Mitochondrial modulation of intracellular Ca(2+) signaling

IP3-mediated Ca(2+) release plays a fundamental role in many cell signaling processes and has been the subject of numerous modeling studies. Only recently has the important role that mitochondria play in the dynamics of intracellular Ca(2+) signaling begun to be considered in experimental work and in computational models. Mitochondria sequester large amounts of Ca(2+) and thus have a modulatory effect on intracellular Ca(2+) signaling, and mitochondrial uptake of Ca(2+), in turn, has a regulatory effect on mitochondrial function. Here we integrate a well-established model of IP3-mediated Ca(2+) signaling with a detailed model of mitochondrial Ca(2+) handling and metabolic function. The incorporation of mitochondria results in oscillations in a bistable formulation of the IP3 model, and increasing metabolic substrate decreases the frequency of these oscillations consistent with the literature. Ca(2+) spikes from the cytosol are communicated into mitochondria and are shown to induce realistic metabolic changes. The model has been formulated using a modular approach that is easy to modify and should serve as a useful basis for the investigation of questions regarding the interaction of these two systems.     

Mitochondria and Ca(2+) signaling

Mitochondria play a central role in cell homeostasis. Amongst others, one of the important functions of mitochondria is to integrate its metabolic response with one of the major signaling pathways - the Ca²⁺ signaling. Mitochondria are capable to sense the levels of cytosolic Ca²⁺ and generate mitochondrial Ca²⁺ responses. Specific mechanisms for both Ca²⁺ uptake and Ca²⁺ release exist in the mitochondrial membranes. In turn, the mitochondrial Ca²⁺ signals are able to produce changes in the mitochondrial function and metabolism, which provide the required level of functional integration. This essay reviews briefly the current available information regarding the mitochondrial Ca²⁺ transport systems and some of the functional consequences of mitochondrial Ca²⁺ uptake     

Ca(2+) signaling in dendritic spines

Recent studies have revealed that Ca(2+) signals evoked by action potentials or by synaptic activity within individual dendritic spines are regulated at multiple levels. Ca(2+) influx through glutamate receptors and voltage-sensitive Ca(2+) channels located on spines depends on the channel subunit composition, the activity of kinases and phosphatases, the local membrane potential and past patterns of activity. Furthermore, sources of spine Ca(2+) interact nonlinearly such that activation of one Ca(2+) channel can enhance or depress the activity of others. These studies have revealed that each spine is a complex and partitioned Ca(2+) signaling domain capable of autonomously regulating the electrical and biochemical consequences of synaptic activity.     

Ca(2+) signaling in dendritic spines

Dendritic spines are cellular microcompartments that are isolated from their parent dendrites and neighboring spines. Recently, imaging studies of spine Ca(2+) dynamics have revealed that Ca(2+) can enter spines through voltage-sensitive and ligand-activated channels, as well as through Ca(2+) release from intracellular stores. Relationships between spine Ca(2+) signals and induction of various forms of synaptic plasticity are beginning to be elucidated. Measurements of spine Ca(2+) concentration are also being used to probe the properties of single synapses and even individual calcium channels in their native environment.     

Ca(2+)-sensor region of IP(3) receptor controls intracellular Ca(2+) signaling

Many important cell functions are controlled by Ca(2+) release from intracellular stores via the inositol 1,4,5-trisphosphate receptor (IP(3)R), which requires both IP(3) and Ca(2+) for its activity. Due to the Ca(2+) requirement, the IP(3)R and the cytoplasmic Ca(2+) concentration form a positive feedback loop, which has been assumed to confer regenerativity on the IP(3)-induced Ca(2+) release and to play an important role in the generation of spatiotemporal patterns of Ca(2+) signals such as Ca(2+) waves and oscillations. Here we show that glutamate 2100 of rat type 1 IP(3)R (IP(3)R1) is a key residue for the Ca(2+) requirement. Substitution of this residue by aspartate (E2100D) results in a 10-fold decrease in the Ca(2+) sensitivity without other effects on the properties of the IP(3)R1. Agonist-induced Ca(2+) responses are greatly diminished in cells expressing the E2100D mutant IP(3)R1, particularly the rate of rise of initial Ca(2+) spike is markedly reduced and the subsequent Ca(2+) oscillations are abolished. These results demonstrate that the Ca(2+) sensitivity of the IP(3)R is functionally indispensable for the determination of Ca(2+) signaling patterns.     

