Voltage Dependent Ca²⁺ (Ca_V) Channels

In many physiological systems, voltage dependent Ca²⁺ (Ca_V) channels are the molecular link between cellular membrane potential and intracellular Ca²⁺ signaling. As such, Ca_V channels are drug targets for several cardiovascular and neuronal conditions and the role they play in other, non-excitable tissues is beginning to emerge.

The Importance of the Ca²⁺ Ion as a Signaling Molecule

In most cells the intracellular concentration of ionized calcium ([Ca²⁺]_in) is actively kept very low, at about 100-200 nM, while the plasma Ca²⁺ concentration ([Ca²⁺]_out) is at about 2 mM. This is achieved by several mechanisms. Firstly, the lipid membrane is impermeable to Ca²⁺, unless a Ca²⁺ conducting channel in the plasma membrane is open, allowing ion diffusion along its electrochemical gradient. Other mechanisms include fast Ca²⁺ buffering either by active transport into organelles such as mitochondria and endoplasmic reticulum (ER) or by binding to cytosolic proteins. Therefore, small and local increases in [Ca²⁺]in trigger the cellular signaling cascades which underlie the basis of many physiological systems¹.

This paper focuses on a set of plasma membrane proteins that respond to membrane potential depolarization by opening a Ca²⁺ selective pore: the voltage dependent/gated/operated Ca²⁺ (Ca_V) channels.

Structure and Naming of Ca_V Channels

The principal protein (α1 subunit) that forms a Ca_V channel is a large (~2000 amino acids) protein that spans the membrane 24 times and includes the Ca²⁺ selective pore, four voltage sensors and large intracellular loops with different regulatory functions. Such proteins are a single polypeptide analogous to the archetypal K_V channel tetramer (Ca_V proteins contain four homologous domains, each resembles a K_V subunit)². Recently, the structures of purified skeletal and cardiac muscle L-type channels was determined by electron microscopy at low resolution^3-5.

In addition, three other protein families are referred to as Ca_V auxiliary subunits: a cytoplasmic β subunit, a membrane anchored extracellular α2δ subunit and the integral membrane γ subunit. All the auxiliary subunits affect the activity of all pore forming α1 subunits differently and to different degrees^6-9. In 2004, the high resolution crystal structures of the core domain of three β subunits as well as of β subunits complexed with part of the Ca_V α1 subunit, were published, shedding light on the intersubunit relationship within a Ca_V complex^10-12,13.

The ten Ca_V α1 subunit genes are divided into three subfamilies on both a functional and sequence homology basis (Table). The Ca_V1 subfamily includes four genes that encode L-type channels and the Ca_V3 subfamily includes three genes that encode T-type channels. The Ca_V2 subfamily contains three isoforms encoding P/Q- , N- and R-type channels, with all three contributing to neurotransmitter release in neurons¹⁴.

The Role of Voltage Activation

The control of Ca²⁺ entry into the cell by selective pathways, which sense the voltage of the plasma membrane, enables fast, localized and brief transduction of electrical signal- into cytosolic Ca²⁺– signal. Hence, the four voltage sensors in each Ca_V channel detect action potentials (and many other voltage signals) and induce a conformational change which allows Ca²⁺ ions to permeate into the cell resulting in a brief Ca²⁺ influx. This elevation of the Ca²⁺ concentration near the channel might activate any Ca²⁺ dependent enzyme or processes, which senses it. Two examples which illustrate such very localized signaling are synaptic transmission and calmodulin modulation of channel function.

The process of neurotransmitter release from neuronal nerve terminals is triggered by action potential invasion of the terminal leading to opening of Ca_V channels. Ca_V2 subfamily channels are localized in nerve terminals in the vicinity of docked synaptic vesicles which contain the neurotransmitter. Upon fusion of the vesicles with the plasma membrane the neurotransmitter is released. The “fusion machinery” consists of a number of SNARE proteins (on both the plasma and vesicle membranes), among them Ca²⁺ dependent proteins. These are activated directly by Ca²⁺ entering via Ca_V channels, which are already complexed with parts of the “machinery” such as syntaxin^15,16. Such a process highlights the critical role played by Ca_V channels in the transformation of an electrical signal in one cell (or cell population) into a chemical signal affecting neighboring cells (synaptic transmission) and remote cells or organs (hormone secretion).

Several Ca_V channels were shown to have an inactive (Ca²⁺ free) calmodulin tethered to a cytoplasmic C-terminus of the protein. Once the channel opens in response to membrane potential depolarization, the tethered calmodulin binds the entering Ca²⁺, is activated, and in many cases enforces channel closure (Ca²⁺dependent inactivation), forming a negative feedback loop on the Ca²⁺ entry activity¹⁷.

