Na+−Ca2+ exchanger: From basics to molecular biology

The plasmalemmal Na⁺−Ca²⁺ exchanger is a coupled Na⁺ and Ca²⁺ transport mechanism that plays an important role in regulation of Ca²⁺ homeoslasis in many cell types. A robust Na⁺−Ca²⁺ exchange system is present in the heart where it comprises essential Ca²⁺ extrusion, as well as Ca²⁺ entry pathways, that significantly contribute to the maintenance of cardiac contractility. The review examines the basic properties of Na⁺−Ca²⁺ exchange, the patterns of its regulation, as well as the latest achievements in the cloning and structure-function studies of a Na⁺−Ca²⁺ exchanger molecule.     

Calcium entry in rat parotid acinar cells

Conclusions While it is generally accepted that Ca²⁺ plays an important regulatory role in the physiology of a number of non-excitable cells, the mechanisms which regulate intracellular [Ca²⁺ are far from well established. Ca²⁺ transporting mechanisms which distribute Ca²⁺ intracellularly as well as those which allow influx of extracellular Ca²⁺ are involved in mediating intracellular Ca²⁺ homestasis. In this paper we have described recent studies on the regulation of the Ca²⁺ influx system in the data, it appears that the process of Ca²⁺ entry is extremely complex and may involve several levels of regulation. Understanding the molecular basis of these regulatory mechanisms presents a challeging problem for future studies.     

Ryanodine Receptor Luminal Ca Regulation: Swapping Calsequestrin and Channel Isoforms

Sarcoplasmic reticulum (SR) Ca²⁺ release in striated muscle is mediated by a multiprotein complex that includes the ryanodine receptor (RyR) Ca²⁺ channel and the intra-SR Ca²⁺ buffering protein calsequestrin (CSQ). Besides its buffering role, CSQ is thought to regulate RyR channel function. Here, CSQ-dependent luminal Ca²⁺ regulation of skeletal (RyR1) and cardiac (RyR2) channels is explored. Skeletal (CSQ1) or cardiac (CSQ2) calsequestrin were systematically added to the luminal side of single RyR1 or RyR2 channels. The luminal Ca²⁺ dependence of open probability (Po) over the physiologically relevant range (0.05-1 mM Ca²⁺) was defined for each of the four RyR/CSQ isoform pairings. We found that the luminal Ca²⁺ sensitivity of single RyR2 channels was substantial when either CSQ isoform was present. In contrast, no significant luminal Ca²⁺ sensitivity of single RyR1 channels was detected in the presence of either CSQ isoform. We conclude that CSQ-dependent luminal Ca²⁺ regulation of single RyR2 channels lacks CSQ isoform specificity, and that CSQ-dependent luminal Ca²⁺ regulation in skeletal muscle likely plays a relatively minor (if any) role in regulating the RyR1 channel activity, indicating that the chief role of CSQ1 in this tissue is as an intra-SR Ca²⁺ buffer.     

Evolution of Ca<Superscript>2+</Superscript>-Signaling Mechanisms. Role of Calcium Ions in Signal Transduction in Lower Eukaryotes

This review summarizes current concepts of Ca²⁺-signaling mechanisms in unicellular eukaryotes. Pathways of activation and transduction of Ca²⁺-signal are analyzed and the role of Ca²⁺ in regulation of cellular physiological processes is considered. A special attention is paid to evolutionary aspects of participation of Ca²⁺ ions and Ca²⁺-receptor proteins in regulation of intracellular processes.     

How Does Regulatory Ca2+ Regulate the Na+-Ca2+ Exchanger?

Spatial and temporal regulation of intracellular Ca²⁺ concentrations is a fundamental requirement for life. The mammalian cardiac Na⁺-Ca²⁺ exchanger serves as the main mechanism for Ca²⁺ efflux after heart contraction. Exchange activity is highly regulated by intracellular Ca²⁺, which binds two regulatory domains (CBD1 and CBD2) and triggers the full activity of the exchanger. We solved the X-ray crystallographic structure of CBD2 in the presence and absence of Ca²⁺. Together with mutational analysis of the Ca²⁺ binding sites, this study reveals the crucial role of one of the two bound Ca²⁺ ions and helps propose hypotheses on the mechanism of regulation of the exchanger.     

