Cav1.2

Cav1.2, encoded by the gene cacna1c, is a calcium channel, voltage-dependent, L type, alpha 1C subunit. It is primarily expressed throughout the organism and is responsible for learning, memory, fear conditioning and pacemaking. Mutations of the channel are the cause of developmental disorders and cardiopathies.

In humans,cacna1c, the gene which encodes Cav1.1, is composed of 57 exons located on chromosome 12 (12p13.33). [2360]

There exist multiple Cav1.2 transcript variants across species as a result of alternative splicing of cacna1c (see Protein Isoform table). Cav1.2 has a particularly abundant number of transcript variants with around 40 splice variations identified in 13 splice loci [2372] [2373]

Alternative splicing occurs in [2373]:

• The NH2 terminus
• The DI-II and DII-DIII linkers
• Between S5 and S6 in DI
• Between S2 and S3 in DIV
• Between S3 and S4 in DIV
• DI-S6
• DII-S2
• DIV-S3,
• The COOH terminus.

Expression of Cav1.2 transcript variants was found to be cell-specific, suggesting a potentially tailoring/fine-tuning of the final proteins properties to role specific physiological outcomes.[2372]
For example Cav1.2 α11.2 was identified in the “smooth muscle” and “cardiac muscle”. α11.2 splice variants differ in at least the following 4 regions: exon 1/1a; exon 8/8a; termed exon 9 and exon 31/32 with differences even between smooth and cardiac muscles. The cardiac muscle form was proposed to consist of splice combination 1a/8a/−9/31; while the proposed smooth muscle splice combination was 1/8/+9*/32 [2372]

A less common feature in the alternative splicing of other channels, Cav1.2 splicing events can be cumulative, with a number of splice events stacking onto each other, creating a potential infinity of exon permutations. Some important exons, often involved in alternative splicing are:

• Exons 8 and 8A are mutually exclusive as they encode the same structural domain (DI/S6), but one must be present to encode a functional channel. Exon 8 represents ≈80% of mRNAs in heart and brain [2374]. Exon 8A is also present in the same areas as exon 8 but at relatively lower levels. [2375]
  • The switch from exon 8 to exon 8a containing CaV1.2 mRNAs has been shown to be controlled by the polypyrimidine tract-binding protein (PTB) and its neu- ronal homolog (nPTB) and to be repressed during neuronal development in mouse brain [2373]
• Exon 9 contains the binding domain for CaVsubunits, whereas the adjacent exon 9A if present, affects channel gating by differentially modulating the interaction with CaV isoforms. Exon 9A, sometimes called exon 9*, is not an extension of exon 9 or alternatively used for exon 9 but an exon in its own right.
  • Fox proteins, another family of splicing factors implicated in the control of alternative splicing during development, were recently shown to repress exon 9A and promote inclusion of exon 33 into CaV1.2 mRNAs expressed in the cortex.
• Three NH2 termini have been reported for CaV1.2. Exon 1a (the long NH2 terminus) is expressed in cardiac myocytes; exon 1b (the short NH2 terminus) is expressed in smooth muscle and brain; and exon 1c (even shorter than exon 1b) is present in resistance cerebral artery. [2373]

There exists a number of protein isoforms that arise from the translation of the aforementioned transcript variants as a result of alternative splicing.

The major form of CaV1.2 subunit in smooth muscle is the CaV1.2b isoform though the 1c isoform has also been detected.. The “smooth muscle” splice variant CaV1.2b is more sensitive to inhibition by DHPs than the heart variant CaV1.2a, because it contains the DHP-sensitive exon 8B. [2373]

However, relatively little research has been done on the specific differences between the isoforms and the canonical Cav1.2 protein.

