Regulation of sodium and calcium channels by signaling complexes

Membrane depolarization and intracellular calcium transients generated by activation of voltage-gated sodium and calcium channels are local signals, which initiate physiological processes such as action potential conduction, synaptic transmission, and excitation-contraction coupling. Targeting of effector proteins and regulatory proteins to ion channels is an important mechanism to ensure speed, specificity, and precise regulation of signaling events in response to local stimuli. In this article, we review recent experimental results showing that sodium and calcium channels form local signaling complexes, in which effector proteins, anchoring proteins, and regulatory proteins interact directly with ion channels. The intracellular domains of these channels serve as signaling platforms, mediating their participation in intracellular signaling processes. These protein-protein interactions are important for efficient synaptic transmission and for regulation of ion channels by neurotransmitters and intracellular second messengers. These localized signaling complexes are essential for normal function and regulation of electrical excitability, synaptic transmission, and excitation-contraction coupling.     

Regulation of Sodium and Calcium Channels by Signaling Complexes

Membrane depolarization and intracellular calcium transients generated by activation of voltage-gated sodium and calcium channels are local signals, which initiate physiological processes such as action potential conduction, synaptic transmission, and excitation-contraction coupling. Targeting of effector proteins and regulatory proteins to ion channels is an important mechanism to ensure speed, specificity, and precise regulation of signaling events in response to local stimuli. In this article, we review recent experimental results showing that sodium and calcium channels form local signaling complexes, in which effector proteins, anchoring proteins, and regulatory proteins interact directly with ion channels. The intracellular domains of these channels serve as signaling platforms, mediating their participation in intracellular signaling processes. These protein-protein interactions are important for efficient synaptic transmission and for regulation of ion channels by neurotransmitters and intracellular second messengers. These localized signaling complexes are essential for normal function and regulation of electrical excitability, synaptic transmission, and excitation-contraction coupling.     

Tuning store-operated calcium entry to modulate Ca2+-dependent physiological processes

The intracellular calcium signaling processes are tightly regulated to ensure the generation of calcium signals with the specific spatiotemporal characteristics required for regulating various cell functions. Compartmentalization of the molecular components involved in the generation of these signals at discrete intracellular sites ensures the signaling specificity and transduction fidelity of the signal for regulating downstream effector processes. Store-operated calcium entry (SOCE) is ubiquitously present in cells and is critical for essential cell functions in a variety of tissues. SOCE is mediated via plasma membrane Ca²⁺ channels that are activated when luminal [Ca²⁺] of the endoplasmic reticulum ([Ca²⁺]_ER) is decreased. The ER-resident stromal interaction molecules, STIM1 and STIM2, respond to decreases in [Ca²⁺]_ER by undergoing conformational changes that cause them to aggregate at the cell periphery in ER-plasma membrane (ER-PM) junctions. At these sites, STIM proteins recruit Orai1 channels and trigger their activation. Importantly, the two STIM proteins concertedly modulate Orai1 function as well as the sensitivity of SOCE to ER-Ca²⁺ store depletion. Another family of plasma membrane Ca²⁺ channels, known as the Transient Receptor Potential Canonical (TRPC) channels (TRPC1-7) also contribute to sustained [Ca²⁺]_i elevation. Although Ca²⁺ signals generated by these channels overlap with those of Orai1, they regulate distinct functions in the cells. Importantly, STIM1 is also required for plasma membrane localization and activation of some TRPCs. In this review, we will discuss various molecular components and factors that govern the activation, regulation and modulation of the Ca²⁺ signal generated by Ca²⁺ entry pathways in response to depletion of ER-Ca²⁺ stores. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.     

Signaling complexes of voltage-gated calcium channels

Voltage gated calcium channels are key mediators of depolarization induced calcium entry into electrically excitable cells. There is increasing evidence that voltage gated calcium channels, like many other types of ionic channels, do not operate in isolation, but instead forms signaling complexes with signaling molecules, G protein coupled receptors, and other types of ion channels. Furthermore, there appears to be bidirectional signaling within these protein complexes, thus allowing not only for efficient translation of calcium signals into cellular responses, but also for tight control of calcium entry per se. In this review, we will focus predominantly on signaling complexes between G protein-coupled receptors and high voltage activated calcium channels, and on complexes of voltage-gated calcium channels and members of the potassium channel superfamily.     

Signaling complexes of voltage-gated calcium channels

Voltage gated calcium channels are key mediators of depolarization induced calcium entry into electrically excitable cells. There is increasing evidence that voltage gated calcium channels, like many other types of ionic channels, do not operate in isolation, but instead forms signaling complexes with signaling molecules, G protein coupled receptors, and other types of ion channels. Furthermore, there appears to be bidirectional signaling within these protein complexes, thus allowing not only for efficient translation of calcium signals into cellular responses, but also for tight control of calcium entry per se. In this review, we will focus predominantly on signaling complexes between G protein-coupled receptors and high voltage activated calcium channels, and on complexes of voltage-gated calcium channels and members of the potassium channel superfamily.     

