Calcium channel regulation and presynaptic plasticity

Voltage-gated calcium (Ca(2+)) channels initiate release of neurotransmitters at synapses, and regulation of presynaptic Ca(2+) channels has a powerful influence on synaptic strength. Presynaptic Ca(2+) channels form a large signaling complex, which targets synaptic vesicles to Ca(2+) channels for efficient release and mediates Ca(2+) channel regulation. Presynaptic plasticity regulates synaptic function on the timescale of milliseconds to minutes in response to neurotransmitters and the frequency of action potentials. This article reviews the regulation of presynaptic Ca(2+) channels by effectors and regulators of Ca(2+) signaling and describes the emerging evidence for a critical role of Ca(2+) channel regulation in control of neurotransmission and in presynaptic plasticity. Failure of function and regulation of presynaptic Ca(2+) channels leads to migraine, ataxia, and potentially other forms of neurological disease. We propose that presynaptic Ca(2+) channels serve as the regulatory node in a dynamic, multilayered signaling network that exerts short-term control of neurotransmission in response to synaptic activity.     

Ion channels in smooth muscle: regulation by the sarcoplasmic reticulum and mitochondria

In smooth muscle, Ca(2+) regulates cell division, growth and cell death as well as providing the main trigger for contraction. Ion channels provide the major access route to elevate the cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) in smooth muscle by permitting Ca(2+) entry across the plasma membrane and release of the ion from intracellular Ca(2+) stores. The control of [Ca(2+)](c) relies on feedback modulation of the entry and release channels by Ca(2+) itself. Local rises in [Ca(2+)](c) may promote or inhibit channel activity directly or indirectly. The latter may arise from Ca(2+) regulation of ionic conductances in the plasma membrane to provide control of cell excitability and so [Ca(2+)](c) entry. Organelles such as mitochondria may also contribute significantly to the feedback regulation of ion channel activity by the control of Ca(2+) or redox status of the cell. This brief review describes the feedback regulation of Ca(2+) release from the internal Ca(2+) store and of plasma membrane excitability in smooth muscle.     

Progress in calcium regulation in myocardial and vascular ischemia-reperfusion injury

Ischemia-reperfusion injury (IRI) has been recognized as a serious problem for therapy of cardiovascular diseases. Calcium regulation appears to be an important issue in the study of IRI. This article reviews calcium regulation in myocardial and vascular IRI, including the calcium overload and calcium sensitivity in IRI. This review is focused on the key players in Ca(2+) handling in IRI, including membrane damage resulting in increase in Ca(2+) influx, reverse-mode of Na(+)-Ca(2+) exchangers leading to increased Ca(2+) entry, the decreased activity of sarcoplasmic reticulum (SR) Ca(2+)-ATPase causing SR Ca(2+) uptake dysfunction, and increased activity of Rho kinase. These key players in Ca(2+) homeostasis will provide promising strategies and potential targets for therapy of cardiovascular IRI.     

T-type calcium channel expression and function in the diseased heart

The regulation of intracellular Ca (2+) is essential for cardiomyocyte function, and alterations in proteins that regulate Ca (2+) influx have dire consequences in the diseased heart. Low voltage-activated, T-type Ca (2+) channels are one pathway of Ca (2+) entry that is regulated according to developmental stage and in pathological conditions in the adult heart. Cardiac T-type channels consist of two main types, Cav3.1 (α1G) and Cav3.2 (α1H), and both can be induced in the myocardium in disease and injury but still, relatively little is known about mechanisms for their regulation and their respective functions. This article integrates previous data establishing regulation of T-type Ca (2+) channels in animal models of cardiac disease, with recent data that begin to address the functional consequences of cardiac Cav3.1 and Cav3.2 Ca (2+) channel expression in the pathological setting. The putative association of T-type Ca (2+) channels with Ca (2+) dependent signaling pathways in the context of cardiac hypertrophy is also discussed.     