Mitochondria, calcium and cell death: A deadly triad in neurodegeneration

Mitochondrial Ca²⁺ accumulation is a tightly controlled process, in turn regulating functions as diverse as aerobic metabolism and induction of cell death. The link between Ca²⁺ (dys)regulation, mitochondria and cellular derangement is particularly evident in neurodegenerative disorders, in which genetic models and environmental factors allowed to identify common traits in the pathogenic routes. We will here summarize: i) the current view of mechanisms and functions of mitochondrial Ca²⁺ homeostasis, ii) the basic principles of organelle Ca²⁺ transport, iii) the role of Ca²⁺ in neuronal cell death, and iv) the new information on the pathogenesis of Alzheimer's, Huntington's and Parkinson's diseases, highlighting the role of Ca²⁺ and mitochondria.     

Activity-dependent changes in voltage-dependent calcium currents and transmitter release

Voltage-dependent Ca²⁺ channels are important in the regulation of neuronal structure and function, and as a result, they have received considerable attention. Recent studies have begun to characterize the diversity of their properties and the relationship of this diversity to their various cellular functions. In particular, Ca²⁺ channels play a prominent role in depolarization-secretion coupling, where the release of neurotransmitter is very sensitive to changes in voltage-dependent Ca²⁺ currents. An important feature of Ca²⁺ channels is their regulation by electrical activity. Depolarization can selectively modulate the properties of Ca²⁺ channel types, thus shaping the response of the neuron to future electrical activity. In this article, we examine the diversity of Ca²⁺ channels found in vertebrate and invertebrate neurons, and their short- and long-term regulation by membrane potential and Ca²⁺ influx. Additionally, we consider the extent to which this activity-dependent regulation of Ca²⁺ currents contributes to the development and plasticity of transmitter releasing properties. In the studies of long-term regulation, we focus on crustacean motoneurons where activity levels, Ca²⁺ channel properties, and transmitter releasing properties can be followed in identified neurons.     

Rem GTPase interacts with the proximal CaV1.2 C-terminus and modulates calcium-dependent channel inactivation

The Rem, Rem2, Rad, and Gem/Kir (RGK) GTPases, comprise a subfamily of small Ras-related GTP-binding proteins, and have been shown to potently inhibit high voltage-activated Ca²⁺ channel current following overexpression. Although the molecular mechanisms underlying RGK-mediated Ca²⁺ channel regulation remains controversial, recent studies suggest that RGK proteins inhibit Ca²⁺ channel currents at the plasma membrane in part by interactions with accessory channel β subunits. In this paper, we extend our understanding of the molecular determinants required for RGK-mediated channel regulation by demonstrating a direct interaction between Rem and the proximal C-terminus of Ca_V1.2 (PCT), including the CB/IQ domain known to contribute to Ca²⁺/calmodulin (CaM)-mediated channel regulation. The Rem2 and Rad GTPases display similar patterns of PCT binding, suggesting that the Ca_V1.2 C-terminus represents a common binding partner for all RGK proteins. In vitro Rem:PCT binding is disrupted by Ca²⁺/CaM, and this effect is not due to Ca²⁺/CaM binding to the Rem C-terminus. In addition, co-overexpression of CaM partially relieves Rem-mediated L-type Ca²⁺ channel inhibition and slows the kinetics of Ca²⁺-dependent channel inactivation. Taken together, these results suggest that the association of Rem with the PCT represents a crucial molecular determinant in RGK-mediated Ca²⁺ channel regulation and that the physiological function of the RGK GTPases must be re-evaluated. Rather than serving as endogenous inhibitors of Ca²⁺ channel activity, these studies indicate that RGK proteins may play a more nuanced role, regulating Ca²⁺ currents via modulation of Ca²⁺/CaM-mediated channel inactivation kinetics.     

Insights on the mechanisms of Ca2+ regulation of connexin26 hemichannels revealed by human pathogenic mutations (D50N/Y)