Nonetheless, some of these splice variants have been associated with a diversity of biophysical properties [2373]:

• A depolarizing shift of activation for alternatively spliced exons 31 to 33 encoding the DIV S3-S4 region
• Faster inactivation kinetics, strong voltage dependence, and variable Ca2-dependent inactivation for exon 41 to 42 variants in the COOH terminus
• A higher DHP sensitivity for exon 8a in DI-S6

Like most mammalian proteins, Cav1.2 is subject to a series of post translational modifications (PTM).

Many phosphorylation sites have been identified within Cav1.2 and occur across species. Cav1.2 is phosphorylated by many protein kinases (PKA, PKC, PKG, and CAMKII) depending on where the channel is expressed. [2376] Phosphorylation tends to increase the activity of the channel, however the mechanisms behind such enhancements is still poorly understood. [2377]

Cav1.2 is targeted for degradation through ubiquitination. UB proteins, RFP2 ubiquitin ligase, contain several lysine residues which participate in the tagging to Cav1.2 for degradation. However, recent studies has demonstrated that Cav1.2’s interaction with Cavβ protects Cav1.2 from proteasomal degradation [2376]

Four potential N-glycosylation sites in rabbit Cav1.2 have been identified: N124, N299, N1359, and N1410. Mutant Cav1.2 channels, lacking these sites, were shown to have positive shifted voltage dependent gating and reductions in peak current. These results suggest a non-negligeable role of N-glycosylation in the proper function Cav1.2. Furthermore, the α2δ subunit, an integral interacting component of Cav1.2, is highly N-glycosylated by a 30-kDa oligosaccharide [2376]

S-glutathionylation is a process in which glutathione forms a disulfide bond with cysteine residues of the target protein, and is a major redox-mediated thiol modulation. S-glutathionylation directly changes the redox state of Cav1.2 and increases calcium influx. However, this process is not specific to Cav1.2.
C543 in the cytoplasmic I-II loop is the major glutathiolation target in hCav1.2. C543S mutation alters post-translational folding and shifts the channel open probability, which may lead to the onset of disease pathology [2376]

Lika most Cav channels, Cav1.2 is made up of a single protein composed of 4 homologous domains (DI-DIV). Each domain is made up of 6 transmembrane subunits (S1-S6) connected by extracellular loops. S1-4 form the voltage sensing domain (VSD) whereas the S5-6 form the pore module (PM). The S4 subunit of each domain contains a series of positively charged residues. When membrane depolarization occurs, these charged residues cause the movement of the S4 subunit. This translocation of the S4 helices causes further conformational rearrangements within CaV1.2 that activate the receptor and open the channel pore. [2376]

The structure of human Cav1.2, in complex with a number of drugs, was resolved via cryo-electron microscopy, giving us a detailed insight into the specific structural features the protein. However, it is worth noting that these experiments may not represent the canonical conformation as the used protein is Cav1.2α1, isoform 1. [2378]

Cav1.2, along with other L-type channels, has 3 broadly-defined gating modes [2362]:

• Mode 0: the closed state of the channel
• Mode 1: brief ~1 ms opening
• Mode 2: longer opening duration due to strong depolarisation or interaction with other actors

Particular to the channel, Cav1.2 typically require a strong depolarization for activation and displays long-lasting activity in contrast to other L-type channels [2373]

There has been no single channel unitary conductance recordings for Cav1.2 nor have any kinetic models been generated

Tissue & cellular

Cav1.2 is found expressed in both the PNS and CNS

Within the PNS, locations including [2376]:

• Heart
• Smooth muscle
• Pancreas
• Adrenal gland brain
• Brain

In the brain, Cav1.2 was identified [2376]:

• For humans: cerebral cortex, the pituitary gland, the amygdala, the basal ganglia, and the cerebellum
• In mice: the olfactory region, the basal ganglia, the hippocampal formation, the amygdala, and the thalamus

The subcellular localization of Cav channels is area specific. It is therefore difficult to generalize and pinpoint the exact cellular locations of these channels.

