Ca2 Influx

The ultimate Ca2+ source for neurons exits outside the neurons. Entry of Ca2+ across the plasma membrane is known to be important in generating neuronal Ca2+ signals, resulting in membrane depolarization and an increased [Ca2+]c. Ca2+ channel expression at the cell surface is regulated by intracellular signaling molecules.13 The latter leads to activation of Ca2+-dependent intracellular signal cascades. There is a large gradient of Ca2+ concentration across the plasma membrane: extracellular Ca2+ ([Ca2+]o) is slightly above 2 mM, while [Ca2+]c is approximately 100 nM. Thus, there is a large driving force for Ca2+ entry into neurons. Ca2+ may enter via either VOCCs (Fig. 1) or receptor-operated Ca2+ channels (ROCCs). Ca2+ efflux from the ER may also trigger a small, but prolonged Ca2+ entry across the plasma membrane through the so-called store-operated Ca2+ channels (SOCCs).

Action potentials reliably evoke Ca2+ transients in axons and boutons through VOCCs.35 The VOCCs are involved in providing the Ca2+ for neural signals underlying learning and memory in neural networks.1 Blocking the L-type VOCCs with nimodipine, a 1,4-dihydropyridine, has been reported to dramatically impair learning and memory, 9 limiting their usefulness as therapeutic agents in various brain and cardiovascular disorders, including brain trauma, hypoxia, ischemia, degenerative disorders, memory decline in normal aging, heart failure, and cardiac arrhythmia. Others, however, reported that these substances prevented the performance deficits in spatial memory in rats with a medial septal lesion.12

Multiple classes of VOCCs have been distinguished on the basis of their pharmacological and electrophysiological properties and are often termed L, N, P/Q, and T-types. VOCCs are multiple subunit membrane complexes. In the central nervous system, the complexes are comprised of at least a1, a2, and P subunits. Transcripts encoding a Y subunit have not been identified in RNA from the brain. The a1 and P subunits are each encoded by a gene family, including at least six distinct genes for a1 subunits and four genes for P subunits. Primary transcripts of each of the a1 genes, the a2 gene and two of the P genes have been shown to yield multiple, structurally distinct subunits via differential mRNA processing. The a1 subunits of Ca2+ channels contain the Ca2+-selective pore, the essential gating machinery, and the receptor sites for the most prominent pharmacological agents. Some of the cloned a1 subunits in fact correspond rather well to native L-type or N-type channels. In contrast to the a1 subunits, Ca2+ channel a2 subunits generally serve as modulatory subunits for the Ca2+ channel complex. Although in some cases a2 subunit coexpression is found also to modulate the rates of activation and inactivation, and the voltage-dependence of inactivation. Functions of the P subunits, on the other hand, more likely depend on their interaction with the a subunits as modulatory subunits, by altering the channel complex properties,98 such as voltage dependence, rate of activation and inactivation, and current magnitude. Interestingly, calmodulin may mediate two opposing effects on individual channels, initially promoting and then inhibiting channel opening. Both require Ca2+-calmodulin binding to a single 'iQ-like' domain on the carboxyl tail of a1A, but are mediated by different domains of calmodulin. Ca2+ binding to the amino-terminal domain selectively initiates channel inactivation, whereas Ca2+ sensing by the carboxyl-terminal lobe induces facilitation.30

Figure 1. A cartoon to illustrate the features of Ca2+ cascades. [Ca2+] c may increase due to Ca2+ influx through plasma membrane channels or intracellular release from ER RyR or IP3R channels. Ca2+ triggers many intracellular responses, such as changes in enzyme activity and receptor/synaptic functions, Ca2+ release, mitochondrial functions, gene transcription, and ROS/A| formation/apoptosis. A|damages neurons and promotes apoptosis by a mechanism involving generation of reactive oxygen species (ROS). ROS promote neuronal apoptosis by damaging various cellular proteins. a, a-secretase; |-secretase; y, y-secretase; AA, arachidonic acid; ACh, acetylcholine; APP, amyloid precursor protein; ATP, adenosine triphosphate; CA, carbonic anhydrase; CE, calexcitin; DAG, diacylglycerol; ER, endoplasmic reticulum; IP3R, inositol 1,4,5-triphosphate receptor; PKC, protein kinase C; RyR, ryanodine receptor; sAPPa, a-secretase-derived secreted APP;