Neurocalcin delta modulation of ROS-GC1, a new model of Ca(2+) signaling

ROS-GC1 membrane guanylate cyclase is a Ca(2+) bimodal signal transduction switch. It is turned "off" by a rise in free Ca(2+) from nanomolar to the semicromolar range in the photoreceptor outer segments and the olfactory bulb neurons; by a similar rise in the bipolar and ganglion retinal neurons it is turned "on". These opposite operational modes of the switch are specified by its Ca(2+) sensing devices, respectively termed GCAPs and CD-GCAPs. Neurocalcin delta is a CD-GCAP. In the present study, the neurocalcin delta-modulated site, V(837)-L(858), in ROS-GC1 has been mapped. The location and properties of this site are unique. It resides within the core domain of the catalytic module and does not require the alpha-helical dimerization domain structural element (amino acids 767-811) for activating the catalytic module. Contrary to the current beliefs, the catalytic module is intrinsically active; it is directly regulated by the neurocalcin delta-modulated Ca(2+) signal and is dimeric in nature. A fold recognition based model of the catalytic domain of ROS-GC1 was built, and neurocalcin delta docking simulations were carried out to define the three-dimensional features of the interacting domains of the two molecules. These findings define a new transduction model for the Ca(2+) signaling of ROS-GC1.     

tcBid promotes Ca(2+) signal propagation to the mitochondria: control of Ca(2+) permeation through the outer mitochondrial membrane

Calcium spikes established by IP(3) receptor-mediated Ca(2+) release from the endoplasmic reticulum (ER) are transmitted effectively to the mitochondria, utilizing local Ca(2+) interactions between closely associated subdomains of the ER and mitochondria. Since the outer mitochondrial membrane (OMM) has been thought to be freely permeable to Ca(2+), investigations have focused on IP(3)-driven Ca(2+) transport through the inner mitochondrial membrane (IMM). Here we demonstrate that selective permeabilization of the OMM by tcBid, a proapoptotic protein, results in an increase in the magnitude of the IP(3)-induced mitochondrial [Ca(2+)] signal. This effect of tcBid was due to promotion of activation of Ca(2+) uptake sites in the IMM and, in turn, to facilitation of mitochondrial Ca(2+) uptake. In contrast, tcBid failed to control the delivery of sustained and global Ca(2+) signals to the mitochondria. Thus, our data support a novel model that Ca(2+) permeability of the OMM at the ER- mitochondrial interface is an important determinant of local Ca(2+) signalling. Facilitation of Ca(2+) delivery to the mitochondria by tcBid may also support recruitment of mitochondria to the cell death machinery.     

Ion channels and transporters in cancer. 4. Remodeling of Ca(2+) signaling in tumorigenesis: role of Ca(2+) transport

The Ca(2+) signal has major roles in cellular processes important in tumorigenesis, including migration, invasion, proliferation, and apoptotic sensitivity. New evidence has revealed that, aside from altered expression and effects on global cytosolic free Ca(2+) levels via direct transport of Ca(2+), some Ca(2+) pumps and channels are able to contribute to tumorigenesis via mechanisms that are independent of their ability to transport Ca(2+) or effect global Ca(2+) homeostasis in the cytoplasm. Here, we review some of the most recent studies that present evidence of altered Ca(2+) channel or pump expression in tumorigenesis and discuss the importance and complexity of localized Ca(2+) signaling in events critical for tumor formation.     

