Introduction

Calcium channels play an essential critical role in a variety of physiological functions in cells. They include all pore-forming membrane proteins that are calcium-permeable and used for the transport of these ions across cell membranes. As an ion, calcium is unique in biological systems; this is because calcium not only functions to generate membrane potentials and electrical signals but also functions as a central cell signaling molecule. Therefore, calcium channels play an even more involved role in the cell by allowing for the generation of a multitude of cellular responses. Calcium channels come in many forms and are incredibly diverse in both structure and function.[1] 

Furthermore, there are a variety of methods by which they may be categorized. To provide an overview of calcium channels, it is necessary to describe the different types of calcium channels, their structures, functions in cellular responses, pharmacological properties, and associated pathologies. This article will attempt to summarize this diverse class of proteins to elucidate their nature at the molecular, cellular, tissue, and organismal levels.

Fundamentals

Ion channels are pore-forming integral membrane proteins that are composed of various combinations of subunits.[2] Embedded in cell membranes, these subunits associate with one another, and together they form a channel in the membrane that permits specific ions to pass through. Ion channels generally consist of three main components, a selectivity filter that provides ion specificity, a gate to control ion flow, and an ion-permeable pore, which provides a pathway for ions through cell membranes. As their name suggests, calcium channels are permeable to calcium ions and play a critical role in the functioning of all eukaryotic cells.[3] Their primary function in the cell is to regulate the concentrations of calcium inside the cell and throughout different cellular compartments. The regulation of calcium ion concentrations is critical for proper cellular functioning since calcium plays a role in almost all cellular processes.[4] It achieves this through a variety of means different means, some of which include acting as a second messenger and contributing to an electrochemical gradient.[5][4]

The concentration of calcium inside the cell can vary widely depending on location.[4] At rest, calcium channels contribute to maintaining an extremely low concentration of cytoplasmic calcium, at 10(-7)M [5], which is about 1000 times lower than extracellular calcium concentrations. Dysfunctional regulation of calcium concentrations can result in serious deleterious effects on cells, including apoptosis and cell death.[5][4][3] Because calcium regulates almost every aspect of cell function, the calcium concentration and its signaling processes in the cell require tight regulation. Calcium channels must balance the influx of calcium into the cell and the efflux of calcium out of the cell.[6] They can be stimulated by a variety of stimuli including, but not limited to, membrane depolarization, signaling molecules (both extracellular and intracellular), and physical forces acting on the membrane itself like stretch or temperature.

A plasma membrane is an important place for calcium channels to carry out their function by regulating the entry and exit of calcium from the cell. One of the most important types of calcium channels found on plasma membranes is voltage-gated calcium channels, which rapidly transport calcium into the cytoplasm. These channels respond to a change in voltage across the cell membrane, which is driven by an electrochemical gradient. They are especially critical for the initiation of cellular processes, including muscle contraction and gene transcription.[4]

Other calcium channels exist on the plasma membrane to transport calcium out of the cell to provide a very low cytosolic concentration. These channels include various calcium channel pumps and exchangers. Among these are the plasma membrane calcium ATPase (PMCA) pump and the sodium/calcium ion exchanger (NCX), which expend large amounts of energy to function.[4] These channels are critical to the cell because a low cytosolic calcium concentration is vital for it to be used as a second messenger.[5] Also, these pumps and exchangers are necessary to terminate calcium-dependent cellular responses. Other calcium channels also exist on the plasma membrane. Specialized cell types, for example, contain unique varieties of calcium channels on their plasma membranes. In neurons, calcium channels open in response to the binding of neurotransmitters to cell surface receptors that are permeable to calcium, such as NMDA and AMPA receptors. [3] Another example is in sensory cells, whose plasma membrane calcium channels open in response to mechanical stimuli like stretch and temperature such as Transient Receptor Potential (TRP) channels.[3] Ultimately, the plasma membrane serves as an essential place for calcium channels and provides both a source of calcium and a place to excrete it.

Calcium is so essential for intracellular processes that the cell stores it in high concentrations, particularly in the endoplasmic reticulum (ER) (sarcoplasmic reticulum (SR) in muscle cells). The ER/SR serves as the main storage site for calcium inside the cell.[6] Like the plasma membrane, calcium channels are embedded in the membranes of the ER/SR to regulate the transfer of calcium between the cytoplasm and the endoplasmic/sarcoplasmic reticulum.[6] Storage in the ER/SR also provides an additional place to transport calcium to keep cytoplasmic calcium concentrations low. A particularly important calcium channel is the sarcoplasmic reticulum calcium ATPase (SERCA) pump, which transports calcium from the cytoplasm to the sarcoplasmic reticulum and complements the actions of PMCA and NCX to decrease cytosolic calcium concentrations.[4][2]

Other channels exist to transport calcium in the opposite direction from the endoplasmic/sarcoplasmic plasmic reticulum to the cytoplasm, such as the inositol triphosphate (IP3) receptor and ryanodine receptor (RyR).[3] By releasing calcium into the cytoplasm, these channels rapidly provide the calcium necessary for the cell to perform various calcium-dependent intracellular processes. By mediating actions like calcium-induced calcium release (CICR) and the activation of intracellular calcium signaling pathways through the macromolecular coupling of receptors, these calcium channels are critical in mediating calcium "on" responses. The transport of calcium across the ER/SR contributes to overall calcium homeostasis and is responsible for regulating various cellular processes, including muscle contraction and cell differentiation.[7]

Once calcium has been released into the cytoplasm, the calcium stored inside the storage organelles of the ER/SR eventually becomes depleted. For the cell to continue performing calcium-dependent responses, these stores need to be rapidly replenished. Special calcium channels exist for this purpose called store-operated calcium channels, which mediate a process called store-operated calcium entry (SOCE). The way these channels function involve a complex series of events that have only recently come to light. They form when concentrations of calcium in the lumen of the ER are low, sensed by a calcium-sensing protein called the stomal interacting molecule (STIM). When calcium becomes low, the STIM protein oligomerizes and moves to the ER membrane, where it interacts with another protein called Orai1, referred to as a calcium-release activated channel (CRAC). The interaction between these two proteins creates a passageway for calcium between the membranes of the endoplasmic reticulum and the plasma membrane. Through the bypassing of these two different membrane systems, a direct passageway for calcium from the cell exterior to the primary storage organelles is made possible. Because the concentration of calcium is generally one thousand or more times greater outside of the cell than inside, it provides a powerful mechanism by which intracellular calcium levels are rapidly replenished.[8]

 All calcium channels play an essential role in calcium homeostasis and signaling in the cell. It is not possible to describe every type of calcium channel in this article. However, the essential calcium channels that contribute to the regulation of calcium concentration in the cell will be discussed. Ultimately, calcium channels are involved in a large number of different processes. They exhibit significant diversity in both structure, function, and pharmacology. The abnormal functioning of calcium channels is associated with a large number of pathological conditions. As a result, a greater understanding of calcium channels is necessary.