Figure 1. A cartoon to illustrate the features of Ca2+ cascades. [Ca2+] c may increase due to Ca2+ influx through plasma membrane channels or intracellular release from ER RyR or IP3R channels. Ca2+ triggers many intracellular responses, such as changes in enzyme activity and receptor/synaptic functions, Ca2+ release, mitochondrial functions, gene transcription, and ROS/A| formation/apoptosis. A|damages neurons and promotes apoptosis by a mechanism involving generation of reactive oxygen species (ROS). ROS promote neuronal apoptosis by damaging various cellular proteins. a, a-secretase; |-secretase; y, y-secretase; AA, arachidonic acid; ACh, acetylcholine; APP, amyloid precursor protein; ATP, adenosine triphosphate; CA, carbonic anhydrase; CE, calexcitin; DAG, diacylglycerol; ER, endoplasmic reticulum; IP3R, inositol 1,4,5-triphosphate receptor; PKC, protein kinase C; RyR, ryanodine receptor; sAPPa, a-secretase-derived secreted APP;

L-type Ca2+ channels represent a subset of high voltage-threshold Ca2+ channel that can generally be distinguished by their persistent activation during a maintained depolarization and by sensitivity to dihydropyridine antagonists and agonists. L-type channels are widely distributed in excitable and nonexcitable cells and are reported that the synaptic transmission between hippocampal CA3 and CA1 neurons does not involve Ca2+ from activation of L-type Ca2+ channels.

N-type Ca2+ channels are found in many central and peripheral neurons and have been proposed to play a role in the release of neurotransmitter at certain synapses. N-type channels can generally be distinguished by the combination of a number of criteria, including activation at potentials more positive than -30 mV (high voltage-threshold), inactivation during a prolonged depolarization, insensitivity to dihydropyridines, and a strong and irreversible block by the neuropeptide toxin ro-conotoxin (ro-CTx)-GVIA. However, this toxin does not block N-type channels exclusively. At micromolar concentrations, ro-CTx-GVIA also reduces currents carried by doe-1, class D L-type channels, and an adrenal chromafin channel that is not the classical N-type.

P-type channels are potently blocked by ro-Aga-IVA with an IC50 of 1-2 nM. In contrast, aiA channels in oocytes are much less sensitive to ro-Aga-IVA, showing an IC50 of about 200 nM. However, at submicromolar concentrations, the toxin also strongly inhibits aiA currents. Agonists of metabotropic glutamate receptors (mGluRs) are also found to suppress a large voltage-activated P/Q-type Ca2+ conductance in the presynaptic terminal, therefore inhibiting synaptic transmitter release at glutamatergic synapses.

R-type channels in cerebellar granule neurons are resistant to blockade by ro-CTx-GVIA, nimodipine (up to 5 ^M), and ro-Aga-IVA (30 nM) at concentrations sufficient to eliminate N-, L-, and P-type channels, respectively.

After blocking N-type channels with ro-Conotoxin GVIA (1-3 ^M), much of the synaptic transmission between hippocampal CA3 and CA1 neurons remains. The pharmacological profile of Ca2+ channels mediating the remaining transmission resembles that of aiA Ca2+ channel subunits expressed in Xenopus oocytes and the Q-type Ca2+ channel current in cerebellar granule neurons. Like the R-type channels, Q-type channels are resistant to ro-CTx-GIVA, nimodipine, or ro-Aga-IVA. The Q-type channels appear to be generated by aiA and aiE sub-units and are completely blocked by 1.5 ^M ro-CTx-MVIIC, and are largely suppressed by ro-Aga-IVA at 1 ^M, a concentration 100 to 1000 times that needed to block P-type channels.

N- and P/Q-type Ca2+ channels are inhibited by G proteins.54,57 Ca2+ can regulate P/Q-type channels through feedback mechanisms,41 probably through an association of Ca2+/calmodulin with P/Q type Ca2+ channels.69 Thus, Ca2+ entry through P/Q-type channels promotes Ca2+/ calmodulin binding to the a1A subunit. The association of Ca2+/calmodulin with the channel accelerates inactivation, enhances recovery from inactivation and augments Ca2+ influx by facilitating the Ca2+ current so that it is larger after recovery from inactivation.69

Low-voltage-activated VOCC channels are called 'T' type because their currents are both transient (owing to fast inactivation) and tiny (owing to small conductance). T-type channels are thought to be involved in pacemaker activity, low-threshold Ca2+ spikes, neuronal oscillations and resonance, and rebound burst firing.