Mitochondrial Ca(2+)-induced Ca(2+) release mediated by the Ca(2+) uniporter

We have reported that a population of chromaffin cell mitochondria takes up large amounts of Ca(2+) during cell stimulation. The present study focuses on the pathways for mitochondrial Ca(2+) efflux. Treatment with protonophores before cell stimulation abolished mitochondrial Ca(2+) uptake and increased the cytosolic [Ca(2+)] ([Ca(2+)](c)) peak induced by the stimulus. Instead, when protonophores were added after cell stimulation, they did not modify [Ca(2+)](c) kinetics and inhibited Ca(2+) release from Ca(2+)-loaded mitochondria. This effect was due to inhibition of mitochondrial Na(+)/Ca(2+) exchange, because blocking this system with CGP37157 produced no further effect. Increasing extramitochondrial [Ca(2+)](c) triggered fast Ca(2+) release from these depolarized Ca(2+)-loaded mitochondria, both in intact or permeabilized cells. These effects of protonophores were mimicked by valinomycin, but not by nigericin. The observed mitochondrial Ca(2+)-induced Ca(2+) release response was insensitive to cyclosporin A and CGP37157 but fully blocked by ruthenium red, suggesting that it may be mediated by reversal of the Ca(2+) uniporter. This novel kind of mitochondrial Ca(2+)-induced Ca(2+) release might contribute to Ca(2+) clearance from mitochondria that become depolarized during Ca(2+) overload.     

Receptor-induced Ca(2+) signaling in cultured myenteric neurons

We studied the effect of excitatory neurotransmitters (10(-5) M) on the intracellular Ca(2+) concentration ([Ca(2+)](i)) of cultured myenteric neurons. ACh evoked a response in 48.6% of the neurons. This response consisted of a fast and a slow component, respectively mediated by nicotinic and muscarinic receptors, as revealed by specific agonists and antagonists. Substance P evoked a [Ca(2+)](i) rise in 68.2% of the neurons, which was highly dependent on Ca(2+) release from intracellular stores, since after thapsigargin (5 microM) pretreatment only 8% responded. The responses to serotonin, present in 90.7%, were completely blocked by ondansetron (10(-5) M), a 5-HT(3) receptor antagonist. Specific agonists of other serotonin receptors were not able to induce a [Ca(2+)](i) rise. Removing extracellular Ca(2+) abolished all serotonin and fast ACh responses, whereas substance P and slow ACh responses were more persistent. We conclude that ACh-induced signaling involves both nicotinic and muscarinic receptors responsible for a fast and a more delayed component, respectively. Substance P-induced signaling requires functional intracellular Ca(2+) stores, and the 5-HT(3) receptor mediates the serotonin-induced Ca(2+) signaling in cultured myenteric neurons.     

Cell cycle control by anchorage signaling

Virtually all the cells constituting solid organs in adult animals require anchorage to the extracellular matrix for their proliferation and survival. When deprived of anchorage, those cells arrest in G(1) phase of the cell cycle and die of apoptosis known as anoikis. However, if malignantly transformed, cells no longer require such an anchorage to proliferate and survive, and it is generally thought that the acquirement of this ability underlies the tumorigenic and metastatic capability of malignant cells. Therefore, for the past two decades, great efforts have been devoted to uncovering the nature of the anchorage signal and the mechanism by which this signal controls the G(1)-S transition in the cell cycle with little progress. However, several critical findings were recently made on anchorage signaling and the control of the cell cycle and cell death by this signaling. This review focuses on the newly emerging understanding and perspective of this highly important cell cycle and cell death regulation.     

Decoding complex Ca2+ signals through the modulation of Ras signaling

Calcium (Ca(2+)) is a universal regulator of a wide variety of cellular processes. For such control to be achieved, information is encoded within spatial and temporal components of the underlying Ca(2+) signal. One pathway through which Ca(2+) signals are decoded is the Ras binary switch. Here I describe some recent advances that have shed light on how cells can decode the spatial and temporal aspects of Ca(2+) signals through the regulation of this important signalling switch.     

