Voltage-GatedCalcium Channels

Brad Groveman

Membrane Biophysics

“Excitable cells translate their electricity into action by Ca2+ fluxes modulated by Voltage-Sensitive, Ca2+ permeable channels.”

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 CDIBa2+ 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 Distal

No CaM BindingCaM BindingIn presence of Ca2+

CaM1234 binding shows CaM binds Cav1.2 and 1.4 at basal Ca2+ conditions

Loss of Calcium Sensor CaM NOT responsible for CDI insensitivity

*Co-IP

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 voltage-dependent 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 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

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 ω-CgTx

What does this have to do with APP?

• Current augmentation caused by up-regulation 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 β-Secretase 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 Hypoxic

γ-Secretase inhibitor shows no effect on Ca2+ currents under normoxic conditions

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

β -Secretase inhibitor shows no effect on Ca2+ currents under normoxic conditions

β -Secretase inhibitor fully prevents Ca2+ currents augmentation by hypoxic conditions

Normoxic Hypoxic

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

ψR

Small subthreshold hump

“Jitters” seen in later spikes indicating a subthreshold

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 burstingBlocks 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 burstingBlocks 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 PDBINaP 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 burstingApical Dendrite application suppressed burstingSDP 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 SDPTTX 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 SDPApplication 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 axo-soma and ICaT driven depolarizations in apical dendrites

“Ping Pong”

1) Somatic spike backpropagation

2 & 4) ICaT driven depolarization3) Spike ADP boost triggered fast spikes

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

Thank you for staying awake