Because of the large size and modest selectivity of the connexin hemichannel aqueous pore, hemichannel opening must be highly regulated to maintain cell viability. At normal resting potentials, this regulation is achieved predominantly by the physiological extracellular Ca²⁺ concentration, which drastically reduces hemichannel activity. Here, we characterize the Ca²⁺ regulation of channels formed by wild-type human connexin26 (hCx26) and its human mutations, D50N/Y, that cause aberrant hemichannel opening and result in deafness and skin disorders. We found that in hCx26 wild-type channels, deactivation kinetics are accelerated as a function of Ca²⁺ concentration, indicating that Ca²⁺ facilitates transition to, and stabilizes, the closed state of the hemichannels. The D50N/Y mutant hemichannels show lower apparent affinities for Ca²⁺-induced closing than wild-type channels and have more rapid deactivation kinetics, which are Ca²⁺ insensitive. These results suggest that D50 plays a role in (a) stabilizing the open state in the absence of Ca²⁺, and (b) facilitating closing and stabilization of the closed state in the presence of Ca²⁺. To explore the role of a negatively charged residue at position 50 in regulation by Ca²⁺, this position was substituted with a cysteine residue, which was then modified with a negatively charged methanethiosulfonate reagent, sodium (2-sulfanoethyl) methanethiosulfonate (MTSES)⁻. D50C mutant hemichannels display properties similar to those of D50N/Y mutants. Recovery of the negative charge with chemical modification by MTSES⁻ restores the wild-type Ca²⁺ regulation of the channels. These results confirm the essential role of a negative charge at position 50 for Ca²⁺ regulation. Additionally, charge-swapping mutagenesis studies suggest involvement of a salt bridge interaction between D50 and K61 in the adjacent connexin subunit in stabilizing the open state in low extracellular Ca²⁺. Mutant cycle analysis supports a Ca²⁺-sensitive interaction between these two residues in the open state of the channel. We propose that disruption of this interaction by extracellular Ca²⁺ destabilizes the open state and facilitates hemichannel closing. Our data provide a mechanistic understanding of how mutations at position 50 that cause human diseases are linked to dysfunction of hemichannel gating by external Ca²⁺.     

Model for the allosteric regulation of the Na+/Ca2+ exchanger NCX

The Na⁺/Ca²⁺ exchanger provides a major Ca²⁺ extrusion pathway in excitable cells and plays a key role in the control of intracellular Ca²⁺ concentrations. In Canis familiaris, Na⁺/Ca²⁺ exchanger (NCX) activity is regulated by the binding of Ca²⁺ to two cytosolic Ca²⁺‐binding domains, CBD1 and CBD2, such that Ca²⁺‐binding activates the exchanger. Despite its physiological importance, little is known about the exchanger's global structure, and the mechanism of allosteric Ca²⁺‐regulation remains unclear. It was found previously that for NCX in the absence of Ca²⁺ the two domains CBD1 and CBD2 of the cytosolic loop are flexibly linked, while after Ca²⁺‐binding they adopt a rigid arrangement that is slightly tilted. A realistic model for the mechanism of the exchanger's allosteric regulation should not only address this property, but also it should explain the distinctive behavior of Drosophila melanogaster's sodium/calcium exchanger, CALX, for which Ca²⁺‐binding to CBD1 inhibits Ca²⁺ exchange. Here, NMR spin relaxation and residual dipolar couplings were used to show that Ca²⁺ modulates CBD1 and CBD2 interdomain flexibility of CALX in an analogous way as for NCX. A mechanistic model for the allosteric Ca²⁺ regulation of the Na⁺/Ca²⁺ exchanger is proposed. In this model, the intracellular loop acts as an entropic spring whose strength is modulated by Ca²⁺‐binding to CBD1 controlling ion transport across the plasma membrane. Proteins 2016; 84:580–590. © 2016 Wiley Periodicals, Inc.     

Ca2+ in quality control

Calcium (Ca²⁺) has long been known as a ubiquitous intracellular second messenger, exploited by cells to control processes as diverse as development, proliferation, learning, muscle contraction and secretion. The spatial and temporal patterns of these Ca²⁺-associated signals, as well as their amplitude, is precisely controlled to create gradients of the ion, varying considerably depending on cell type and function. Tuning of intracellular Ca²⁺ is achieved in part by the buffering role of mitochondria, whose unperturbed function is essential for maintaining cellular energy balance. Quality of mitochondria is ensured by the process of targeted autophagy or mitophagy, which depends on a molecular cascade driving the catabolic process of autophagy toward damaged or deficient organelles for elimination via the lysosomal pathway. Nonspecific and targeted autophagy are highly regulated processes fundamental to cell growth and tissue homeostasis, allowing resources to be reallocated in nutrient-deprived cells as well as being instrumental in the repair of damaged organelles or the elimination of those in excess. Given the role of Ca²⁺ signaling in many fundamental cellular processes requiring precise regulation, the involvement of Ca²⁺ in autophagy is still somewhat ill-defined, and only in the past few years has evidence emerged linking the two. This mini-review aims to summarize recent work implicating Ca²⁺ as an important regulator of autophagy, outlining a role for Ca²⁺ that may be even more critical in the regulation of targeted mitochondrial autophagy.     