In rat hippocampus, Cav1.2 was found present in the soma and both in the synapses [2373]

Cav1.2 channels are located in the cardiovascular system, the nervous system, and endocrine glands where they serve important physiopathological functions. Cav1.2 participates in learning and memory, drug addiction, and neuronal development [2376]

Learning, memory & fear conditioning

Given its location in the brain, it is reasonable to assume that Cav1. plays a role in the cerebral function. Indeed, Cav1.2 deletion in the hippocampus and neocortex-specific induced a defect in long LTP, spatial learning, and induction of nuclear gene transcription [2373] [2379] Further genetic manipulation studies also highlighted that Cav1.2 subunit of the L-type Ca2+ channel in the ACC is required for social fear learning. Mice with an ACC-specific deletion of Cav1.2 (Cav1.2ACC/Cre mice) exhibited impaired social fear learning and reduced pain responses but had normal anxiety, innate fear, object recognition, and classical fear conditioning. This suggests different neural mechanisms for observational social fear behavior compared to anxiety or innate fear. The role of Cav1.2 in observational fear is still unclear, potentially linked to reduced excitability in the ACC. [2380]

Smooth muscle contraction

Ca2+ influx through L-type Ca2 channels is the major source for the increase in [Ca2]i leading to contraction. Cav1.2 plays a particularly important role in smooth muscle [2373]

Deletion of CaV1.2 in smooth muscle in mice led to [2373]: * Death within 21 days due to paralytic ileus. * Significant decrease (~30 mmHg) in arterial blood pressure. * Loss of myogenic tone development in response to intravascular pressure. * Reduced resistance to phenylephrine-induced constriction in hindlimb perfusion experiments. * Lacked rhythmic intestinal contractions and diminished hormone-induced contractions and reduced feces excretion.

Because of this crucial role within smooth muscle, Cav1.2 is essential for proper cardiovascular function. Complete Cav1.2 knock-out mice resulted in embryonic death whereas conditional KO lead to severe hypotension and related smooth muscle dysfunction and death one month after Cav1.2 channel expression ablation [2372]

Neural development

Pharmacological and genetic experiments have revealed the critical importance of the Cav1.2 in the development of the immature mouse brains. Cav1.2 knock outs resulted in significant alterations of neurite outgrowth. [2381] Cav1.2 also plays a role in the proper function of other neural cells. Deletion Cav1.2 in mice Oligodendrocyte progenitor cells led to reduced myelination in brain areas, such as the corpus callosum. This deletion hindered OPC proliferation, decreased mature oligodendrocyte production, and slowed OPC migration. These findings suggest Cav1.2 is essential for normal myelination by regulating calcium influx in OPCs. [2382]

Channelopathies

As Cav1.2 plays in important role in properper brain function, mutations to the protein is the cause of a number of psychiatric disorcers. These include [2383]:

• Bipolar disorder
• Schizophrenia
• Major depressive disorder
• Timothy syndrome
• Chronic unpredictable stress [2384]

Other developmental disorder

Cav1.2 role in smooth muscle, particularly cardiac muscle, is also the cause of channelopathies in case of degeneration. Deregulation of Cav1.2 was shown to cause:

• Long QT syndrome [2385]
• Ischemic stroke [2386]
• Familial sudden cardiac death syndrome [2387]
• Brugada syndrome
• ST-segment elevation, short QT intervals, and sudden cardiac death [2387]

During ageing, increased activity of Cav1.2 channels raises intracellular calcium levels, which may affect amyloid precursor protein processing and promote Alzheimer's disease (AD) pathogenesis. The calcium hypothesis of AD suggests that disrupted calcium balance leads to the formation of amyloid plaques and neurofibrillary tangles, impairing synaptic plasticity and causing neuron death. Additionally, calcium imbalance enhances tau phosphorylation and disrupts neuronal autophagy. Elevated endoplasmic reticulum stress and tau hyperphosphorylation are also linked to chronic traumatic encephalopathy. [2376]