Recent advances in Ca(2+)-dependent Ras regulation and cell proliferation

Our understanding of the mechanisms whereby growth factors stimulate cell proliferation through the Ras pathway stems largely from studies of the canonical pathway involving recruitment of Ras activators and inhibitors to the vicinity of receptor tyrosine kinases via phosphotyrosine-binding adaptor proteins. Ca(2+) has seldom joined the party, despite the identification of phospholipase Cgamma and Ca(2+) entry as receptor tyrosine kinase-dependent signals. Mechanisms by which Ca(2+) can directly influence Ras activity have remained relatively elusive. Similarly, the mechanisms whereby Ca(2+) modulates the cell cycle have been equally murky, and yet there are some interesting parallels in the role of Ras and Ca(2+) in cell cycle re-entry. This review focuses on a number of novel mechanisms that link Ca(2+) with the regulation of Ras activity and signaling output. Their collective discovery adds to the complexities of Ras regulation and raises further questions about the role of Ca(2+) signals in Ras-dependent cell proliferation.     

Factors that determine Ca2+ sensitivity of photoreceptor guanylyl cyclase. Kinetic analysis of the interaction between the Ca2+-bound and the Ca2+-free guanylyl cyclase activating proteins (GCAPs) and recombinant photoreceptor guanylyl cyclase 1 (RetGC-1)

We explored the possibility that, in the regulation of an effector enzyme by a Ca(2+)-sensor protein, the actual Ca(2+) sensitivity of the effector enzyme can be determined not only by the affinity of the Ca(2+)-sensor protein for Ca(2+) but also by the relative affinities of its Ca(2+)-bound versus Ca(2+)-free form for the effector enzyme. As a model, we used Ca(2+)-sensitive activation of photoreceptor guanylyl cyclase (RetGC-1) by guanylyl cyclase activating proteins (GCAPs). A substitution Arg(838)Ser in RetGC-1 found in human patients with cone-rod dystrophy is known to shift the Ca(2+) sensitivity of RetGC-1 regulation by GCAP-1 to a higher Ca(2+) range. We find that at physiological concentrations of Mg(2+) this mutation increases the free Ca(2+) concentration required for half-maximal inhibition of the cyclase from 0.27 to 0.61 microM. Similar to rod outer segment cyclase, Ca(2+) sensitivity of recombinant RetGC-1 is strongly affected by Mg(2+), but the shift in Ca(2+) sensitivity for the R838S mutant relative to the wild type is Mg(2+)-independent. We determined the apparent affinity of the wild-type and the mutant RetGC-1 for both Ca(2+)-bound and Ca(2+)-free GCAP-1 and found that the net shift in Ca(2+) sensitivity of the R838S RetGC-1 observed in vitro can arise predominantly from the change in the affinity of the mutant cyclase for the Ca(2+)-free versus Ca(2+)-loaded GCAP-1. Our findings confirm that the dynamic range for RetGC regulation by Ca(2+)/GCAP is determined by both the affinity of GCAP for Ca(2+) and relative affinities of the effector enzyme for the Ca(2+)-free versus Ca(2+)-loaded GCAP.     

Bradykinin and ATP accelerate Ca(2+) efflux from rat sensory neurons via protein kinase C and the plasma membrane Ca(2+) pump isoform 4.

Modulation of Ca(2+) channels by neurotransmitters provides critical control of neuronal excitability and synaptic strength. Little is known about regulation of the Ca(2+) efflux pathways that counterbalance Ca(2+) influx in neurons. We demonstrate that bradykinin and ATP significantly facilitate removal of action potential-induced Ca(2+) loads by stimulating plasma membrane Ca(2+)-ATPases (PMCAs) in rat sensory neurons. This effect was mimicked in the soma and axonal varicosities by phorbol esters and was blocked by antagonists of protein kinase C (PKC). Reduced expression of PMCA isoform 4 abolished, and overexpression of isoform 4b enhanced, PKC-dependent facilitation of Ca(2+) efflux. This acceleration of PMCA4 underlies the shortening of the action potential afterhyperpolarization produced by activation of bradykinin and purinergic receptors. Thus, isoform-specific modulation of PMCA-mediated Ca(2+) efflux represents a novel mechanism to control excitability in sensory neurons.     