ROCCs mediate major classes of signal processing throughout the brain network. L-Glutamate is the major neurotransmitter in the principal pathways that connect the major cell groups in the hippocampus and cortex. Activation of glutamate receptors (GluR) increases Ca2+ entry into the neurons. It acts through either mGluRs (coupled to G proteins) or ionotropic receptors (iGluRs; ligand-gated ion channels). iGluR subunits are further subdivided into NMDA, AMPA, and kainate subtypes. When sufficient membrane potential changes are elicited by activation of ROCCs, VOCCs might also be activated, providing additional Ca2+ influx. The Ca2+ influx initiates intracellular events including intracellular Ca2+ release, alterations in gene transcription, and modifications of synaptic strengths. Through the Ca2+ signal cascades, glutamatergic activity dramatically alters neuronal activity, which in the hippocampal place cells encode spatial information. Individual hippocampal pyramidal cells demonstrate reliable place field correlates, increasing their discharge rates in selected places within an environment and becoming virtually silent in other places. Excessive activation of the glutamate receptors, however, results in increased Ca2+ influx and may cause oxidative stress.

Forming assembling complexes provides a mechanism that ensures specific and rapid signaling through ROCCs. For instance, the P2-adrenergic receptor is directly associated with one of its ultimate effectors, the class C L-type Ca2+ channel Cav1.2,2 generating highly localized signal transduction from the receptor to the channel.

Intracellular Release and Storage

Other than Ca2+ entry through the plasma membranes, rapid changes in [Ca2+]c can be induced through Ca2+ release from intracellular stores (Fig. 1). Intracellular Ca2+ release is generally viewed as a mechanism to amplify and prolong Ca2+ influx signals.48 The release mechanisms are widely used by neurons in signaling. The intracellular Ca + stores include the ER, mitochondria, and less well defined nuclear store. The involvement of mitochondria in the Ca2+ release for Ca2+ signaling, however, remains controversial.

The ER is a continuous network that extends throughout the axon, soma, dendrites, and spines and is therefore uniquely placed to generate Ca + signals in every compartment of a neuron. Ca2+ is released from the ER via inositol 1,4,5-triphosphate receptors (IP3Rs) or ryanodine receptors (RyRs). IP3Rs are synergistically triggered by IP3 and Ca2+, while RyRs respond to [Ca +]c and the intracellular messenger cyclic ADP ribose. Since the ER has a large capacity, it can function as a Ca2+ sink to generate a large number of spikes, but as its load increases the intracellular channels will become increasingly excitable, and Ca2+ may be released back into the cytoplasm through the process of Ca2+-induced Ca2+ release. Ca + waves can be generated by first enhancement then inhibition. In Purkinje cells of the cerebellum, Ca2+ elevation is required for the IP3R/channel to open. concentrations well below 0.25 ^M, increasing [Ca2+]c increases the open probability of the IP3R/channel. For [Ca2+]c higher than 0.25 ^M, however, the open probability decreases. The hippocampal pyramidal cells, on the other hand, have complex dendritic arbors, receiving on the order of 10,000 synapses largely on dendritic spines. These dendrites contain a complex ER that reaches into a majority of large spines. In contrast to Purkinje spines, the ER of the hippocampal pyramidal cells is studded with RyRs in dendrites and spines, while IP3Rs appear to exist largely in dendritic shafts.107

The ER can function as an integrator or "memory" depot of neuronal activity. By absorbing and storing the brief pulses of Ca + associated with each action potential, the ER may keep track of neuronal activity and be able to signal this information to the nucleus through periodic bursts of Ca2+. For example, brief bursts of neuronal activity generate small localized pulses of Ca2+ that are rapidly buffered, but prolonged firing may charge up the ER sufficiently for it to transmit regenerative global signals to the nucleus to initiate gene transcription.

