Cav3.2

Cav3.2, encoded by the gene cacna1h, is a calcium channel, voltage-dependent, T type, alpha 1H subunit. It is highly expressed in primary afferent neurons and plays a crucial role in sensory perception, but it can be found throughout the body. Mutations of the channel are the cause of pain disorders and other endocrinopathies.

In humans, cacna1g, the gene which encodes Cav3.1, is composed of 36 exons located on chromosome 16 at position 13. (16p13.3).

Like most Cav channels, cacna1g is subject to alternative splicing. 12-14 potential alternative splice sites have been identified in the ORF [2406]:

• Exon 17 (ID2-3 region): corresponds to a segment of a cytoplasmic domain. Segment contains conserved phosphorylation sites for neuromodulation. Deletion can impair neuromodulation.
• Exons 25C and 26: codes for the intracellular loop between domains III and IV (III-IV linker). These segments are often mutually exclusive or deleted, creating multiple functional variants. Inclusion of 25C introduces a protein kinase A phosphorylation site, affecting the channel’s regulation.
• Exon 35A: codes for area of the C-terminus which modulates gating properties. Deletion leads to significant shifts in window current magnitude and midpoint voltages.
• Exon 9B: represents a section of D1-2 region. Mutations to this area result in premature chain termination that, when not destroyed, lead dysfunctional proteins
• Exon 31A/31A’: represents a membrane-spanning region. Deletion of this region results in premature chain termination
• i23, i27B, i32B: Under normal expression conditions, these are considered introns but their inclusion introduces new coding sequences. For example i23 results in the extension of an extracellular domain, in turn potentially affecting interactions with other molecules
• D9B: encompasses 26 amino acids of cytoplasmic ID1– 2. Its inclusion shifts the open reading frame and produces a premature chain termination

The human Cav3.2 protein is composed of 2353 amino acid (aa) and has a molecular weight of 259 Kda.

As discussed, the CACNA1H gene undergoes extensive alternative splicing, leading to channels with subtle differences. However, not all splice variants have been characterized. Most studies have focused on the isoforms resulting from the alternative splicing of exons 25B and 26. The different identified permutations of exons are [109]:

• +25B/∆26: higher expression levels in the uterus during pregnancy.
• +25B/+26: higher expression levels in the uterus during pregnancy.
• ∆25B/+26: the canonical α1H-a isoform, more common in the ovary and non-pregnant uterus
• ∆25B/∆26: coined the α1H-b, more common in the pregnant uterus and testis

Versions with exon 25B (+25B/∆26 or +25B/+26) had faster activation and inactivation at lower voltages compared to those without exon 25B. Inclusion of exon 26 did not change the kinetic properties, but having a higher positive charge (like in ∆25B/∆26, +25B/∆26, and +25B/+26) made recovery from inactivation quicker. These behaviors highlight that these structural differences in the III-IV linker region affect how the channel activates and inactivates. [109]

Like most mammalian proteins, cav3.2 is subject to a series of post translational modifications (PTM).

Cav3.2 is regulated by phosphorylation via protein kinase C (PKC), cAMP-dependent protein kinase, Rho-associated kinase (ROCK), and cyclin dependent kinase 5 (CDK5). These kinases modulate the channel’s activity and expression and may play a crucial role in the mechanisms underlying chronic pain. [2507] [2482] [2508]

Cav3.2 is ubiquitinated by WWP1, a plasma-membrane-associated ubiquitin ligase that binds to the intracellular domain III-IV linker region of the Cav3.2 T-type channel and modifies specific lysine residues in this region. Absence of ubiquitination lead to increases in pain sensitivity and associated pain conditions [2509]

A number of important N-Glycosylation Sites, in the extracellular loops, have been identified. These contribute to the stability and function of Cav3.2. deglycosylation of the channels resulted in a reduction in T-type current and a relief in neuropathic pain. [2511] [2457][2508]

Cav3.2 can also be affected by reducing agents, like the sulfur-containing amino acid L-cysteine, which specifically enhance T-type currents in thalamic neurons and recombinant Cav3.2, but not in other T-type channels (Cav3.1 or Cav3.3). Mice lacking Cav3.2 channels show no modulation by reducing agents, confirming Cav3.2’s role in redox regulation. Oxidizing agents, however, inhibit all T-type currents non-selectively. This selective redox regulation of Cav3.2 is important for controlling neuronal excitability and sensory information processing under both normal and pathological conditions. [2512]

Like most Cav channels, Cav3.2 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-S6 act as the selectivity filter and form the pore module (PM). The S4 subunit of each domain contains a series of positively charged residues. [2511]

The structure of a Cav3.2 isoform was resolved via electron-microscopy, to a resolution of 2.8 Å angstrom, allowing for a detailed insight into the specific structural features of the protein. Overall, the structure of Cav3.2 is very similar to that of Cav3.1 and Cav3.3 with a few unique features that confer the channel its specific properties.