Mitochondrial Ca2+ and the heart

It is now well established that mitochondria accumulate Ca(2+) ions during cytosolic Ca(2+) ([Ca(2+)](i)) elevations in a variety of cell types including cardiomyocytes. Elevations in intramitochondrial Ca(2+) ([Ca(2+)](m)) activate several key enzymes in the mitochondrial matrix to enhance ATP production, alter the spatial and temporal profile of intracellular Ca(2+) signaling, and play an important role in the initiation of cell death pathways. Moreover, mitochondrial Ca(2+) uptake stimulates nitric oxide (NO) production by mitochondria, which modulates oxygen consumption, ATP production, reactive oxygen species (ROS) generation, and in turn provides negative feedback for the regulation of mitochondrial Ca(2+) accumulation. Controversy remains, however, whether in cardiac myocytes mitochondrial Ca(2+) transport mechanisms allow beat-to-beat transmission of fast cytosolic [Ca(2+)](i) oscillations into oscillatory changes in mitochondrial matrix [Ca(2+)](m). This review critically summarizes the recent experimental work in this field.     

Dynamic features of allosteric Ca2+ sensor in tissue-specific NCX variants

The Na(+)-Ca(2+) exchanger (NCX) mediated Ca(2+) fluxes are essential for handling Ca(2+) homeostasis in many cell-types. Eukaryotic NCX variants contain regulatory CBD1 and CBD2 domains, whereas in distinct variants the Ca(2+) binding to Ca3-Ca4 sites of CBD1 results either in sustained activation, inhibition or no effect. CBD2 contains an alternatively spliced segment, which is expressed in a tissue-specific manner although its impact on allosteric regulation remains unclear. Recent studies revealed that the Ca(2+) binding to Ca3-Ca4 sites results in interdomain tethering of CBDs, which rigidifies CBDs movements with accompanied slow dissociation of "occluded" Ca(2+). Here we investigate the effects of CBD2 variants on Ca(2+) occlusion in the two-domain construct (CBD12). Mutational studies revealed that both sites (Ca3 and Ca4) contribute to Ca(2+) occlusion, whereas after dissociation of the first Ca(2+) ion the second Ca(2+) ion becomes occluded. This mechanism is common for the brain, kidney and cardiac splice variants of CBD12, although the occluded Ca(2+) exhibits 20-50-fold difference in off-rates among the tested variants. Therefore, the spliced exons on CBD2 affect the rate-limiting step of the occluded Ca(2+) dissociation at the primary regulatory sensor to shape dynamic features of allosteric regulation in NCX variants.     

Intracellular sodium modulates mitochondrial calcium signaling in vascular endothelial cells

We have investigated the role of extramitochondrial Na(+) for the regulation of mitochondrial Ca(2+) concentration ([Ca(2+)](m)) in permeabilized single vascular endothelial cells. [Ca(2+)](m) was measured by loading the cells with the membrane-permeant Ca(2+) indicator fluo-3/AM and subsequent removal of cytoplasmic fluo-3 by surface membrane permeabilization with digitonin. An elevation of extramitochondrial Ca(2+) resulted in a dose-dependent increase in the rate of Ca(2+) accumulation into mitochondria (k(0.5) = 3 microm) via the mitochondrial Ca(2+) uniporter. In the presence of 10 mm extramitochondrial Na(+) ([Na(+)](em)), repetitive application of brief pulses of high Ca(2+) (2-10 microm) to simulate cytoplasmic [Ca(2+)] oscillations caused transient increases of [Ca(2+)](m) characterized by a fast rising phase that was followed by a slow decay. Removal of extramitochondrial Na(+) or inhibition of mitochondrial Na(+)/Ca(2+) exchange with clonazepam blocked mitochondrial Ca(2+) efflux and resulted in a net accumulation of Ca(2+) by the mitochondria. Half-maximal activation of mitochondrial Na(+)/Ca(2+) exchange occurred at [Na(+)](em) = 4.4 mm, which is well within the physiological range of cytoplasmic [Na(+)]. This study provides evidence that Ca(2+) efflux from the mitochondria in vascular endothelial cells occurs solely via Na(+)/Ca(2+) exchange and emphasizes the important role of intracellular Na(+) for mitochondrial Ca(2+) regulation.     