Cellular Level

Calcium channels perform functions that act not only on a molecular scale through the transportation of calcium ions, but on a cellular level by participating in whole-cell processes that affect an entire organism. As a result, the regulation of calcium is exceptionally precise. As stated previously, the concentration of calcium during resting conditions is kept extremely low by the actions of various calcium pumps, channels, and exchangers. The purpose of this is so that calcium may act as an intracellular signal, which may be initiated or modified by calcium channels on the plasma membrane or in membranes of intracellular organelles. These rapid calcium fluxes exhibit characteristics that provide the cell with additional information than signal stimulus intensity. These signals also give information that shows both spatial and temporal properties.[9] 

Furthermore, calcium signals may be self-regulatory, whereby calcium channels are regulated by calcium itself. As a result, not only does calcium act as a signal itself, but it can modify its own signal through its actions on calcium channel function. Because of the unique features of calcium channels, they can participate in functions much more complicated than merely the transportation of ions. Each cell type can carry out specific physiological functions by the presence of unique combinations of calcium channels on their cell membranes. Together, they provide information concerning the precise duration, frequency, timing, and amplitude of a calcium signal to control a particular calcium-dependent signaling process.

All calcium channels generate brief pulses or transients of calcium by their opening.[10] By their nature, these calcium transients can generate a variety of different kinds of signals via the temporal characteristics of calcium release existing on different possible time scales. At the smallest end of the time scale, for example, calcium transients generated by fast calcium channels can mediate processes such as exocytosis of neurotransmitters and hormones on the order of milliseconds by creating rapid calcium fluxes. Effector systems are present in the cell to respond to these brief calcium transients to perform processes that must occur rapidly. Moving up the time scale, repeated activation of transient-generating calcium channels may generate slightly longer, more robust calcium signals if occurring over an extended duration in the form of calcium "waves."

Calcium waves spread throughout the cytoplasm and occur on the order of seconds to minutes. At this time scale, ryanodine receptors, for example, mediate a self-regenerative calcium amplifying process known as calcium-induced calcium release (CICR) through positive feedback, which is essential for muscle contraction. Calcium channel-generated calcium waves are also involved in modifying the actions of various calcium-dependent proteins such as CaMKII, MAP-kinases, and CREB, which can modify intracellular processes.[5] Further up the time scale, the repeated generation of calcium waves over an extended period can result in the transformation of these calcium waves into calcium "oscillations." Calcium oscillations occur in the order of minutes to hours and carry implications in a variety of complex cellular processes such as cellular proliferation, axonal growth, and cell migration.[10] In total, calcium channels influence cellular processes through the generation of temporal signals, which allow them to participate in the activation, modification, and regulation of complex cellular processes.

Calcium channels also generate signals that are spatial in nature that dependent on highly localized signaling structures and processes. For example, the release of calcium by a single calcium channel generates a localized increase in calcium just adjacent to the channel. This localized increase in calcium may create elementary calcium signals referred to as calcium "puffs" or "sparks," depending on the channel and can initiate highly localized responses; this is made possible by the ability of some calcium channels such as IP3 and ryanodine receptors to form organized and highly localized macromolecular complexes with other proteins such as receptors (i.e., G-protein coupled receptors or receptor tyrosine kinases) or calcium-binding/calcium-dependent proteins.[10] 

These organized macromolecular signaling processes are the bases of calcium signaling "units," which can function autonomously. These calcium signaling units can then be summed, multiplied, or otherwise calculated together to create even more complex cellular responses. Examples of processes that involve these organized macromolecular complexes include the synaptic integration of information by single neurons. Input-specific calcium-dependent synaptic modifications occur during the processes of learning and memory on individual dendritic spines, and they depend on localized processes that occur adjacent to single calcium channels.[11]

Calcium channels do not simply open and close to allow the passage of calcium. Calcium channels can open and close in intricate patterns that contain specific temporal and spatial characteristics to produce complex cellular responses. The cell has utilized calcium channels not only to provide calcium for calcium-dependent cellular processes but to encode information that accompanies the release of calcium to produce specific intracellular signals. Through this, calcium channels mediate processes that affect the entire cell addition to the affecting processes at the molecular level.

An example of a whole-cell process controlled almost entirely by the actions of calcium channels is in the regulation of cardiac muscle contraction in cardiac myocytes. Calcium channels are the structural components of cardiac cells that provide a mechanism to modulate the force of contraction. One of the ways that this occurs is through beta-adrenergic receptor (b-AR) stimulation to cause a positive inotropic response that is regulated by protein kinase A (PKA). The initiation of a cardiac muscle contraction is mediated by the entry of calcium through L-type voltage-gated calcium channels on the membrane of the sarcoplasmic reticulum.

During each action potential, cAMP is produced by the Ga(S) subunit of the B-adrenergic receptor upon activation by norepinephrine. PKA becomes activated by cAMP resulting in widespread changes in calcium channel function. As a kinase, it displays phosphorylative activity to modify the actions of calcium channels, specifically L-type VGCCs, SERCA, and RyRs. When L-type VGCCs are phosphorylated, each action potential can produce a larger flux of calcium resulting in potentiation of channel currents. Secondly, PKA phosphorylates phospholamban, which disinhibits the actions of the SERCA pump, which results in more calcium available in the sarcoplasmic reticulum for muscle contraction. Thirdly, phosphorylation of ryanodine receptors by PKA increases their ability to generate the calcium sparks necessary for calcium-induced calcium release (CICR), which is required to initiate muscle contraction. Ultimately, this process is an example where calcium channel activity can be modified to produce different responses by the sympathetic nervous system through kinase-mediated phosphorylation.[12][13]