• Selectivity Filter (EEDD motif): The EEDD motif in Cav3.2 plays a critical role in calcium ion selectivity. This motif is conserved across Cav3s, but small differences in nearby residues contribute to Cav3.2's distinct ion conductance and pharmacology.
• N-Glycosylation Sites: Cav3.2 has two key glycosylation sites (Asn345 and Asn1466) in its extracellular loops (ECLs I and III). These sites are critical for Cav3.2’s surface expression and stability, more so than in Cav3.1 or Cav3.3.
• Drug Binding Sites (Fenestrations): Cav3.2 has specific fenestrations between its domains (II-III and IV-I), where drugs bind differently than in Cav3.1 or Cav3.3.
• α-to-π helical transition: α-to-π helical transition in the S6II segment, alters the conformation of Cav3.2’a intracellular gate. This results in a stronger state-dependent inhibition to certain drugs compared to other Cav3s
• Lipid Binding sites: An endogenous lipid found in the central cavity of Cav3.2 stabilizes the binding of certain antagonists and may enhance Cav3.2’s drug selectivity and efficacy.

Cav3.2 channels generate significant calcium currents by allowing entry of calcium into the cell in response to action potentials. [2484]

CaV3.1, CaV3.2, and CaV3.3 are characterized by their activation at low membrane potentials, close to the resting membrane potential, and transient single-channel conductance and are thus designated as the low-voltage-activated (LVA) or T-type channels. This is in contrast to Cav1 and Cav2, which are high-voltage-activated and conduct larger, long-lasting conductance. [2485] (31766050)

The low voltage threshold for activation of T-type channels drives their opening in response to relatively small positive changes in membrane potential. They are therefore important tuners of cell excitability [2514] Further, an overlap in the membrane potentials at which T-type channels open, close, and inactivate gives them a particular property known as a “window current” whereby a basal inward flux of calcium ions can occur near the resting potential [2484]

Cav3.2 also has its own particular gating properties [2484]:

• Activation and Inactivation Kinetics: Cav3.2 channels exhibit faster activation and inactivation kinetics compared to Cav3.3, while their activation is slower and inactivation kinetics are similar to those of Cav3.1.
• Voltage Dependence: Cav3.2 channels open and close at similar membrane potentials as Cav3.1 channels, but at more hyperpolarized potentials compared to Cav3.3.
• Deactivation Kinetics: Cav3.2 is the slowest channel to deactivate (close).
• Recovery from Inactivation: Cav3.2 is also the slowest channel to recover from inactivation.

Single channel unitary conductance

The single channel unitary conductance values differ based on experimental recording conditions. Single channel unitary conductanceCav3.2 was measure between 1.7 pS and 5.3±0.3 pS [2510] [1240]

Model

To date, no Cav3.2 model has been made.

Tissues & Cellular

Cav3.2 has been identified throughout the body.

In the CNS location include [2415]: * The brain cortex *Amygdala * Caudate nucleus * Putamen * Excitatory and inhibitory interneurons [2508]

In the PNS, Cav was identified in:

• Primary afferent neurons or the dorsal root ganglia [2509] [2457]
• Predominantly within small and medium-size cells, corresponding to C and Aδ-fibers, respectively.
• Cav3.2 is also present in D-hair mechanoreceptors [2508]

Other areas of expression are:

• Liver [2415]
• Heart [2415]
• Vascular smooth muscle cells (VSMCs) [2513]
• Endothelial cells (ECs) of small arteries [2513]
• Kidney [2508]

Developmental

Cav3.2 transcripts were shown to be 10-40 times more abundant in fetal brain compared to the adult human brain [2406]

Subcellular distribution of Cav3.2 depends on the localisation of the channel. In rodent primary affect neurons, It was predominantly identified in the somata. [2508]

Cav3.2’s role is to allow the entry of Ca2+ into the cell in a voltage dependent manner. Depending on its expressed location, the channel fulfils a number of function

Sensing & Nociception

Given its predominant location in primary afferent sensory neurons, Cav3.2 plays a crucial role in sensory perception. Cav3.2 conducts almost 80% of all t-type calcium current in the DRG neurons [2507]. Furthermore, the channel is responsible for innocuous touch, noxious mechanical cold, and chemical pain in low-threshold mechanoreceptors [2514] In D-hair mechanoreceptors, Cav3.2 KO leads to 50% less fewer spikes as a results of the increase in mechanical threshold [2515]
Cav3.2 antisense mRNA knockdown experiments resulted in major antinociceptive, anti-hyperalgesic, and anti-allodynic effects, highlighting cav3.2 role in perception and pro-nociceptive role in acute and chronic pain conditions. [2485] Conversely, Conversely, enhanced Cav3.2 activity increases chronic pain perception [2514]
Furthermore, in KO mice experiments, mice showed decreased pain responses to acute mechanical, thermal and chemical pain tests, and tonic noxious stimuli compared to wild-type mice. [2516]