IP3 Receptors

The IP3Rs consist of three isoforms. Each has a special role in the cell. The IP3RI showed a bell-shaped activity in response to [Ca2+]c. This property, however, is not intrinsic to the receptor (its pure form is not inhibited by up to 200 ^M Ca2+), rather it is mediated by calmodulin132 through a negative regulation by binding to calmodulin or a cGMP kinase substrate.101 The IP3R3 forms Ca2+ channels with single-channel currents that are similar to those of IP3R1 at low [Ca2+]c; however, the open probability of the IP3R3 isoform increases monotonically with increased [Ca2+]c (ref. 50) and channels are more active even at 100 ^M [Ca2+]c, whereas the IP3R1 isoform has a bell-shaped dependence on maximum channel activity at 250

nM [Ca2+]c and complete inhibition at 5 ^M [Ca2+]c. The properties of IP3R3 provide positive feedback as Ca2+ is released; the lack of negative feedback allows complete Ca2+ release from intracellular stores. Thus activation of IP3R3 in cells that express only this isoform results in a single transient, but globally increased [Ca2+]c, that is better suited to signal initiation. The bell-shaped Ca2+-dependence curve of IP3R1 is, however, ideal for supporting Ca2+ oscillations and the frequency of Ca2+ transients can be modulated when IP3 concentrations are increased.

Ryanodine Receptors

The RyRs correspond to the sarcoplasmic reticulum calcium channels and bind specifically the plant alkaloid ryanodine. All known members of RyR family, namely, skeletal muscle type RyR1, cardiac muscle type RyR2, and brain type RyR3, are abundantly expressed in the central nervous system. They include about 5000 (4872-5037) amino acid residues and are coded by three different genes, which are located on chromosomes 1, 15, and 19, respectively, in humans. The functional receptor is thought to be a homotetramer, which has a quarterfoil shape and a size of 22 to 27 nm on each side. The center of the quarterfoil includes a pore, with a diameter of 1 to 2 nm, which likely represents the Ca2+ channel. Near its cytoplasmic end, the channel appears to be blocked by a mass, sometimes referred to as the "plug", which might be involved in the modulation of channel conductance. Hippocampal CA1 pyramidal cells express all three types of RyRs and, compared with other central neurons, have the highest level of the RyR3, in greater abundance than the IP3Rs. Moreover, in these neurons, RyRs are ex-

pressed in the axon, soma, and dendrites, including spines107 and thus occupy strategically important position for synaptic signaling and integration.

Activation of RyR requires Ca2+, which is therefore thought to be the "physiological" channel activator, since other ligands cannot activate the channel in the absence of Ca2+, or they require Ca2+ for maximum effect. Activation of RyR may involve a global conformational change including rotation of channel domain relative to the cytoplasmic domain and appearance of a porelike structure within the channel domain preceding Ca2+ release (for review see ref. 58). In the heart cells, a cleft of roughly 12 nm is formed between the cell surface and sarcoplasmic membrane and local Ca2+ signal produced by a single opening of an L-type Ca2+ channel can trigger about 4-6 RyR receptors to generate a existence of other endogenous RyR activators, such as calexcitin2,10,17,87,89 or calexcitin-like mammalian proteins, has been proposed. The RyR is activated by caffeine43 and many other substances.112,113,116,131 Activation of RyR typically requires large [Ca2+]c (~1 ^M), incompatible with the small bulk NMDAR-mediated Ca2+ signals. However, local [Ca2+]c is more likely to provide sufficient Ca2+ for the receptor activation (see below). The RyR is a substrate of several protein kinases, namely cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), protein kinase C (PKC), and calmodulin-dependent protein kinase II (CaMKII). These pathways may be activated in combination to evoke specific functions. The involvement of RyRs in spatial memory is suggested by an increased expression of RyR2 in the rat hippocampus after training.21,129