Molecular Level

Both PMCA and SERCA are P-type ATPases. All P-type ATPases exhibit a similar structure and mechanism of action. Both of these pumps use the energy derived from ATP to pump calcium against their concentration gradients.)[14] The PMCA and SERCA pump both contain a 10-transmembrane spanning domain called the "M" (membrane) domain. Also, they contain several cytosolic domains, including an "A" (actuator) domain, an "N" (nucleotide-binding) domain, and a "P" (Phosphorylation) domain.[15] The amino carboxy-terminal of these peptides both face the cytosolic compartment.[15][16][9] The active site of the PMCA pump is located between membrane segments 4 and 5, which forms a cytosolic loop and contains the ATP-binding and phosphorylation site. The PMCA pump expresses in all tissues and cell types. It exhibits a 1:1 Ca/ATP stoichiometry.[17][9][18] In contrast, the SERCA pump has a 2:1 Ca/ATP stoichiometry.[14]

VGCCs are located on the plasma membrane and open in response to a change in voltage across the plasma membrane. Currently, there are ten known subtypes of voltage-gated calcium channels, each with a different molecular structure and variations in subunit composition.[19][20] All voltage-gated calcium channels contain one of ten different a1 subunits. The a1 subunit that a particular VGCC channel contains determines to which subtype the channel belongs. In addition to the a1 subunit, there are also four auxiliary subunits: A beta (B), a2, delta, and a gamma subunit, with the a2 and delta subunits connected by a disulfide bond.[21][19][22] The auxiliary subunits are not present in every subtype of voltage-gated calcium channels. Instead, different subtypes are composed of different combinations of these subunits. Furthermore, there are different subtypes of the auxiliary subunits, with ten distinct a1 subunits, four distinct beta subunits, and four distinct a2d subunits that have so far been identified.[19][20] Voltage-gated calcium channels subdivide into three main classes: Cav1, Cav2, Cav3, which represent L-types (Cav1.1-1.4), P/Q-type (Cav2.1), N-Type (Cav2.2), R-type (Cav2.3), and T-type (Cav3.1–3.3) based on the particular a1 subunit. For example, the Cav2.2 N-type VGCC contains the Cav2.2 a1 subunit. The a1 subunit present in a particular subtype of VGCC is the main determining factor that dictates a channel's biophysical and pharmacological properties.[20][23]  

The a1 subunit contains the channel gate, which controls ion flow, the voltage sensor which controls channel activation, and the ion-conducting pore for calcium ions to flow through.[10] The a1 subunit is also the most common site of regulation by proteins, second messengers, toxins, and pharmacological agents. Each VGCC produces a current that is distinct in amplitude, the voltage of activation, and the voltage of inactivation based on its a1 subunit and auxiliary subunit composition. The structure of the a1 subunit consists of 4 homologous groups of six transmembrane-spanning segments, where each group represents a different domain (labeled I-IV). The transmembrane segments (labeled S1-S6) are present in each group. In-between the transmembrane segments S5 and S6 for each domain is a membrane-associated peptide loop continuous with these two segments extruding from the extracellular side of the membrane. Another peptide loop extrudes into the cytosolic side of the membrane connecting S6 of the previous domain to S1 of the next domain.[24][12][19][25] The pore itself consists of the S5 and S6 transmembrane segments along with the P-loop.[19] The transmembrane-spanning domains S1-S4 are linked to pore and form the voltage sensor.[22] Finally, the amino and carboxyl terminals provide a regulatory function of channel function through interaction with regulatory proteins.[24]

L-Type VGCCs are found in both excitable and non-excitable cells and are present throughout the body.[26] There are four types, Cav1.1 Cav1.2, Cav1.3, and Cav1.4.[19] They are especially present in muscle cells, including skeletal, cardiac, and smooth muscle. Also, they are in endocrine and kidney cells.[25][23] Skeletal muscle contains the Cav1.1, whereas the Cav1.2 channel is prevalent in cardiac muscle. Furthermore, the Cav1.3 is prevalent in the sinoatrial node of the heart, and the Cav1.4 channel is prevalent in the retinal cells.[23] This class of channels is most often associated with the beta and a2d subunits.[20] A distinguishing feature of L-type voltage-gated calcium channels is that voltage-dependent inactivation.[19] Also, show high sensitivity to calcium channel blockers.[27] T-Type calcium channels are also referred to as low-voltage activated channels. They contain the Cav3 family of a1 subunits. They are distinguished by their voltages of activation, which occur at negative membrane potential.[19] They are particularly important in the cardiovascular system, where they can be found in cardiac myocytes of the sinoatrial node. Also, they are especially prominent in the thalamus and other areas of the brain.[19][23] Due to difficulties in distinguishing between P and Q-type calcium channels, researchers have instead classified them as P/Q-type VGCCs. These calcium channels contain the Cav2.2 a1 subunit. P-type VGGCs are highly sensitive to a toxin called v-agatoxin IVA, which is found in spiders. This sensitivity provides a method for their identification. Q-type VGCC is also sensitive to this toxin, but with lower affinity.[12] In contrast to L and T-type VGCCs, N-Type VGCCs activate at intermediate membrane voltages contain voltages of inactivation that are more negative than L-type and more positive than T- Type.[12] They are associated with only one a1 subtype, the Cav2.2 subtype.[19] The kinetics of N-type VGCCs vary considerably in different cells.[12] Lastly, the R-type VGCCs are distinguishable by their resistance to a variety of different organic and peptide calcium channel blockers. They are associated with the Cav2.3 a1 subunit.[12]

 Most subtypes of voltage-gated calcium channels contain some combination of auxiliary subunits, principally the beta and A2sd subunits. All of the subunits take part in modifying channel gating, assembly, and insertion of the channels into the plasma membrane, among other functions.[22][28] The subunits undergo extensive alternative splicing, which is a source of functional differences between channels.[19]