Brain function & memory
Cav3.2 channels are responsible for the retrieval of memory, and plays a major role in short-term plasticity. Mutations in the identified calcium genes related to ID/GDD, intellectual disability (ID)/global developmental delay (GDD), can affect neurotransmitter release [2415] Other impairments to Cav3.2 channels were shown to decrease sleep duration [2490]

Heart function
Cav3.2 present in arterial vascular smooth muscle cells (VSMCs) and pits structure of caveolae are tightly coupled with ryanodine receptors (RyRs). The interaction between the channel and receptors provides an additional Ca2+ source for Ca2+ spark generation in mesenteric and arterial smooth muscle arteries, allowing for vasodilation [2517]

Secretion
As it is present in secreting organs such as the liver and kidney, Cav3.2 is thought to also contribute to proper secretion, namely of hormones such as aldosterone [2415] [2518]

Channelopathies

Given the importance of Cav3.1’s role through the body, mutations to coding gene or disruption to the protein are responsible for a number of pathologies

Pain Syndromes
Given its important and predominant expression in sensory neurons, aberrant Cav3.2 function leads to a number of pain disorders. As previously discussed, knockdown and knockout experiments are known to alleviate pain caused by acute mechanical, thermal and chemical pain tests, and tonic noxious stimuli. Additionally, increased expression of Cav3.2 results in increased pain perception. [2485] [2514] A wealth of studies showcase that Cav3.2 is upregulated within rodent primary afferent neurons in many chronic inflammatory and neuropathic pain conditions. These studies highly implicate primary afferent neuron Cav3.2 as being pronociceptive and contributing to hyperexcitability of nociceptive circuitry in chronic pain conditions [2508] Nerve injury and painful diabetic neuropathy (PDN) are known to induce changes to Cav3.2, namely increases in channel expression, increasing neuronal excitability. Neuropathic pain resulting from these conditions can be alleviate by STZ treatment, a Cav3.2 blocker [2457]

Developmental disorders

Given its function in the brain, aberrant Cav3.2 function leads to a number of cognitive impairment. These include [2487]:

• Epileptogenesis: mutations to Cav3.2 are associated with different forms of idiopathic generalized epilepsy. This is due to increased excitability as a result of changes to the channel’s biophysical properties or its surface expression [2519] Interestingly, development of epilepsy was absent in epilepsy mouse models lacking Cav3.2. [2520]
• Autism spectrum disorder: CACNA1G was identified as a candidate gene for autism spectrum disorder (ASD) in a subset of cases [2460]

Endocrinopathies

In secreting organs, impaired Cav3.2 channels lead to increases in intracellular Ca(2+), a signal for aldosterone production. [2518]

Whole exome sequencing identified CACNA1H mutations linked to [2521]:

• Early-onset primary aldosteronism (PA),
• Familial hyperaldosteronism (FH)
• Aldosterone-producing adenomas (APA)

Cancer

Aberrant expression of cacna1H is common in a broad range of cancers. The channel is one of the top downregulated genes in kidney and lung cancer. [2440] Aberrant expression of Cav3.2 can also be responsible for Glioblastoma (GBM), the most common primary malignant brain tumor, as increases in intracellular calcium, regulated by Cav3.2 expression, has been shown to regulate GBM cell proliferation [2522]

Ca2+ plays a number of other important roles and functions in the body, besides those mediated by calcium channels, in many cascade reactions and interactions. The above list of pathologies are those specific to ones associated with Cav3.2 but deregulation of Ca2+ trafficking could have further impacts that were not discussed here.

Cav3.2 interacts with β subunits intracellularly. These subunits play a critical roles in cell surface expression and in the modulation of the gating properties of the α1 subunit. [2507]

Cav3.2 also interacts with G-protein betagamma subunits, namely beta2gamma2. The subunit binds to the intracellular loop between DII and DIII resulting in the inhibition of the channel’s activity in a voltage independent manner. [2523]

Paclitaxel is a chemotherapeutic agents utilized as a treatment for various types of cancer. The drug is known the enhance T-type calcium currents. Paclitaxel treatment resulted in a 75% increase in Cav3.2 expression in DRG neurons. Furthermore the activation curves are left shifted and the inactivation curves showed an upward flattened shift. These changes indicate that the channels open more readily and are harder to inactivate [2507]

Cav3.2 was also found to be highly sensitive to nickel, around 20 fold times more sensitive compared to other Cav3

Aside from compounds and subunits, Cav3.2 also interacts indirectly with other ion channels, namely HCN1. HCN1 subunits are colocalized alongside Cav3.2 in the active zone of mature synaptic terminals of cortical layer III pyramidal neurons. HCN channels suppress the activity of Cav3.2 channels, leading to the inhibition glutamate synaptic [2524]

Contributors: Rajnish Ranjan, Michael Schartner