Characterization of Mg2+ inhibition of mitochondrial Ca2+ uptake by a mechanistic model of mitochondrial Ca2+ uniporter

Ca(2+) is an important regulatory ion and alteration of mitochondrial Ca(2+) homeostasis can lead to cellular dysfunction and apoptosis. Ca(2+) is transported into respiring mitochondria via the Ca(2+) uniporter, which is known to be inhibited by Mg(2+). This uniporter-mediated mitochondrial Ca(2+) transport is also shown to be influenced by inorganic phosphate (Pi). Despite a large number of experimental studies, the kinetic mechanisms associated with the Mg(2+) inhibition and Pi regulation of the uniporter function are not well established. To gain a quantitative understanding of the effects of Mg(2+) and Pi on the uniporter function, we developed here a mathematical model based on known kinetic properties of the uniporter and presumed Mg(2+) inhibition and Pi regulation mechanisms. The model is extended from our previous model of the uniporter that is based on a multistate catalytic binding and interconversion mechanism and Eyring's free energy barrier theory for interconversion. The model satisfactorily describes a wide variety of experimental data sets on the kinetics of mitochondrial Ca(2+) uptake. The model also appropriately depicts the inhibitory effect of Mg(2+) on the uniporter function, in which Ca(2+) uptake is hyperbolic in the absence of Mg(2+) and sigmoid in the presence of Mg(2+). The model suggests a mixed-type inhibition mechanism for Mg(2+) inhibition of the uniporter function. This model is critical for building mechanistic models of mitochondrial bioenergetics and Ca(2+) handling to understand the mechanisms by which Ca(2+) mediates signaling pathways and modulates energy metabolism.     

Dose-dependent and isoform-specific modulation of Ca2+ channels by RGK GTPases

Although inhibition of voltage-gated calcium channels by RGK GTPases (RGKs) represents an important mode of regulation to control Ca(2+) influx in excitable cells, their exact mechanism of inhibition remains controversial. This has prevented an understanding of how RGK regulation can be significant in a physiological context. Here we show that RGKs-Gem, Rem, and Rem2-decreased Ca(V)1.2 Ca(2+) current amplitude in a dose-dependent manner. Moreover, Rem2, but not Rem or Gem, produced dose-dependent alterations on gating kinetics, uncovering a new mode by which certain RGKs can precisely modulate Ca(2+) currents and affect Ca(2+) influx during action potentials. To explore how RGKs influence gating kinetics, we separated the roles mediated by the Ca(2+) channel accessory beta subunit's interaction with its high affinity binding site in the pore-forming alpha(1C) subunit (AID) from its other putative contact sites by utilizing an alpha(1C)*beta3 concatemer in which the AID was mutated to prevent beta subunit interaction. This mutant concatemer generated currents with all the hallmarks of beta subunit modulation, demonstrating that AID-beta-independent interactions are sufficient for beta subunit modulation. Using this construct we found that although inhibition of current amplitude was still partially sensitive to RGKs, Rem2 no longer altered gating kinetics, implicating different determinants for this specific mode of Rem2-mediated regulation. Together, these results offer new insights into the molecular mechanism of RGK-mediated Ca(2+) channel current modulation.     

Molecular identity and functional properties of the mitochondrial na+/ca2+ exchanger

The mitochondrial membrane potential that powers the generation of ATP also facilitates mitochondrial Ca(2+) shuttling. This process is fundamental to a wide range of cellular activities, as it regulates ATP production, shapes cytosolic and endoplasmic recticulum Ca(2+) signaling, and determines cell fate. Mitochondrial Ca(2+) transport is mediated primarily by two major transporters: a Ca(2+) uniporter that mediates Ca(2+) uptake and a Na(+)/Ca(2+) exchanger that subsequently extrudes mitochondrial Ca(2+). In this minireview, we focus on the specific role of the mitochondrial Na(+)/Ca(2+) exchanger and describe its ion exchange mechanism, regulation by ions, and putative partner proteins. We discuss the recent molecular identification of the mitochondrial exchanger and how its activity is linked to physiological and pathophysiological processes.     