The beta subunit can be found in both the Cav1 (L-type) and Cav2 (P/Q, N, and R-type) families of voltage-gated calcium channels.[19][20][23] It influences both membrane trafficking and the expression of voltage-gated calcium channels.[19][20][23] The beta subunit is localized on the cytoplasmic side of the membrane at the intracellular surface and interacts with domains I and II of the a1 subunit.[21][19] Like the beta subunit, the a2d subunit contributes to membrane trafficking and overall expression of the Cav1 and Cav2 classes of VGCCs.[20][19][23] The beta and a2d subunits appear to act synergistically in this function[20]. This subunit is composed of an a2 subunit and delta subunit linked by a disulfide bond.[20] It is located on the extracellular side of the membrane and contains a single transmembrane domain.[12][24] The a2d subunit of voltage-gated calcium channels can be found in both the Cav1 (L-type) and Cav2 (P/Q, N, and R-type) families of voltage-gated calcium channels.[19] The gamma subunit is a membrane-bound protein containing four transmembrane segments.[19] This subunit is thought to influence VGCC channel function and gating. It also regulates membrane trafficking and interacts with the voltage-sensor of the a1 subunit via a linker segment.[24][19]

IP3 channels are consist of four subunits that make up a tetrameric channel embedded in the membrane of the endoplasmic and sarcoplasmic reticulum.[29] The IP3R monomers have a binding pocket, a regulatory domain in the cytosol, and a pore region. The pore region is composed of a six-transmembrane spanning helix located near the C-terminus through which calcium flows. The ligand-binding pocket is located near the N-terminal and binds to IP3.[10] An essential aspect of these channels is the various scaffolding and cytoskeletal proteins it associates with to form macromolecular complexes with other receptors and provide stability. Furthermore, various adaptor proteins link the IP3 receptor to different other molecules in the cell.[30] Finally, various calcium-binding proteins also have binding sites on the channel to modulate their function.[30] Its ligand-binding site for inositol triphosphate (IP3) is located near the N-terminal of the protein.[31] The ligand-binding site must be occupied on all four monomers by IP3 to induce the necessary conformational change that sensitizes the channel to calcium.[29] Once the channel undergoes this conformational change and calcium has bound to its binding site, the channel opens. Calcium then passes through from the ER or SR to the cytoplasm. This sequence of events often follows the activation of another receptor, such as a G-protein-coupled receptor or receptor tyrosine kinase on the plasma membrane to which the IP3 receptor couples. These cell surface receptors activate an intracellular signaling pathway that leads to the activation of phospholipase C (PLC), which produces IP3 as one of its end-products.

The ryanodine receptor displays some structural similarities to the IP3 receptors. It is also composed of four subunits, which form a homotetrameric channel.[32][33][34] There are multiple subtypes of ryanodine receptors, with the RyR1 subtype being prominent in skeletal muscle cells and RyR2 being prominent in cardiac muscle cells.[32][35][33] Interestingly, ryanodine receptors are the largest known ion channel.[32][35] This receptor can also be found in other cell types, including but not limited to neurons, epithelial cells, and endocrine cells.[33]

Finally, The Orai subunit of store-operated calcium channels is a four transmembrane-domain spanning protein containing two extracellular loops, one intracellular loop, and cytosolic N and C-terminals.[36][37][38] Unlike its partner, STIM, it is embedded into the plasma membrane and is the pore-forming subunit.[37][38] In contrast, STIM is a single transmembrane protein localized on the ER membrane and contains a calcium-binding EF-hand motif on the lumenal side of the ER to sense calcium levels.[36][38][39] It expresses ubiquitously in cells.[37][36][38][40]

Function

The primary functional role of the PMCA pump is to transport calcium against its concentration gradient out from the cytoplasm out of the cell through the plasma membrane.[9] It plays an essential role in "fine-tuning" calcium levels rather than creating large-scale calcium concentration decreases.[9] This is because although it has a high affinity for calcium, its capacity for ion transport is low.[18][9] Therefore, it is an important protein involved in maintaining long-term rather than short-term calcium homeostasis by removing excess calcium that has entered the cell.[17][9] Furthermore, its role as a fine-tuner of calcium levels is vital for calcium-dependent signal transduction pathways, as the cell is extremely sensitive to calcium concentration.[9]

The main functional role of SERCA is to pump calcium from the cytoplasm back into the lumen of the sarcoplasmic reticulum, which is essentially the endoplasmic reticulum of muscle cells.[12][7] It ensures the maintenance of low levels of cytosolic calcium by pumping it into the lumen of the sarcoplasmic reticulum after calcium has been released into the cytoplasm by various calcium-release channels in the cell. In this capacity, it serves as the main controller of cytoplasmic calcium levels in muscle cells.[18] it transports two calcium ions per 1 ATP hydrolyzed. Also, one of its most important functions is to prevent the buildup of charge across the sarcoplasmic reticular membrane.[7][12] 

The Na+/Ca+ exchanger also is responsible for keeping cytosolic calcium levels low. Unlike PMCA and SERCA, it has a low affinity for calcium but a high transport capacity.[11] As a result, it rapidly exports large amounts of calcium and has significant actions on intracellular calcium concentrations.[11]

Voltage-gated calcium channels play a crucial role in various cellular processes. In particular, they act as key signal transducers by influencing the electrical excitability of multiple cell types, such as neurons and endocrine cells. They convert electrical signals in the form of action potentials or depolarization into the form of a calcium transient inside the cell by generating rapid calcium fluxes into the cell. These rapid fluxes of calcium are important for mediating fast cellular processes such as muscle contraction, initiation of neurotransmission, and secretion in muscle cells, neurons, and endocrine cells, respectively. In other cell types, the influx of calcium into the cytosol via VGCCs regulates various biochemical processes, such as enzyme activity and gene expression.

Different subtypes of  VGCCs have different electrophysical properties in terms of current and voltages of activation. As such, these channels mediate the activation of various signal transduction pathways and are involved in diverse cellular processes. For example, in cardiac and smooth muscle cells, large influxes of calcium into the cytoplasm by voltage-gated calcium provide the calcium required to initiate the process of calcium-induced calcium-release (CICR) in ryanodine receptors of the sarcoplasmic reticulum, which is essential for initiating muscle contraction. In skeletal muscle cells, VGCCs are mechanically linked to ryanodine receptors and control their activation directly via conformational changes.[12] 