Mitochondrial calcium channels

Mitochondrial Ca²⁺ handling plays an important role in energy production and various cellular signaling processes. Mitochondrial Ca²⁺ uptake is regulated by the mitochondrial Ca²⁺ uniporter (MCU), at least one non-MCU Ca²⁺ channel and possibly a mitochondrial ryanodine receptor. Two distinct mechanisms mediate Ca²⁺ outward transport, the Na⁺-dependent (mNCX) and the Na⁺-independent Ca²⁺ efflux. In recent years we gained more insight into the regulation and function of these different Ca²⁺ transport mechanisms. However, the precise physiological role and the molecular structure of all mitochondrial Ca²⁺ transporters and channels still has to be determined.     

Calmodulin stimulation of calcium uptake and (Ca2+-Mg2+)-ATPase activities in microsomes from canine tracheal smooth muscle

The role of calmodulin in the regulation of microsomal ⁴⁵Ca²⁺ transport in canine tracheal smooth muscle was studied. Calmodulin stimulated ATP-dependent ⁴⁵Ca²⁺ uptake and (Ca²⁺Mg²⁺)-ATPase activities in microsomes treated with 0.5 mM EDTA and 0.5 mM EGTA. Oxalate also stimulated ATP-dependent ⁴⁵Ca²⁺ uptake and (Ca²⁺Mg²⁺)-ATPase activities and the stimulation was additive to the effects of calmodulin. The (Ca²⁺Mg²⁺)-ATPase and ATP-dependent ⁴⁵Ca²⁺ uptake activities are probably related as they exhibited similar [Ca²⁺]_free- and [calmodulin]-dependencies. These results indicate that calmodulin may play a role in the control of the cytosolic [Ca²⁺]_free in canine tracheal smooth muscle.     

Properties of calcium channels in cardiac muscle and vascular smooth muscle

The voltage-dependent slow channels in the myocardial cell membrane are the major pathway by which Ca²⁺ ions enter the cell during excitation for initiation and regulation of the force of contraction of cardiac muscle. The slow channels have some special properties, including functional dependence on metabolic energy, selective blockade by acidosis, and regulation by the intracellular cyclic nucleotide levels. Because of these special properties of the slow channels, Ca²⁺ influx into the myocardial cell can be controlled by extrinsic factors (such as autonomic nerve stimulation or circulating hormones) and by intrinsic factors (such as cellular pH or ATP level). The slow Ca²⁺ channels of the heart are regulated by cAMP in a stimulatory fashion. Elevation of cAMP produces a very rapid increase in number of slow channels available for voltage activation during excitation. The probability of a slow channel opening and the mean open time of the channel are increased. Therefore, any agent that increases the cAMP level of the myocardial cell will tend to potentiate I_si, Ca²⁺ influx, and contraction. The myocardial slow Ca²⁺ channels are also regulated by cGMP, in a manner that is opposite to that of CAMP. The effect of cGMP is presumably mediated by means of phosphorylation of a protein, as for example, a regulatory protein (inhibitory-type) associated with the slow channel. Preliminary data suggest that calmodulin also may play a role in regulation of the myocardial slow Ca²⁺ channels, possibly mediated by the Ca²⁺-calmodulin-protein kinase and phosphorylation of some regulatory-type of protein. Thus, it appears that the slow Ca²⁺ channel is a complex structure, including perhaps several associated regulatory proteins, which can be regulated by a number of extrinsic and intrinsic factors.VSM cells contain two types of Ca²⁺ channels: slow (L-type) Ca²⁺ channels and fast (T-type) Ca²⁺ channels. Although regulation of voltage-dependent Ca²⁺ slow channels of VSM cells have not been fully clarified yet, we have made some progress towards answering this question. Slow (L-type, high-threshold) Ca²⁺ channels may be modified by phosphorylation of the channel protein or an associated regulatory protein. In contrast to cardiac muscle where cAMP and cGMP have antagonistic effects on Ca²⁺ slow channel activity, in VSM, cAMP and cGMP have similar effects, namely inhibition of the Ca²⁺ slow channels. Thus, any agent that elevates cAMP or cGMP will inhibit Ca²⁺ influx, and thereby act to produce vasodilation. The Ca²⁺ slow channels require ATP for activity, with a K_0.5 of about 0.3 mM. C-kinase may stimulate the Ca²⁺ slow channels by phosphorylation. G-protein may have a direct action on the Ca²⁺ channels, and may mediate the effects of activation of some receptors. These mechanisms of Ca²⁺ channel regulation may be invoked during exposure to agonists or drugs, which change second messenger levels, thereby controlling vascular tone.     