Auxiliary subunit

Cav1.2 channels typically exist as multi-subunit complexes comprised of the main pore-forming α1 subunit, as previously described, and auxiliary subunits α2δ-2, β2 subunits [2373]

• Cavβ was shown to increase Cav1.2 membrane expression by preventing the entry of the channels into the endoplasmic reticulum-associated protein degradation (ERAD) complex. [2388]

Calcium & CaM

Cav1.2 allows the passage of Ca2+ ions in and out of the cell. However, it is itself sensitive to the fluctuation concentrations of the ion and can undergo Ca2+ dependent facilitation or Ca2+ dependent inactivation thanks to its interaction with certain proteins.

Ca2+ dependent facilitation (CDF) has been observed particularly in cardiac tissue. During repeated activity, CaV1.2 channels allow more Ca2+ to enter. The process depends on CaM and is blocked by Ca2+-insensitive CaM or by inhibiting another protein, CaMKII. This influx of Ca2+ activated Calmodulin-dependent protein kinase II (CamKII), which binds the C-terminus of Cav1.2. CamKII causes a signaling cascade but also modulates channel gating mechanics, resulting in frequent, long channel openings. Both main α1 CaV1.2 subunits and its β2 subunits are modified by CaMKII at specific locations. [2373] [2376]

CaM binds to the IQ motif of the CaV1.2 channel that is located at amino acids 1624–1635 (exon 42) of the CaV1.2 COOH terminus. CaM-mediated Ca2+ dependent inactivation (CDI ) of these channels has been attributed to the interaction via the C-lobe [2389]. Disruption of this domain prevents CDI [2373]

Dihydropyridine

3 large classes of drugs are known to bind to Cav1.2: phenylalkylamines (PAAs), benzothiazepines (BTZ), and dihydropyridines (DHP). Although all three classes of drugs bind to the α11.2 subunit of CaV1.2 channels, they block the channels through different mechanisms. PAAs and BTZs block the channels in a use-dependent manner, meaning the channels must be open first. In contrast, DHPs block the channels in a state-dependent manner by binding preferentially to inactivated channels, with binding increasing as the membrane becomes more depolarized. Studies have shown that specific segments (IIIS5, IIIS6, IVS6) determine the binding of DHPs to the α11.2 subunit, while IIIS6 and IVS6 are involved in the binding of PAAs and BTZs. Additionally, the IS6 segment, encoded by exons 8/8a, affects the channel's sensitivity to isradipine and may also be involved in DHP binding. Recent research indicates that the IIIS5 segment also plays a role in PAA inhibition of CaV1.2 channels. [2372]

Other proteins

Rad is a member of the Rad, Rem, Rem2, and Gem/Kir (RGK) family of Ras-like GTP-binding proteins, known for their capacity to inhibit all high-voltage-activated Ca2+ channels 8,9, and is a potential PKA target [2390]

APP physically interacts with Ca(v)1.2.Lloss of APP leads to an inappropriate accumulation and aberrant activity of Ca(v)1.2. [2391]

AKAP5 signalling module that regulates L-type Ca2+ channel activity and vascular reactivity upon elevated glucose. [2392]

Second Messenger-activated Protein Kinases
Ca2+ channels are regulated primarily by second messenger-activated protein kinases. [503]

kir/Gem
The small G-protein kir/Gem inhibits L- type Ca2+ channel activities by interacting directly with the Ca2+ channel β subunit [504].

COP9 signalosome subunit 5
COP9 signalosome subunit 5 (CSN5)/Jun activation domain-binding protein 1 (Jab1) interacts with the II–III linker of the α1C subunit. Inhibi- tion of CSN5 expression by siRNA enhanced the L-type Ca2+ currents. CSN5 regulates the cardiac L-type Ca2+ channel through protein–protein interactions. [226]

Contributors: Katherine Johnston, Rajnish Ranjan, Michael Schartner