Voltage-Gated Calcium Channels Notes

Voltage-Gated Calcium Channels (CaV Channels)

Structure and Function of Voltage-Gated Calcium Channels

Classification of different voltage-gated $Ca^{2+}$ (CaV) channels, including their structure.
CaV channel inactivation & modulation.
CaV channels & exocytosis, including CaV channel pharmacology.

Classification of CaV Channels

High-Voltage Activated (HVA):
- Activate at > -20mV.
- Subtypes include L-type, N-type, P/Q-type, and R-type.
Low-Voltage Activated (LVA):
- Activate at > -70mV.
- T-type channels are low-threshold, low-conductance variants.

Kinetic Properties of CaV Channels

L-type and Q-type $Ca^{2+}$ currents can be described by a two-exponential function.
The early fast phase of current decay is governed by $Ca^{2+}$ -dependent inactivation (CDI).

CaV Channel Structure: The α1 Subunit

CaV channel $α1$ subunits have 24 transmembrane α-helices, organized into four homologous repeats (Domains I–IV).
The fourth transmembrane segment of each repeat (S4) has five positively charged amino acids and, together with S1, S2, and S3, comprises the voltage-sensing domain of the channel.
S5 and S6 comprise the pore-forming domain.

CaV Channel Structure (Continued)

Comprised of a single $α1$ subunit; ~2000 amino acids (~170-250 kDa), 10 genes.
Like NaV channels, CaVs are also tetramer-mimicking structures.
Auxiliary Subunits:
- $α2δ$ dimer: 170Da, 4 isoforms
- $β$ subunit: ~55KDa, 4 isoforms
- $γ$ subunit: 8 isoforms, only for CaV1.1 channels
- Auxiliary subunits modulate the membrane trafficking, current kinetics, and gating properties of CaV channels and mediate the regulation of the $α1$ subunit by a variety of signals.

Role of Auxiliary Subunits

High voltage-activated calcium channels are heteromultimers of $α1$ , $β$ , $α2δ$ and $γ$ subunits, whereas low voltage-activated channels contain only a single $α1$ subunit.
Auxiliary subunit functions:
1. Increase membrane expression of $α1$ subunit.
2. Facilitate channel opening.
3. Affect the affinity of $Ca^{2+}$ channels for channel blockers.
The consequences of $β$ subunit expression on the function of the $α1$ subunit include an increase in current density, modulation of activation and inactivation kinetics, effects on pharmacological properties, and interactions with second-messenger regulation.

Key Protein Interaction Sites

N-terminal calmodulin association site in L-type channels.
CaV $β$ interaction domain in all HVA CaV channels.
Synaptic protein interaction site (synprint) present in CaV2 channels.
PreIQ-IQ and IQ motifs in CaV1 and CaV2 channels that associate with calmodulin.
Scaffolding protein interaction sites in CaV2 channels.

Structure of VSDs and Channel Pore

The bundle-crossing region at the lower third of the S6 segments forms the activation gate.
In the closed state, the pore-lining S6 helices converge at the intracellular side to obstruct ion flow through the pore.

Pore Domain of CaV Channels

The negatively-charged selectivity filter vestibule is formed by the side chains of the essential EEEE residues (Glu292, Glu614, Glu1014, and Glu1323 in the four repeats, respectively).

Voltage Sensing Domain of CaV Channels

VSDs sense depolarization by virtue of a signature motif of positively charged arginine or lysine at every third position of helix S4, which rearranges in response to depolarization.
Segments S1–S4 form a voltage-sensing domain (VSD), whereas segments S5 and S6 contribute to the $Ca^{2+}$ -conductive pore.
The VSDs surround the central pore.
The $α1$ subunit consists of four repeated motifs (I–IV), each consisting of six membrane-spanning helical segments (S1–S6).

Movement of the S4 Helix

At rest, the voltage sensors (VSs) are pulled into a down position by the electrical field. In their down state, the VSs lock the channel in its closed state.
Membrane depolarization releases the VSs, resulting in their upward movement, which in turn releases the closed channel gates.
Channel opening and inactivation are enabled when all four S4 segments have left their resting position.
The arginine and lysine residues align at the same point on each bend in the coil of S4 forming a positively charged band along the length of the alpha helix.