Ca(2+) sensors of L-type Ca(2+) channel

Ca(2+)-induced inactivation of L-type Ca(2+) is differentially mediated by two C-terminal motifs of the alpha(1C) subunit, L (1572-1587) and K (1599-1651) implicated for calmodulin binding. We found that motif L is composed of a highly selective Ca(2+) sensor and an adjacent Ca(2+)-independent tethering site for calmodulin. The Ca(2+) sensor contributes to higher Ca(2+) sensitivity of the motif L complex with calmodulin. Since only combined mutation of both sites removes Ca(2+)-dependent current decay, the two-site modulation by Ca(2+) and calmodulin may underlie Ca(2+)-induced inactivation of the channel.     

Cell cycle control by Ca2+ in Saccharomyces cerevisiae

We established an experimental system suitable for study of cell cycle regulation by Ca2+ in the yeast Saccharomyces cerevisiae. Systematic cell cycle analysis using media containing various concentrations of Ca2+, a Ca2(+)-ionophore (A23187), and a Ca2(+)-chelator [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) revealed that simultaneous addition of 10 microM A23187 and 10 mM EGTA to cells growing in a Ca2(+)-deficient medium at 22 degrees C caused rapid decrease in intracellular Ca content and resulted in transient G1 arrest followed by block mostly at G2/M, as revealed by flow cytometry. Recovery from G1 arrest was not due to coordinated initiation of DNA synthesis and bud emergence: unbudded cells with S or G2/M DNA were observed. Examination of terminal phenotype suggested that Ca2+ was required at all the stages of the cell cycle except for the initiation of DNA synthesis. The intracellular cAMP level decreased within 10 min of addition of A23187 and EGTA. No significant transient G1 arrest was observed in cells incubated with 8-Br-cAMP, or RAS2val19 and delta bcy1 mutants, which produce a high level of cAMP and have constitutively activated cAMP-dependent protein kinase, respectively. These results indicate that Ca2+ is essential for cell cycle progression and suggest that Ca2+ may regulate the cAMP level. This system will be useful for genetic and molecular studies on cell cycle events regulated by Ca2+.     

Ca(V)1 and Ca(V)2 channels engage distinct modes of Ca(2+) signaling to control CREB-dependent gene expression

Activity-dependent gene expression triggered by Ca(2+) entry into neurons is critical for learning and memory, but whether specific sources of Ca(2+) act distinctly or merely supply Ca(2+) to a common pool remains uncertain. Here, we report that both signaling modes coexist and pertain to Ca(V)1 and Ca(V)2 channels, respectively, coupling membrane depolarization to CREB phosphorylation and gene expression. Ca(V)1 channels are advantaged in their voltage-dependent gating and use nanodomain Ca(2+) to drive local CaMKII aggregation and trigger communication with the nucleus. In contrast, Ca(V)2 channels must elevate [Ca(2+)](i) microns away and promote CaMKII aggregation at Ca(V)1 channels. Consequently, Ca(V)2 channels are ~10-fold less effective in signaling to the nucleus than are Ca(V)1 channels for the same bulk [Ca(2+)](i) increase. Furthermore, Ca(V)2-mediated Ca(2+) rises are preferentially curbed by uptake into the endoplasmic reticulum and mitochondria. This source-biased buffering limits the spatial spread of Ca(2+), further attenuating Ca(V)2-mediated gene expression.     