The Cav1 family of VGCCs plays an important role in the nervous system by contributing to the integration of synaptic input and transmission at sensory synapses. Furthermore, they play important regulatory roles in gene expression, secretion, and muscle contraction in skeletal and cardiac muscles.[12] L-type voltage-gated calcium channels are located in the cell membranes of transverse tubules of skeletal muscle. They are mechanically linked to ryanodine receptors, where their activation leads to ryanodine receptor channel opening and initiation of skeletal muscle contractions.[27] In endocrine cells, VGCCs are responsible for mediating the secretion of hormones. Cav1.1 channels are also involved in the fight or flight response, which is initiated by the actions of catecholamines, which bind to b-adrenergic receptors. When this occurs, the force of contraction is increased in both skeletal and cardiac muscles. In cardiac muscle, the excitation-contraction coupling is initiated by Cav1.2 channels, which mediate PKA mediates calcium influx and the increase in the force of contraction. PKA-mediated phosphorylation of Cav1.2 channel proteins by b-adrenergic receptor-induced cAMP signaling occurs upon activation of the b-adrenergic receptor, which increases L-type calcium channel currents.[12] L-type calcium channels, specifically Cav1.2 and 1.3, are known to be implicated in several nervous system processes, including the acquisition of fear, long-term potentiation, and the expression of certain emotional behaviors.[41] L-type calcium channels of the Cav1.3 and 1.4 a1 subtypes are involved in neurotransmitter release.[41]

P/Q-type calcium channels (Cav2.1) play a crucial role in neurotransmission. They are involved in linking neuronal excitation in pre-synaptic neurons to the secretion of neurotransmitters by mediating the increase in local calcium concentration in the pre-synaptic cell.[28]

IP3 receptors have a diverse variety of functions in the cell. It is a component of one of the many calcium signaling pathways in the cell, which is highly versatile.[29] IP3 receptors are involved in regulating processes that occur in a wide variety of cell types, including neurons, liver cells, muscle cells, glands, and many others. Such processes include synaptic transmission, metabolism, muscle contraction, secretion, and development, among others.[29]

P3 receptors function as a hub for cell signaling activity by forming macromolecular complexes with various other molecules to create signaling cascades and activate cellular pathways.[30][31] It amplifies signals generated at the plasma membrane by stimuli such as neurotransmitters, hormones, and growth factors by coupling to their receptors to generate an intracellular response.[30][31][29] Another very important function of IP3 receptors is to generate temporal calcium signals in the form of oscillations. Calcium is normally released by IP3 channels as brief transients but may transform into oscillations after an extended period.[30][29]

IP3 receptor channels also play a critical role in various processes during development. In neurons, they play a role in synaptic plasticity and neuronal growth. For example,  IP3 receptors are necessary for calcium release in dendritic spines during long-term potentiation. Furthermore, the guidance of axons to neuronal targets and the formation of neuronal growth cones are dependent on IP3 receptors. They are also necessary to regulate cell proliferation.[30][31][29] After development, their oscillations contribute to the maintenance and regulation of various brain functions, including the regulation of brain rhythms.

Ryanodine receptors play a crucial role in the normal functioning of muscle cells. In muscle cells, the cytoplasmic domain of RyRs is coupled to L-type voltage-gated calcium channels on the cell membrane. Electromechanical coupling between these two receptors allows depolarizations in the cell membrane to directly control the gating of the ryanodine receptor.

Critical for excitation-contraction coupling in both skeletal and cardiac muscle.[32] Mediate calcium-induced calcium release, which causes a rapid rise in intracellular calcium levels for calcium-dependent intracellular reactions. In endocrine cells, they are involved in the secretion of hormones. In the nervous system, they are involved in signal transduction and are implicated in synaptic plasticity.

One of the most important functions of ryanodine receptors is to play a critical role in excitation-contraction coupling, which links electrical signals to calcium release during the initiation of muscle contraction.[32] Interestingly, the channel's main ligand is calcium, which is also its permeating ion.[33][34] This allows it to participate in a process called calcium-induced calcium release, which amplifies calcium release in response to the release of calcium through voltage-gated calcium channels.[35][34] Also, the ryanodine receptor has endoplasmic/sarcoplasmic reticular calcium-sensing ability.[33] Not only is it involved in muscle contraction, but it plays other important roles in various processes, including apoptosis, secretion, and nervous system processes.[35] Like the IP3 receptor, the ryanodine receptor provides a platform for the formation of large macromolecular complexes, which contributes to its extremely large size.[35] As such, it, therefore, plays an important role in various signal transduction processes.[32] Overall, ryanodine receptors are critical for maintaining calcium homeostasis in the cell.[34]

Store-operated calcium channels are located on the membrane of the endoplasmic/sarcoplasmic reticulum. They play an essential role in maintaining a constant store of calcium when required by the cell. Calcium continually leaks out of the endoplasmic reticulum into the cytosol during normal cellular processes. To avoid depletion of the cell's calcium stores, the cell utilizes store-operated calcium channels. Although the SERCA pump provides the initial storage of calcium, upon generation of cellular response and the utilization of second messengers, of which calcium is an important messenger, calcium gets used by the cell for such processes, and eventually, the calcium stores become depleted. As such, store-operated channels function to avoid the complete emptying of calcium. Essentially, store-operated calcium channels act as mediators of communication for the cell between the endoplasmic reticulum and the plasma membrane and provide vital information regarding the status of calcium homeostasis at any given time.

Mechanism

SERCA and PMCA belong to a group of ion pumps called P-type ATPases. P-type ATPases exist in two conformational states: E1 and E2. In the E1 conformation, the pump exhibits a high affinity for calcium on the cytoplasmic side of the cell. In the E2 conformational state, the transporter’s affinity for calcium rapidly decreases and subsequently leads to the release of calcium to the other side of the membrane.[42] Calcium ATPase transporters use the energy directly from ATP to pump calcium against its concentration gradient. This is known as primary active transport.[11][15] 

When calcium concentrations are low in the cytosol, the PMCA pump is in the resting position, elicited by the blocking of the phosphorylation site by the C-terminal auto-inhibitory domain. A calcium-sensing protein called cAM binds to calcium when concentrations of cytosolic calcium increase. This causes a conformational change driven by the interaction of Ca-cAM and the autoinhibitory site, which dissociates from the active site. Calcium is transported to the extracellular environment when it ATP-phosphorylates a conserved aspartate residue.[9]

The voltage sensor for the voltage-gated calcium channels is located at the S4 segments of each a1 homologous domain. Under the influence of an electrical field, these segments undergo conformational changes that cause them to move outward and rotate, causing the pore to open. The lining of the pore within the channel is composed of segments S5 and S6. Required for calcium selectivity is a glutamate residue located inside the pore loop. At the S6 segments are the binding sites for the organic calcium channel antagonists.