Vascular smooth muscle mitochondria at the cross roads of Ca(2+) regulation

Mitochondria play an essential role in the regulation of vascular smooth muscle Ca(2+) signaling being simultaneously integrated in the regulation of ion channels and Ca(2+) transporters, oxygen radical production, metabolite recycling and intracellular redox potential. Mitochondria buffer Ca(2+) from cytoplasmic microdomains to alter the spatio-temporal pattern of Ca(2+) gradients following Ca(2+)-influx and Ca(2+)-release, and thus control site-specific, Ca(2+)-dependent ion channel activation and inactivation. The sub-cellular localization of mitochondria in conjunction with tissue-specific channel expression is fundamental to vascular heterogeneity. The mitochondrial electron transport chain recycles metabolic intermediates that modulate cellular redox potential and produces oxygen radicals in proportion to oxygen tension. Perturbation of specific complexes within the transport chain can affects NADH:NAD and ATP:ADP ratios and radical production, which can in turn influence second messenger metabolism, ion channel gating and Ca(2+)-transporter activity. Mitochondria thus provide the common ground for cross-talk between these regulatory systems that are mutually sensitive to one another. This cross-talk between signaling systems provides a means to render the physiological regulation of vascular tone responsive to complex stimulation by paracrine and endocrine factors, blood pressure and flow, tissue oxygenation and metabolic state.     

Tyrosine phosphatase PTP1B interacts with TRPV6 in vivo and plays a role in TRPV6-mediated calcium influx in HEK293 cells

This study investigates the role of tyrosine phosphorylation and dephosphorylation in the regulation of the Ca(2+) permeant TRPV6 channel. HEK293 cells co-transfected with TRPV6 and the tyrosine phosphatase PTP1B show a constitutive Ca(2+) entry which was independent of tyrosine phosphorylation under resting conditions. Following depletion of intracellular Ca(2+) stores, TRPV6-mediated Ca(2+) entry could be increased in the presence of a tyrosine phosphatase inhibitor (bis-(N,N-dimethyl-hydroxamido) hydroxo-vanadate; DMHV). Inhibition of Src-kinases completely abolished DMHV-induced increase in TRPV6-mediated Ca(2+) influx. Co-transfection with Src led to tyrosine phosphorylation of TRPV6 which could be dephosphorylated by PTP1B. In vivo interaction of TRPV6 with PTP1B was visualized using the bimolecular fluorescence complementation (BiFC) method and proved by co-immunoprecipitation of both proteins. These data indicate that tyrosine phosphorylation is involved in the regulation of the TRPV6 channel protein.     

Caffeine stores and dopamine differentially require Ca(2+) channels in goldfish somatotropes

The regulation of growth hormone (GH) secretion by intracellular Ca(2+) stores was studied in dissociated goldfish somatotropes. We characterized a caffeine-activated intracellular store that had been shown to mediate GH release in response to gonadotropin-releasing hormone. The peak response of caffeine stimulation was reduced by approximately 28% by 100 microM ryanodine in a use-dependent manner suggesting that the first 10 min of GH release is partially mediated by a caffeine-activated ryanodine receptor. The temporal sensitivities of caffeine- and dopamine-evoked GH release to blockade of Cd(2+)-sensitive Ca(2+) channels were compared. We demonstrated that the initial phase of dopamine-evoked release was dependent on Ca(2+) channels, whereas the initial phase of caffeine-evoked release was sensitive only to pretreatment blockade. This would suggest that the maintenance of one class of caffeine-activated intracellular stores requires entry of Ca(2+) through Cd(2+)-sensitive Ca(2+) channels. This differential temporal requirement for Ca(2+) channels in Ca(2+) signaling may be a mechanism to segregate intracellular signaling pathways of multiple neuroendocrine regulators in the teleost pituitary.     