Mechanism Underlying CaV Channel Inactivation

Voltage-dependent inactivation
Calcium-dependent inactivation

Voltage- vs Calcium-Dependent Inactivation

Membrane depolarizations trigger a conformational change in the channel that opens the pore.
Prolonged depolarization triggers a further conformational change that repositions the inactivation shield to expose a docking site for the inactivation gate.
$Ca^{2+}$ influx creates a $Ca^{2+}$ domain by the inner pore.
$Ca^{2+}$ binds to calmodulin, which in turn causes it to change configuration, leading to an inactivated conformation of the channel.

Voltage-Dependent Inactivation of CaVs

The domain I–II linker might act as an inactivation particle that physically occludes the pore of the channel by interacting, at least in part, with the domain II and III S6 regions of the channel.
The $Ca^{2+}$ channel $β$ subunit associates with the domain I–II linker to modify inactivation properties.

CDI Depends on CaM Interactions with CaVs

The interaction of calmodulin (CaM) with the CaV is required for CDI.
At rest, the amino-terminal lobe of CaM is tethered to the 1-8-14-binding motif, whereas both the amino- and the carboxy-terminal lobes are tethered to the CB region.
Following the influx of $Ca^{2+}$ , the $Ca^{2+}$ -binding sites of the carboxy-terminal lobe of CaM become occupied, and the carboxy-terminal lobe binds the IQ motif.
Subsequently, processes that lead to channel closure are induced.

Calcium-Dependent Inactivation of CaVs

Calcium-dependent inactivation (CDI) is essential for voltage-gated calcium channel (CaV) autoregulation.
One possible mechanism is that the CaV selectivity filter forms the CDI gate, suggesting an SF-based inactivation paradigm shared with other voltage-gated ion channel (VGIC) superfamily members, like KVs.
Closed channels are activated by depolarization that includes voltage sensor domain (VSD) activation and pore opening.
Flow of $Ca^{2+}$ through the channel leads to $Ca^{2+}$ binding to CaM that initiates CDI.
CDI results in a selectivity filter (SF) conformational change that obstructs ion flow.

Modulation of CaV Channels

Modulation of L-Type CaV Channels by PKA

The cAMP activator, forskolin, enhances current flow through L-type CaVs via activation of PKA.
A-kinase anchoring proteins (or AKAPs) direct PKA to the channel.
L-type CaVs interact directly with AKAP79/150, which binds both PKA and calcineurin (CaN).
CaN strongly opposes L channel phosphorylation by PKA.

Modulation of CaV Channels by G-Proteins

Schematic representation of a presumed G-protein $βγ$ -binding pocket within the CaV2 subunits most likely responsible for the variable sets of G $βγ$ -mediated modulation of the channel.
The carboxy-terminal region of the CaV2 $α$ subunit also contributes in some cases to the direct biochemical coupling of the channel with the GPCR.
Ag = agonist.

Auxiliary Subunit Modulation of CaVs

Example of peak CaV2.2 current at 0mV in the absence of any $β$ subunit and presence of $β1b$ .
Example of peak CaV2.2/ $β1b$ current at 0 mV in the absence of $α2δ$ and presence of $α2δ$ .
Effect of different $α2δ$ subunits on inactivation.

What do Voltage-Gated Calcium Channels do?

CaVs are located both pre- & postsynaptically
While each type of CaV is expressed in the brain, only N- and P/Q-types are located presynaptically.
Presynaptic $Ca^{2+}$ coming through CaVs is known to interact with the SNARE complex facilitating neurotransmitter release.
Additionally, postsynaptic activation of AMPARs can depolarize cells leading to $Ca^{2+}$ entry through NMDARs, as well as via postsynaptic CaVs.