Voltage-gated calcium channels are highly regulated, specifically through G-protein coupled receptors and second messengers.[43] Voltage-gated calcium channels and G-protein coupled receptors can form large macromolecular complexes with one another, which leads to the regulation of the expression and activity of voltage-gated calcium channels.[44][20] Also, voltage-gated calcium channels are sensitive to phosphorylation, which provides an important mechanism for their regulation. Specifically, the beta subunit of voltage-gated calcium channels is targeted by protein kinase A (PKA), CaM-KII, and the PI3K/Akt, which serve to modulate both the electrophysical properties of voltage-gated calcium channels and their expression.[20] N and P/Q-type calcium channels become inhibited by the (b)(y) subunit complex of certain subtypes of GPCRs, which dissociate from the receptor and bind to voltage-gated calcium channels. GPCRs make up many different types of receptors in the body, including the brain. D1, for example, are GPCRs that interact directly with N-type voltage-gated calcium channels to influence the expression of voltage-gated calcium channels on the cell surface, resulting in changes in neurotransmission.[20][43]

The NCX does not use the energy from ATP directly. Instead, its energy derives from the sodium concentration gradient, which is used to move calcium against its concentration gradient. This process is called secondary active transport.

The formation of IP3 occurs in response to various stimuli, including neurotransmitters, hormones, and growth factors. These stimuli act to stimulate IP3 production via activation of either GPCRs and RTKs, which are coupled to phospholipase C. Upon the binding of these ligands to their receptors, a downstream signaling pathway is activated via the hydrolysis of the G-alpha and G-beta/gamma subunits of G-protein coupled receptors causing the activation of phospholipase C. PLC converts a molecule called PIP2 into IP3 and DAG. Phospholipase C then produces IP3.[30][29][14]

A variety of means perform the regulation of the IP3 receptor function. Central to its functions in cell signaling are inositol triphosphate (IP3) and calcium. Both of these are the primary controllers of IP3 receptors and are its two main ligands, of which calcium is its molecule of transport.[29] Its functioning is also affected by phosphorylation by various kinases such as PKA and PKC, various calcium-binding proteins such as CaMKII, and calcium itself.[30] Calcium both directly and indirectly affects IP3 receptor functioning.[30] It has direct potentiating effects on IP3 receptors when it is present in low concentrations and an inhibitory effect when present in high concentrations.[30][29] Calcium released by the IP3 receptor also activates various kinases, such as protein kinase C, which sensitizes the IP3 receptor to further calcium release and ultimately influences downstream cellular events, including synaptic transmission, exocytosis, and apoptosis.[30] Furthermore, protein kinase-A mediated phosphorylation of IP3 has been linked to muscarinic receptor pathways.[30]

The expression of IP3 receptors is triggered by an increase in intracellular calcium levels via the activation of different receptors, including voltage-gated calcium channels, NMDA receptors, and D1 receptors.[29]

Calcium oscillations are generated by the IP3/calcium signaling pathway when the receptor produces calcium transients/spikes repeatedly over an extended period.[29] These calcium oscillations generate signals in the form of frequency. The control of the frequency of these calcium oscillations is dependent on the intensity of the stimulus that initiated them.[29] The oscillatory activity of IP3 receptors is regulated in part by CAM-KII, which is an important calcium-binding protein.[30]

The oscillations generated by both IP3 and ryanodine receptors enhance the activity of the sodium/calcium exchanger by releasing pulses of calcium, which depolarizes pacemaker cells by creating an inward calcium current. The SERCA pump refills calcium stores in the sarcoplasmic reticulum, which is relied upon for the periodic release of calcium from the SR by IP3 and RyR receptors. Ryanodine receptors are sensitized to release periodic calcium sparks upon the slow accumulation of calcium in the lumen of the endoplasmic reticulum by the SERCA pump. Toward the end of the pacemaker cell action potential, these calcium sparks begin to develop as a result of IP3 receptor calcium signaling. Immediately preceding and contributing to the pacemaker cell depolarization, the action potential is triggered when calcium is suddenly released by ryanodine receptors. The frequency and amplitude of these ryanodine-receptor mediated calcium sparks driving the pacemaker potential are increased by IP3 channel calcium release, which in turn are activated by beta-adrenergic receptor stimulation.[29]

IP3 channels mediate the oscillatory calcium signals that drive smooth muscle contraction and are essential for the maintenance of smooth muscle tone. These IP3 channels become activated by various neurotransmitters, including serotonin and norepinephrine. Signals in the form of oscillatory frequency vary according to neurotransmitter concentration. Vascular tone is driven by oscillations generated from the release of calcium stores, which in turn is driven by the presence of these neurotransmitters, whose concentration determines the extent of vascular smooth muscle contraction.

Ryanodine receptors are critical for both skeletal and cardiac muscle contraction. In these cells, it is involved in excitation-contraction coupling. This process is slightly different in skeletal muscle and cardiac muscle. In skeletal muscle, calcium currents through L-type voltage=gated calcium channels open ryanodine receptors via a mechanical coupling.[32] This occurs when the depolarization of transverse tubules of muscle cells consisting of invaginations of the plasma membrane leads to the opening of L-type voltage-gated calcium channels. In response, mechanical coupling to ryanodine receptors in skeletal muscle opens them, leading to a massive release of calcium from the sarcoplasmic reticulum and eventually triggering muscle contraction.[35][32][33] In cardiac cells, transverse tubule membrane depolarization causes the opening of L-type voltage-gated calcium channels, which triggers calcium-sensing ryanodine receptors to perform calcium-induced calcium release in response.[33] In both skeletal and cardiac muscle, the result is massive calcium release through ryanodine receptor channels, leading to muscle contraction.[35][33][32]

The mechanism by which SOCE channels operate is unique.[39][40] They are most often activated in response to activation of IP3 receptors, which leads to depletion of calcium stores in the endoplasmic reticulum.[38] Therefore, the activation of G-proteins and RTKs ultimately precedes SOCE channel activation as a result of phospholipase-C activation.[39][29][36] Additionally, activation of PM calcium influx channels.[38]