Tissue Distribution

Although all Ca_V channels are mostly studied in excitable tissues, i.e. nerve and muscle, their presence and roles in other tissues (such as hormonal glands,^18,19 immune system cells,²⁰ kidney,²¹ tumors²² etc.) are important in a physiological and pathological context. Ca_V channels control key points in the Ca²⁺ activation of processes such as neurotransmitter release, skeletal, cardiac and smooth muscle contraction, gene expression, apoptosis and proliferation.

Some Ca_V specific isoforms or splice variants are to a large extent cell type specific. For example, Ca_V1.1 is expressed almost specifically in skeletal muscle and the protein functions as the main L-type channel in these cells, while the Ca_V1.4 isoform is expressed mainly in the retina²³ and T-lymphocytes²⁰ and underlies their L-type currents. In contrast, Ca_V1.2 is widely expressed, and this gene product underlies L-type currents in both cardiac and smooth muscle myocytes, in many neurons and pancreatic β cells. However, different gene products (splice variants) of the Ca_V1.2 gene might be selectively expressed in one tissue compared to another^24,25. Ca_V1.3 is also expressed in several different tissues and knockout mice are affected with hearing loss and cardiac arrhythmia²⁶ as well as with impaired glucose metabolism²⁷.

The Ca_V2 family’s most notable function is in linking membrane potential depolarization to neurotransmitter release in presynaptic nerve terminals. As such, these channels are key regulators of neuronal communication. Within various nerve cell populations, Ca_V2 family members are differentially expressed, sometimes with overlapping patterns²⁸. Here as well, splice variants may be specifically expressed. A most interesting example is related to pain control with regard to an N-type channel (Ca_V2.2) variant, which is selectively expressed in sensory neurons in DRG²⁹. Ca_V2 channels are modulated by G-protein coupled receptors, a cellular pathway that is most important to physiological processes such as hormonal regulation of neuronal activity as well as in clinical interventions such as morphine-induced analgesia³⁰.

The three T-type (Ca_V3.1-3) channel isoforms are also widely and differentially distributed and for members of this subfamily as well specific expression of splice variants was demonstrated³¹.

Channelopathies, Toxins and Clinical Ca_V Channel Blockers

Mutations in Ca_V channel genes are linked to several inherited neuronal (Ca_V2.1) and muscular diseases (Ca_V1.1)³² as well to a multisystemic syndrome involving autism (Ca_V1.2)³³. The latter exemplifies the general role played by this L-type channel in regulating Ca²⁺ metabolism in excitable as well in non-excitable tissues (such as bone and lung). Although these mutations are rare, pharmacological interventions that target Ca_V channels are widely used in the clinic,^34-37 among them antihypertensive drugs acting on smooth muscle Ca_V1.2 channels or anticonvulsant drugs that target brain T-type channels. Recently, the FDA has approved Prialt^TM (Ziconotide) a synthetic version of ω-Conotoxin MVIIA, as a painkiller for some indications^38,39.

One of the first methods used to differentiate among voltage dependent Ca²⁺ channels in assessing their particular physiological roles, is based on differential pharmacological sensitivities. Particularly, the differentiation between the presynaptic Ca_V2 subfamily isoforms was facilitated due to specific toxin sensitivities. The cone snail peptides ω-Conotoxin GVIA and ω-Conotoxin MVIIA⁴⁰ inhibit the Ca_V2.2 channel potently and specifically, while the spider toxin ω-Agatoxin IVA⁴¹ is specific for Ca_V2.1. Later, the tarantula peptide, SNX-482⁴² was shown to be a specific blocker of Ca_V2.3, which was associated with the toxin resistant R-type current. These toxins were used to block transmission in different synapses^43-45.

Other compounds that block Ca_V channels are very useful in differentiating between subfamilies but have little selectivity toward specific isoforms within the subfamily. The Ca_V1 subfamily proteins are often referred to as DHP receptors, as this group of chemical compounds interacts specifically with Ca_V1 channels either as antagonists (such as Nimodipine) or agonists (i.e. (S)(-)-Bay K8644). Other examples include, the scorpion peptide kurtoxin, that blocks all three T-type channels with similar potencies, but has little effect on other Ca_V channels^46,47. In a similar manner Calcicludine^48,49 and Calciseptine^50,51, toxins purified from the venom of the green and black mamba respectively, block L-type channels specifically, but little is known regarding their selectivity between Ca_V1 subfamily channels.

Acknowledgements: We thank Annette C. Dolphin, Ph.D. from Department of Pharmacology, UCL, UK for her helpful comments.

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