Crystal Structures of Progressive Ca2+ Binding States of the Ca2+ Sensor Ca2+ Binding Domain 1 (CBD1) from the CALX Na+/Ca2+ Exchanger Reveal Incremental Conformational Transitions

Na⁺/Ca²⁺ exchangers (NCX) constitute a major Ca²⁺ export system that facilitates the re-establishment of cytosolic Ca²⁺ levels in many tissues. Ca²⁺ interactions at its Ca²⁺ binding domains (CBD1 and CBD2) are essential for the allosteric regulation of Na⁺/Ca²⁺ exchange activity. The structure of the Ca²⁺-bound form of CBD1, the primary Ca²⁺ sensor from canine NCX1, but not the Ca²⁺-free form, has been reported, although the molecular mechanism of Ca²⁺ regulation remains unclear. Here, we report crystal structures for three distinct Ca²⁺ binding states of CBD1 from CALX, a Na⁺/Ca²⁺ exchanger found in Drosophila sensory neurons. The fully Ca²⁺-bound CALX-CBD1 structure shows that four Ca²⁺ atoms bind at identical Ca²⁺ binding sites as those found in NCX1 and that the partial Ca²⁺ occupancy and apoform structures exhibit progressive conformational transitions, indicating incremental regulation of CALX exchange by successive Ca²⁺ binding at CBD1. The structures also predict that the primary Ca²⁺ pair plays the main role in triggering functional conformational changes. Confirming this prediction, mutagenesis of Glu⁴⁵⁵, which coordinates the primary Ca²⁺ pair, produces dramatic reductions of the regulatory Ca²⁺ affinity for exchange current, whereas mutagenesis of Glu⁵²⁰, which coordinates the secondary Ca²⁺ pair, has much smaller effects. Furthermore, our structures indicate that Ca²⁺ binding only enhances the stability of the Ca²⁺ binding site of CBD1 near the hinge region while the overall structure of CBD1 remains largely unaffected, implying that the Ca²⁺ regulatory function of CBD1, and possibly that for the entire NCX family, is mediated through domain interactions between CBD1 and the adjacent CBD2 at this hinge.     

Stimulation by calmodulin of Ca2+ uptake and (Ca2+-Mg2+) ATPase activity in membrane fractions from ox neurohypophyses

Calmodulin stimulated ⁴⁵Ca²⁺ uptake into a plasma membrane enriched fraction from ox neurohypophysial nerve endings and into a microsome fraction. The ⁴⁵Ca²⁺ uptake and the (Ca²⁺-Mg²⁺) ATPase activity in the plasma membrane fraction exhibited similar pCa and calmodulin sensitivities, suggesting that the enzyme activity is the biochemical expression of a high affinity Ca²⁺ pump. Calmodulin thus seems to play a role in regulation of the intracellular free Ca²⁺ concentration in the neurohypophysis.     

Motility processes in Acantharia (protozoa) III. Calcium regulation of the contraction - relaxation cycles of in vivo myonemes

The myonemes in the marine pelagic protozoa Acantharia are contractile organelles involved in buoyancy regulation. It was previously shown that they can perform three kinds of movement: rapid contraction, slow undulation and slow relaxation. They consist of a periodically striated bundle of 2–4 nm nonactin filaments that are twisted in pairs and shortened by a coiling mechanism. After permeabilization or demembranation, contraction and relaxation can still be performed by varying Ca²⁺ concentration and ATP is not needed. In the present paper, we have studied the role of Ca²⁺ and inhibitors of energy production in intact cells. Our data suggest that; (i) the in vivo rapid contraction subsequent to mechanical or electrical stimulation is triggered by Ca²⁺ influx across the cell membrane; (ii) the slow contraction that takes place during the undulating movement depends on Ca²⁺ release provided by internal calcium stores; (iii) the rapid contraction as well as the progressive shortening that occurs during the slow undulating movement are caused by Ca²⁺ binding to the myoneme filaments; (iv) ATP is not directly involved in the saturation by Ca²⁺ of Ca²⁺ sensitive sites located along the myoneme microstrands; (v) regulation of the movements of Ca²⁺ within the cytoplasm depends mainly upon the alternative pathway of ATP production; (vi) calmodulin is presumably involved in this regulation. A tentative cytophysiologic interpretation of the mechanism of contractility is proposed.