When calcium concentration in the endoplasmic reticulum is low, this is sensed by the EF-hand domain on STIM. The release of calcium from the EF-Hand domain promotes its oligomerization into dimers and more complex oligomers due to the exposure of hydrophobic surfaces. STIM then redistributes via diffusion into clusters to sites near the plasma membrane at junctional sites that form between the PM and ER membranes.[37] Conformational changes in STIM mediate this process.[38][37] At these membrane junctions, when STIM and Orai become close enough to each other, STIM binds to Orai, leading to the formation of a calcium channel called a calcium release-activated channel (CRAC), which allows the entry of calcium from an extracellular milieu into the endoplasmic reticulum.[40][39] Calcium then flows through the channel, and once the store becomes refilled, the two proteins unbind from each other and diffuse away from the junctional sites back to their resting position.[40]

Pathophysiology

Calcium channels appear to be involved in the pathophysiology of chronic pain. Synaptic nerve terminals located in the dorsal horn of the spinal cord receive action potentials propagating through primary afferent fibers, which occur upon activation of peripheral nociceptors in different organs and the skin. Pain is perceived when neurons activate in higher brain areas upon receiving an excitatory synaptic transmission from these dorsal horn synaptic terminals. The neuronal excitability of the afferent fibers is known to be regulated by Cav3.2 T-type VGCCs. Furthermore, neurotransmission in dorsal horn synapses is controlled by both Cav2.2 and Cav3.2 channels. In chronic pain conditions, these calcium channel subtypes have been shown to be upregulated.[21]

Voltage-gated calcium channels are thought to be involved in the pathophysiology of seizures, including absence seizures. Cav3.1 and Cav3.2 channels, for example, are expressed on thalamocortical and reticular thalamic neurons, and alterations in their activity have been shown to result in absence seizures, which may occur as a result of various mutations in these channels. Specific seizure disorders have links with specific calcium channels. For example, juvenile myoclonic epilepsy and juvenile absence epilepsy are associated with Cav3.2 (T-type) channel mutations. Importantly, more than 30 mutations have been found in just the Cav3.2 a1 subunit gene alone that have correlations with various types of epilepsies. Also, gain-of-function mutations have been found to affect the gating activity and plasma membrane trafficking of these channels. Increased seizure susceptibility associated with overexpression or overactivity of these channels also makes them an important target for treating absence seizures. Absence seizures are one of the hallmarks of idiopathic generalized epilepsy, which accounts for one-third of all epilepsies.[21]

All addictive drugs share a common underlying pathophysiology in addiction in that they all can increase dopamine in the mesolimbic dopamine system and change CREB-dependent gene expression in the ventral segmental area and nucleus accumbens, whether it be through activation of opioid receptors (opioids), nicotinic receptors (nicotine), GABA receptors (alcohol, barbiturates, benzodiazepines) or dopamine receptors (stimulants). Voltage-gated calcium channels are involved in these processes, which lead to several important neurobiological and behavioral changes associated with addiction. For example, changes in neuroplasticity associated with stimulant abuse appear to involve the CaV1 family of voltage-gated calcium channels. Increased glutamatergic activity on the VTA occurs upon activation of Cav1.3 channels resulting from chronic exposure to psychostimulants.

The activity of Cav1.3 channels, along with increased glutamatergic activity in the VTA, causes NMDA and AMPA receptor-dependent changes in gene expression, which involves the upregulation of Cav1.2 receptors in VTA neurons. Since the firing behavior of neurons in the VTA is differentially regulated by Cav1.2 and Cav1.3 channels, dopamine release is increased onto the D1 receptors of the NAc. This activity leads to the insertion of AMPA receptors onto the NAc neuron plasma membranes and mediates long-term changes in gene expression. Ultimately, these activities have been linked to behavioral sensitization to psychostimulants.[21]

Mutations in STIM and Orai are both associated with pathological diseases. Both gain and loss of function mutations lead to numerous pathologies.[40] For example, defects in the ability to form the CRAC channel are associated with severe combined immunodeficiency (SCID). The formation of Store-operated calcium channels is essential for T-cell lymphocyte function. Mutations in these channel proteins cause a reduction in the ability of T-cells to produce cytokines and may result in serious infections during infancy.[37] Other pathologies associated with abnormal or inactive SOCE function are myopathies and ectodermal dysplasia.[40]

Adrenergic receptors are important physiological mediators of both cardiovascular and smooth muscle activity. They are especially significant pharmacologically as they are the targets of a variety of drug classes, such as beta-blockers, alpha-blockers, and alpha-adrenergic receptor agonists. Epinephrine, a general adrenergic receptor activator, has a significant influence on cardiovascular and smooth muscle activity. This activity occurs in part through its actions on both the plasma membrane and intracellular calcium channels. In cardiac muscle, beta-adrenergic receptors are targeted by epinephrine and produce positive chronotropic, inotropic, and dromotropic physiological effects, which are mediated by their ability to increase the opening time of voltage-gated calcium channels. In vascular smooth muscle, both contraction and vascular tone are increased by norepinephrine by alpha-receptor activation due to its ability to increase cytosolic calcium levels through a depolarization-independent IP3-receptor-mediated mechanism.[45][46][47]

Clinical Significance

Calcium channel blockers have a variety of uses in medicine and are used to treat a variety of conditions, including but not limited to cardiac arrhythmia and hypertension. Also, there are currently several drugs used for bipolar disorder that block calcium channels.[28] Among the difficulties in utilizing calcium channel blockers for several diseases where they have implications is the development of calcium channel blockers that are selective for specific subtypes.[28]

Because of the involvement of Cav2.2 channels in chronic pain states, they serve as a potentially important target for treating this condition. As stated previously, voltage-gated calcium channels are regulated by G-protein coupled receptors, and Cav2.2 channels are no exception. Both the GABAb receptor and various subtypes of opioid receptors affect the functioning of this channel. Morphine's ability to reduce the symptoms of chronic pain appears to be associated with its ability to mitigate primary afferent neuron activity by inhibiting the activity of Cav2.2 channels, resulting in a decrease in neurotransmitter release. Another group of drugs called the gabapentinoids also affects the activity of Cav2.2 channels but through a different mechanism. Overexpression of Cav2.2 channels in chronic pain states is thought to occur as a result of the upregulation of a2D subunits, which influence the cell-surface expression of these channels. By interfering with a2D subunit functioning, gabapentinoids appear to decrease presynaptic plasma membrane Cav2.2 channel density, thereby decreasing neurotransmitter release.[48]