Analysis of cellular calcium fluxes in cardiac muscle to understand calcium homeostasis in the heart

Central to controlling intracellular calcium concentration ([Ca(2+)](i)) are a number of Ca(2+) transporters and channels with the L-type Ca(2+) channel, Na(+)-Ca(2+) exchanger and sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) being of particular note in the heart. This review concentrates on the regulation of [Ca(2+)](i) in cardiac muscle and the homeostatic mechanisms employed to ensure that the heart can operate under steady-state conditions on a beat by beat basis. To this end we discuss the relative importance of various sources and sinks of Ca(2+) responsible for initiating contraction and relaxation in cardiac myocytes and how these can be manipulated to regulate the Ca(2+) content of the major Ca(2+) store, the sarcoplasmic reticulum (SR). We will present a simple feedback system detailing how such control can be achieved and highlight how small perturbations to the steady-state operation of the feedback loop can be both beneficial physiologically and underlie changes in systolic Ca(2+) in ageing and heart disease. In addition to manipulating the amplitude of the normal systolic Ca(2+) transient, the tight regulation of SR Ca(2+) content is also required to prevent the abnormal, spontaneous or diastolic release of Ca(2+) from the SR. Such diastolic events are a major factor contributing to the genesis of cardiac arrhythmias in disease situations and in recently identified familial mutations in the SR Ca(2+) release channel (ryanodine receptor, RyR). How such diastolic release arises and potential mechanisms for controlling this will be discussed.     

Regulation of the epithelial Ca2+ channels TRPV5 and TRPV6 by 1alpha,25-dihydroxy Vitamin D3 and dietary Ca2+

Active, transepithelial Ca(2+) transport is a pivotal process in the regulation of Ca(2+) homeostasis and consists of three sequential steps: apical Ca(2+) influx, diffusion towards the basolateral membrane and subsequent extrusion into the blood compartment. TRPV5 and TRPV6 (renamed after ECaC1 and ECaC2/CaT1, respectively) constitute the rate-limiting influx step of transepithelial Ca(2+) transport and these highly selective Ca(2+) channels are controlled by several factors. This review focuses on the regulation of TRPV5 and TRPV6 abundance and/or activity by 1alpha,25-dihydroxyVitamin D(3) (1alpha,25(OH)(2)D(3)), dietary Ca(2+) and the auxiliary protein pair S100A10/annexin 2. Finally, the implications for our understanding of transcellular Ca(2+) transport will be discussed.     

Calcium-myristoyl Tug is a new mechanism for intramolecular tuning of calcium sensitivity and target enzyme interaction for guanylyl cyclase-activating protein 1: dynamic connection between N-fatty acyl group and EF-hand controls calcium sensitivity

Guanylyl cyclase-activating protein 1 (GCAP1), a myristoylated Ca(2+) sensor in vision, regulates retinal guanylyl cyclase (RetGC). We show that protein-myristoyl group interactions control Ca(2+) sensitivity, apparent affinity for RetGC, and maximal level of cyclase activation. Mutating residues near the myristoyl moiety affected the affinity of Ca(2+) binding to EF-hand 4. Inserting Phe residues in the cavity around the myristoyl group increased both the affinity of GCAP1 for RetGC and maximal activation of the cyclase. NMR spectra show that the myristoyl group in the L80F/L176F/V180F mutant remained sequestered inside GCAP1 in both Ca(2+)-bound and Mg(2+)-bound states. This mutant displayed much higher affinity for the cyclase but reduced Ca(2+) sensitivity of the cyclase regulation. The L176F substitution improved affinity of myristoylated and non-acylated GCAP1 for the cyclase but simultaneously reduced the affinity of Ca(2+) binding to EF-hand 4 and Ca(2+) sensitivity of the cyclase regulation by acylated GCAP1. The replacement of amino acids near both ends of the myristoyl moiety (Leu(80) and Val(180)) minimally affected regulatory properties of GCAP1. N-Lauryl- and N-myristoyl-GCAP1 activated RetGC in a similar fashion. Thus, protein interactions with the central region of the fatty acyl chain optimize GCAP1 binding to RetGC and maximize activation of the cyclase. We propose a dynamic connection (or "tug") between the fatty acyl group and EF-hand 4 via the C-terminal helix that attenuates the efficiency of RetGC activation in exchange for optimal Ca(2+) sensitivity.