Refilling of the ER is mediated by ER Ca2+-ATPases since it is blocked by cyclopiazonic acid. Even without prior store depletion, the caffeine-induced Ca2+ transients disappear after 6-minute exposure to cyclopiazonic acid,43 suggesting that ryanodine-sensitive Ca2+ stores are maintained at rest by continuous Ca2+ sequestration. In addition, the store does not refill in Ca2+-free saline, suggesting that the refilling of the stores depends upon Ca2+ influx, probably through a 'capacitative-like' transmembrane influx pathway, or store-operated Ca2+ channels,76 at resting membrane potential, a process that depends on a spatial cytoskeleton rearrangement between cell membrane and the ER structures.47 One possible mechanism underlying neuronal injury by low [Ca2+]c is a disturbance of ER Ca2+ homeostasis. As mentioned previously, low ER Ca2+ loading is also neurotoxic. This toxicity may result from other biological activity in the ER that depends on high Ca2+ levels. Besides functioning as a major intracellular Ca2+ store, the ER plays a pivotal role in the folding, processing, and excretion of membrane and secretory proteins, processes that depend on Ca2+ concentration. Depletion of ER Ca2+ stores thus is a severe form of stress that blocks the folding and processing of membrane proteins.73 The involvement of mitochondria in intracellular Ca2+ signaling remains controversial, particularly signaling that requires physiological Ca2+ release from mitochondria. It is well established, however, that physiological Ca2+ levels are associated with significant movement of Ca2+ and Ca2+ uptake into mitochondria (Fig. 1). With a bacterial evolutionary origin, mitochondria maintain a modicum of independence from the host cell in some respects (maintaining their own DNA while also deriving many important proteins from the nuclear DNA of the host cell). Nevertheless, they are critical for the life of almost all eukaryotic cells. The primary functions of the mitochondria involve oxidative phosphorylation and ATP supply (Fig. 1). The major targets of mitochondrial Ca2+ uptake are the dehydrogenases of the Krebs cycle. Increases in mitochondrial [Ca2+] ([Ca2+]m) participate in activation of the respiratory chain through stimulation of Ca2+-sensitive mitochondrial dehydrogenases (isocitrate, oxoglutarate, and pyruvate dehydrogenases), thereby ensuring adequate ATP synthesis to match the increased energetic demand of stimulated cells. 3 The activation of dehydrogenases stimulates mitochondrial respiration leading to an increase in A^m, driving an increase in ATP production (for review see ref. 33). Thus, [Ca2+]c oscillations, through their effect on mitochondrial Ca2+ uptake, are represented by long-term activation of mitochondrial metabolism. Interestingly, a significant portion of the Ca + entering mitochondria may not appear as free ionized Ca2+ in the matrix, but might rather be present either bound to phosphate or to phospholipids.27

Mitochondrial Ca2+ uptake may also exert subtle effects on the spatiotemporal characteristics of the [Ca2+]c in micro-domains through the cell (see below).

Buffering and Sequestration

Buffering and sequestration of Ca2+ play an important role in Ca2+ homeostasis, involving plasma membrane Na+-Ca2+ exchange, extrusion by plasma membrane Ca2+-ATPase, and uptake into mitochondria and/or the ER. Extrusion through the ATP-dependent Ca2+ pump, energized by the mitochondria, across the plasma membrane is the dominant form of Ca + removal from the bipolar cell synaptic terminals.127 These mechanisms are, however, vulnerable to energy shortage as occurs in various disease states.

Sequestration of cytosolic Ca2+ by intracellular Ca2+ stores (ER and mitochondria) contributes substantially to Ca2+ clearance in neurons. In permeabilized cells, mitochondria can buffer moderate levels of [Ca2+]—the so-called mitochondrial 'set point'—at around 1 ^M (for review see ref. 96). The peak [Ca2+]m of highly responsive mitochondria can be as high as a few hundred ^M. Mitochondrial Ca2+ accumulation results from the close apposition of the organelles to either ER Ca2+ release channels or to plasma membrane Ca2+ channels (for review see ref. 100). Mitochondria take up Ca2+ primarily through a uniporter,33 an electrogenic process. The ability to remove Ca2+ from local cytosol enables mitochondria to regulate the [Ca2+] in micro-domains close to ER Ca2+-release channels. The sensitivity of the IP3R/RyR-channels to Ca2+ means that, by regulating local [Ca2+]c, mitochondrial Ca2+ uptake modulates the rate and extent of propagation of [Ca2+]c waves in a variety of cell types.

Two observations suggest that intracellular ER Ca2+ stores may also act as a buffering system for intracellular Ca2+. First, KCl-induced increase in [Ca2+]c in bullfrog sympathetic neurons is reported to be substantially attenuated after depletion of ryanodine-sensitive Ca2+ stores by prolonged caffeine application. Second, blockers of ER Ca2+-ATPases have been found to prolong the depolarization-induced increases in dendritic [Ca2+] c in rat neo-cortical layer V pyramidal neurons in slices.133

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