Because of the involvement of T-type calcium channels in the absence seizure pathophysiology, pharmaceutical agents that target their activity may act as potent anti-epileptic drugs. The ability of T-type calcium channels to increase seizure susceptibility in the thalamus makes them an important clinical target for treating absence seizures. Drugs that inhibit specific subtypes of T-type calcium channel subunits would likely decrease absence seizure activity by reducing channel activity and expression. Mouse models of absence seizures exhibit resistance to drug-induced seizure activity when crossed with Cav3.1 T-type channel knockout mice, serving as an important clinical indicator of the importance of targeting this calcium channel in treating absence seizures.[21]

 The variety of different calcium channels in myocardial cells contribute to different physiological activities associated with cardiovascular functioning, and mutations in these channels contribute to various diseases of the cardiovascular system. For example, the SERCA pump plays a vital role in mediating the positively chronotropic effects of beta-adrenergic stimulation because beta-adrenergic generates cAMP, which activates a protein kinase that phosphorylates phospholamban. When phospholamban is phosphorylated, it disinhibits the SERCA pump, increasing the rate of cytosolic uptake of calcium into the sarcoplasmic reticulum and decreasing the relaxation time of the heart during increased physical exertion. Mutations in the SERCA pump have been linked to dilated cardiomyopathy.[49][46] 

The Na+/Ca+ exchanger is extremely important for healthy cardiovascular functioning. It exchanges sodium for calcium in a 3 to 1 ratio. This electrogenic action contributes to the voltage of the cardiac cell membrane and the subsequent direction of calcium flux, which favors calcium influx during systole and calcium efflux diastole.[46] The intracellular concentration has both inotropic and chronotropic implications on cardiac activity. Mutations in the Na+/Ca+ pump have been linked to heart failure and are an indirect target of drugs like digitalis. Finally, L-type calcium channels are also critical for normal cardiovascular functioning. It is responsible for initiating the cardiac action potential by providing the calcium influx to depolarize the cell. Also, they are involved in excitation-contraction coupling, which links calcium release to muscle contraction. L-type calcium channels are important targets for treating a variety of different cardiovascular diseases, such as ischemia, coronary vasospasm, and ischemia.[50][45][51]

Calcium channel blockers are useful for the treatment of angina and related ischemic conditions because they ultimately exert a variety of physiological responses that affect the balance between myocardial oxygen supply and myocardial oxygen demand. DHP calcium channel blockers cause selective vasodilation of the coronary arteries and inhibit vascular smooth muscle contraction, which reduces vascular resistance, increases blood flow, and increases myocardial oxygen supply. Calcium channel blockers also decrease myocardial oxygen demand, determined by the contractile state of the heart, blood pressure, and heart rate. All calcium channel blockers reduce blood pressure, and this is achieved by peripheral vasodilation, which in turn reduces blood pressure. Non-DHP calcium channel blockers are active at the heart's nodal areas, where they decrease calcium influx and reduce the rate of depolarization in cardiac pacemaker cells, thereby reducing heart rate.[45][51][52]

The dromotropic effects of calcium channel blockers like verapamil and diltiazem can be linked to their ability to interact within the SA and AV nodal cardiac cells. They cause blockage of L-type voltage-gated calcium channels at these sites, which reduces pacemaker cell depolarization and subsequent firing rate. This effect is beneficial during the treatment of angina and related ischemic conditions because it reduces the heart's oxygen demand. Because this pacemaker cell depolarization depends on T-type calcium channels as well, they do not achieve total depression of sinal rhythmic activity. In contrast, short-acting nifedipine-like calcium channel blockers, when administered in short-acting formulations, may increase heart rate as a result of reflex tachycardia, which occurs when significant vascular smooth muscle vasodilation causes a rapid decrease in blood pressure, which, in turn, increases sympathetic activity. As a result, they may exacerbate the symptoms of angina and related ischemic conditions of the cardiovascular system by increasing the heart's oxygen demand.[45] These drugs are particularly prone to cause dizziness due to the rapid decrease in blood pressure that occurs upon their administration.[51][53][51]

 The inotropic action of calcium channel blockers plays an essential role in improving symptoms of angina because it reduces oxygen demand. However, this is also responsible for their ability to exacerbate conditions such as heart failure, in which the heart does not pump sufficient amounts of blood. For short-acting DHP calcium channel blockers such as nifedipine, their negative inotropic effect may be counteracted by reflex sympathetic stimulation due to rapidly reduced systemic vascular resistance. Sympathetic stimulation directly enhances cardiac contractility by increasing the calcium influx and, therefore, the availability of calcium for the heart's contractile apparatus. In angina, a decrease in cardiac contractility is particularly beneficial as it increases the heart's oxygen supply by reducing metabolic demand. Finally, concerning the negative chronotropic effect of the heart by calcium channel blockers, it is important to note that their ability to depress heart rate depends on their ability to inhibit adrenergic transmitters release and the ability of the heart to adjust sympathetic tone reflexively.[45][50][53]

 In addition to affecting both myocardial oxygen supply and demand, calcium channel blockers are beneficial for certain diseases of the cardiovascular system because they reduce the heart's workload. Vasodilation of the systemic veins, for example, reduces pre-load. Afterload is also reduced because vascular resistance to blood flow decreases. Two separate mechanisms are responsible for this, inhibition of myogenic tone in vascular smooth muscle and a decrease in responsiveness to vasoconstricting substances like norepinephrine.[45] The latter two effects are particularly prevalent in drugs like verapamil and nifedipine. The workload of the heart is also reduced by restricting the entry of calcium into myocardial cells, which decreases ventricular contraction frequency and thereby decreases the ventricular pressure at the end of diastole and also decreases the pressure on the ventricular walls.[45][50][53]

 In diseases that restrict coronary blood flow, calcium channel blockers exert several beneficial effects. By decreasing the heart rate, the ventricular filling time of the heart is increased, which increases the efficiency of the pumping of blood by the heart. Furthermore, The reduction of ventricular wall tension that occurs due to vascular smooth muscle dilation works by improving perfusion. During anoxic conditions, calcium channel blockers prevent the exaggerated accumulation of calcium by inhibiting calcium influx, which increases the redistribution of blood to ischemic regions. Patients with angina benefit from the inhibition of vascular smooth muscle contraction via blockade of L-type voltage-gated calcium channels, which dilate the coronary vessels and increase blood flow. The reduction of coronary vasospasm is a significant clinical effect of CCBs because it is the primary cause of ischemia in those with angina.[50][51][53][51]