium.ion.ucl.ac.uk/calcium-channels.html
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Accessory Subunits
http//calcium.ion.ucl.ac.uk/calcium-channels.html
http//www.sigmaaldrich.com
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Accessory Subunits

• ß - Contains Guanylate Kinase domain and
• SH3 domain
• GK domain binds a1I-II intracellular loop
• Stabilizes a1 and helps to traffic to membrane
• Allows more current (higher amplitudes) for
  smaller depolarizations (HVA)
• Shifts towards negative membrane potentials

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Accessory Subunits

• a2d- co-expressed, linked by disulfide bond.
• a2 extracellularly glycosylated
• d has a single transmembrane region
• Co-expression enhances a1 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

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Accessory Subunits

• ?- 4 transmembrane
• helices
• Found in skeletal muscles
• May have an inhibitory effect on calcium currents
• Interact with AMPA and Glutamate receptors

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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)

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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.

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Pathologies

• Neuropathic pain
• Epilepsy
• Congestive heart failure
• Familial hemiplegic migraine
• Several cerebellar ataxias

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• 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

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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

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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
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CaM binding in C-Terminal
Proximal Distal
No CaM Binding
CaM Binding In presence of Ca2
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
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CDI masked by inhibitory domain?
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
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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
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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

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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

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Is inactivation of Cav1.2 rendered insensitive by
Cav1.4 CT?

• Generated Cav1.2/1.4 Chimeras

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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

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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 voltage-dependent with
kinetics intrinsic to channel core
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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

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Amyloid Precursor Protein
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Chronic Hypoxia

• Chronic Obstructive Pulmonary Disease
• Arrhythmia
• Stroke
• Reduction of Oxygen in brain

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Previous Studies

• APP expression increased following cerebral
  hypoxia or ischemia
• Prolonged hypoxia enhances Ca2 influx in PC12
  cells apparently dependent on Aß enhanced
  expression
• Suggested Aß composed Ca2 pores as well as
  up-regulation of L-Type Ca2 channels
• THIS CANNOT BE EXTRAPOLATED TO CENTRAL NEURONS!!!

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Mean Current Density vs Voltage
RelationshipsCurrents 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

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Mean Current Density vs Voltage
RelationshipsL-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 ?-CgTx
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What does this have to do with APP?

• Current augmentation caused by up-regulation in
  L-Type Ca2 Channels
• Immunohistochemical studies show increase in Aß
  in hypoxic cells
• This increase is abolished to normoxic conditions
  in presence of either ? or ß-Secretase inhibitors

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To beat a dead horse

• Hypoxia up-regulates L-Type Ca2 Channels
• Hypoxia increases Aß production
• But are they related?

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Blocking Aß production by ?-Secretase inhibitor
abolishes hypoxia effect
Normoxic
Hypoxic
?-Secretase inhibitor fully prevents Ca2
currents augmentation by hypoxic conditions
?-Secretase inhibitor shows no effect on Ca2
currents under normoxic conditions
In presence of N-Type channel blockers
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Blocking Aß production by ß -Secretase inhibitor
abolishes hypoxia effect
Normoxic
Hypoxic
ß -Secretase inhibitor fully prevents Ca2
currents augmentation by hypoxic conditions
ß -Secretase inhibitor shows no effect on Ca2
currents under normoxic conditions
In presence of N-Type channel blockers
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Conclusions

• Hypoxia increases formation of Aß in primary
  culture neurons
• Functional expression of L-Type Channels
  increased
• Dependent on Aß
• Aß do not form Ca2 permeable pores

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Status Epilepticus

• Single episode can be evoked using chemical or
  electrical stimulation to mesial temporal lobe.
  ltPilocarpinegt
• 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

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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

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Purpose

• Contribution of ICaT vs ICaR
• Subcellular localization of ICaT
• Contribution of INaP

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Bursting in early epileptogenesis driven by Ni2
Sensitive Ca2 Current
Jitters seen in later spikes indicating a
subthreshold
?R
Small subthreshold hump
Ni2 suppresses bursting into single
spike T-Type Ca2 channels are blocked by Ni2
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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

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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
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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

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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

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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
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INaP activation not mandatory for bursting
Same effects seen as with PDB
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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

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Axo-Soma application had no effect on
burstingApical Dendrite application suppressed
burstingSDP was unaffected by Ni2 application
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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
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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

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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
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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

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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
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Intradendritic Recordings
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Truncated Dendrites
High-threshold busting
Breif stimuli evoked single spike
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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 axo-soma and
  ICaT driven depolarizations in apical dendrites

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Ping Pong
2 4) ICaT driven depolarization
3) Spike ADP boost triggered fast spikes
1) Somatic spike backpropagation
End) opposing slow K currents
repolarize neuron
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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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