CaV-Mediated Exocytosis

Calcium entry through presynaptic CaVs links membrane depolarization to the exocytosis of synaptic vesicles at the presynaptic terminal.
The amount of neurotransmitter or neuropeptide that is released is highly dependent on presynaptic $Ca^{2+}$ concentrations, such that increases or decreases in $Ca^{2+}$ influx can powerfully alter neurotransmission.

Synaptic Transmission

In the active zone of the presynaptic terminal, synaptic vesicles cluster against the plasma membrane.
A reserve pool of synaptic vesicles lies nearby, and the postsynaptic density lies opposite the active zone within a dendritic spine.

Exocytosis of Neurotransmitter

Actin helps move (or traffic) vesicles to the active zone.
Several proteins are involved with attaching the vesicle to the presynaptic membrane.
Complex of SNARE proteins docks vesicles to membrane.
Fusion between vesicle and membrane requires an increase of calcium ( $Ca^{2+}$ ) in the cytosol.

Exocytosis is Calcium Dependent

Vesicle is docked along inside of presynaptic membrane.
An action potential depolarizes terminal opening voltage-gated $Ca^{2+}$ channels.
$Ca^{2+}$ rapidly floods into the terminal (10,000 times greater concentration outside the neuron!).
$Ca^{2+}$ interacts with SNARE proteins causing the vesicle to fuse with the presynaptic membrane and spill its contents (exocytosis).

The vesicle membrane is recycled (endocytosis).

SNARE Proteins, Fusion, and Exocytosis

Calcium binds to synaptotagmin (a calcium sensor) which then stimulates the v- and t-SNAREs to combine into an α-helical-shaped complex.
The SNARE complex forces the two membranes together (fusion) and then pulls them apart to spill the vesicle contents into the synaptic cleft (exocytosis).

CaVs are Anchored Close to Synaptic Vesicles

Rab3, synaptotagmin (the major $Ca^{2+}$ sensor) and synaptobrevin are proteins known to be involved in anchoring voltage-gated $Ca^{2+}$ (CaV) channels of type CaV2 sufficiently near to synaptic vesicles to form a $Ca^{2+}$ nanodomain within the presynaptic active zone.

Modulation of CaV Channels by G-Proteins

Some GPCRs inhibit CaV channel activity via their G $βγ$ subunits.
This requires the presence of a conserved RAR motif in the N-terminal sequence (N) of the channel and involves CaV $β$ binding to the I–II linker.
GPCRs coupled to Gq/11 inhibit CaV channels by reducing levels of PIP2 and activating PKC, which phosphorylates the $α1$ -interaction domain (AID) and elsewhere.
Modulation of the C-terminal domain (C) increases channel open probability and CDI.

Pharmacology of CaV Channels

1. Dihydropyridines

L-type $Ca^{2+}$ channel blockers:
Dihydropyridines (or DHP), such as Nifedipine, block L-type CaV channels by binding to the pore domain formed by domains I S6, III S5, and IV S6.

2. Phenylalkylamines & Benzothiazepines

Phenylalkylamines (verapamil) and benzothiazepines:
These compounds bind to the central cavity of the pore. Specifically, to the interface of domains III S6 and IV S6 in CaV1.2.
State-dependent block of L-type $Ca^{2+}$ channels.
Blocker enters the intracellular mouth of the pore through the open activation gate and binds to a specific receptor site in a protonated positively charged form.

3. ω-Conotoxins

N-type $Ca^{2+}$ channel blockers, including ω-Conotoxins (from cone snails):
Block CaV2.2 that mediate neurotransmitter release in primary afferent sensory neurons.
Bind to the outer vestibule of $Ca^{2+}$ channels and block ion conductance through pore.
Potent analgesics in neuropathic pain.

4. ω-Agatoxins

P/Q-type $Ca^{2+}$ channel blockers, mainly ω-Agatoxins (spider venoms):
Blocks presynaptic $Ca^{2+}$ channels and reduces release of neurotransmitter.
Binds reversibly to the outside of the pore region (S3-S4 linker) of the channel.
Causes spasms leading to progressive paralysis, which eventually leads to death in insects.
ω-Agatoxins modify CaV gating
ω-Conotoxins are pore blockers