Connexin mimetic peptides reversibly inhibit Ca(2+) signaling through gap junctions in airway cells

The effect of peptides with sequences derived from connexins, the constituent proteins of gap junctions, on mechanically stimulated intercellular Ca(2+) signaling in tracheal airway epithelial cells was studied. Three peptides with sequences corresponding to connexin extracellular loop regions reversibly restricted propagation of Ca(2+) waves to neighboring cells. Recovery of communication began within 10 min of removal of the peptides, with inhibition totally reversed by 20-40 min. The peptides were shown to be more effective in inhibiting Ca(2+) waves than glycyrrhetinic acid or oleamide. Inhibition of intercellular Ca(2+) waves by connexin mimetic peptides did not affect the Ca(2+) response to extracellular ATP. Although the intracellular Ca(2+) response of tracheal epithelial cells to ATP was greatly reduced by either pretreatment with high doses of ATP or application of apyrase, mechanically stimulated intercellular Ca(2+) signaling was not affected by these agents. We conclude that connexin mimetic peptides are effective and reversible inhibitors of gap junctional communication of physiologically significant molecules that underlie Ca(2+) wave propagation in tracheal epithelial cells and propose a potential mechanism for the mode of action of mimetic peptides.     

A new type of Ca(2+) channel blocker that targets Ca(2+) sensors and prevents Ca(2+)-mediated apoptosis.

Calmodulin (CaM), as well as other Ca(2+) binding motifs (i.e., EF hands), have been demonstrated to be Ca(2+) sensors for several ion channel types, usually resulting in an inactivation in a negative feedback manner. This provides a novel target for the regulation of such channels. We have designed peptides that interact with EF hands of CaM in a specific and productive manner. Here we have examined whether these peptides block certain Ca(2+)-permeant channels and inhibit biological activity that is dependent on the influx of Ca(2+). We found that these peptides are able to enter the cell and directly, as well as indirectly (through CaM), block the activity of glutamate receptor channels in cultured neocortical neurons and a nonselective cation channel in Jurkat T cells that is activated by HIV-1 gp120. As a consequence, apoptosis mediated by an influx of Ca(2+) through these channels was also dose-dependently inhibited by these novel peptides. Thus, this new type of Ca(2+) channel blocker may have utility in controlling apoptosis due to HIV infection or neuronal loss due to ischemia.     

Bimodal control of a Ca(2+)-activated Cl(-) channel by different Ca(2+) signals

Ca(2+)-activated Cl(-) channels play important roles in a variety of physiological processes, including epithelial secretion, maintenance of smooth muscle tone, and repolarization of the cardiac action potential. It remains unclear, however, exactly how these channels are controlled by Ca(2+) and voltage. Excised inside-out patches containing many Ca(2+)-activated Cl(-) channels from Xenopus oocytes were used to study channel regulation. The currents were mediated by a single type of Cl(-) channel that exhibited an anionic selectivity of I(-) > Br(-) > Cl(-) (3.6:1.9:1.0), irrespective of the direction of the current flow or [Ca(2+)]. However, depending on the amplitude of the Ca(2+) signal, this channel exhibited qualitatively different behaviors. At [Ca(2+)] < 1 microM, the currents activated slowly upon depolarization and deactivated upon hyperpolarization and the steady state current-voltage relationship was strongly outwardly rectifying. At higher [Ca(2+)], the currents did not rectify and were time independent. This difference in behavior at different [Ca(2+)] was explained by an apparent voltage-dependent Ca(2+) sensitivity of the channel. At +120 mV, the EC(50) for channel activation by Ca(2+) was approximately fourfold less than at -120 mV (0.9 vs. 4 microM). Thus, at [Ca(2+)] < 1 microM, inward current was smaller than outward current and the currents were time dependent as a consequence of voltage-dependent changes in Ca(2+) binding. The voltage-dependent Ca(2+) sensitivity was explained by a kinetic gating scheme in which channel activation was Ca(2+) dependent and channel closing was voltage sensitive. This scheme was supported by the observation that deactivation time constants of currents produced by rapid Ca(2+) concentration jumps were voltage sensitive, but that the activation time constants were Ca(2+) sensitive. The deactivation time constants increased linearly with the log of membrane potential. The qualitatively different behaviors of this channel in response to different Ca(2+) concentrations adds a new dimension to Ca(2+) signaling: the same channel can mediate either excitatory or inhibitory responses, depending on the amplitude of the cellular Ca(2+) signal.