Presynaptic calcium stores and synaptic transmission

Following the gradual recognition of the importance of intracellular calcium stores for somatodendritic signaling in the mammalian brain, recent reports have also indicated a significant role of presynaptic calcium stores. Ryanodine-sensitive stores generate local, random calcium signals that shape spontaneous transmitter release. They amplify spike-driven calcium signals in presynaptic terminals, and consequently enhance the efficacy of transmitter release. They appear to be recruited by an association with certain types of calcium-permeant ion channels, and they induce specific forms of synaptic plasticity. Recent research also indicates a role of inositoltrisphosphate-sensitive presynaptic calcium stores in synaptic plasticity.     

Bioengineering plant resistance to abiotic stresses by the global calcium signal system

Considerable progresses have taken place both in the methodology available to study changes in intracellular cytosolic calcium and in our understanding of calcium signaling cascades. It is generally accepted that the global calcium signal system functions importantly in coping with plant abiotic stresses, especially drought stress, which has been proved further by the recent transgenic and molecular breeding reports under soil water deficits. In plant cells, calcium plays roles as a universal transducer coupling a wide range of extracellular stimuli with intracellular responses. Different extracellular stimuli trigger specific calcium signatures: dynamics, amplitude and duration of calcium transients specify the nature, implication and intensity of stimuli. Calcium-binding proteins (sensors) play a critical role in decoding calcium signatures and transducing signals by activating specific targets and corresponding metabolic pathways. Calmodulin (CAM) is a calcium sensor known to regulate the activity of many mammalian proteins, whose targets in plants are now being identified. Higher plants possess a rapidly growing list of CAM targets with a variety of cellular functions. Nevertheless, many targets appear to be unique to higher plant cells and remain characterized, calling for a concerted effort from plant and animal scientists to elucidate their functions. To date, three major classes of plant calcium signals encoding elements in the calcium signal system, including calcium-permeable ion channels,Ca(2)+/ H(+) antiporters and Ca(2)+-ATPases, are responsible for drought stress signal transduction directly or indirectly. This review summarizes the current knowledge of calcium signals involved in plant abiotic stresses and presents suggestions for future focus areas of study.     

Inositol trisphosphate receptors in smooth muscle cells

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are a family of tetrameric intracellular calcium (Ca(2+)) release channels that are located on the sarcoplasmic reticulum (SR) membrane of virtually all mammalian cell types, including smooth muscle cells (SMC). Here, we have reviewed literature investigating IP(3)R expression, cellular localization, tissue distribution, activity regulation, communication with ion channels and organelles, generation of Ca(2+) signals, modulation of physiological functions, and alterations in pathologies in SMCs. Three IP(3)R isoforms have been identified, with relative expression and cellular localization of each contributing to signaling differences in diverse SMC types. Several endogenous ligands, kinases, proteins, and other modulators control SMC IP(3)R channel activity. SMC IP(3)Rs communicate with nearby ryanodine-sensitive Ca(2+) channels and mitochondria to influence SR Ca(2+) release and reactive oxygen species generation. IP(3)R-mediated Ca(2+) release can stimulate plasma membrane-localized channels, including transient receptor potential (TRP) channels and store-operated Ca(2+) channels. SMC IP(3)Rs also signal to other proteins via SR Ca(2+) release-independent mechanisms through physical coupling to TRP channels and local communication with large-conductance Ca(2+)-activated potassium channels. IP(3)R-mediated Ca(2+) release generates a wide variety of intracellular Ca(2+) signals, which vary with respect to frequency, amplitude, spatial, and temporal properties. IP(3)R signaling controls multiple SMC functions, including contraction, gene expression, migration, and proliferation. IP(3)R expression and cellular signaling are altered in several SMC diseases, notably asthma, atherosclerosis, diabetes, and hypertension. In summary, IP(3)R-mediated pathways control diverse SMC physiological functions, with pathological alterations in IP(3)R signaling contributing to disease.     

Trafficking of L-type calcium channels mediated by the postsynaptic scaffolding protein AKAP79

Accurate calcium signaling requires spatial and temporal coordination of voltage-gated calcium channels (VGCCs) and a variety of signal transduction proteins. Accordingly, regulation of L-type VGCCs involves the assembly of complexes that include the channel subunits, protein kinase A (PKA), protein kinase A anchoring proteins (AKAPs), and beta2-adrenergic receptors, although the molecular details underlying these interactions remain enigmatic. We show here, by combining extracellular epitope splicing into the channel pore-forming subunit and immunoassays with whole cell and single channel electrophysiological recordings, that AKAP79 directly regulates cell surface expression of L-type calcium channels independently of PKA. This regulation involves a short polyproline sequence contained specifically within the II-III cytoplasmic loop of the channel. Thus we propose a novel mechanism whereby AKAP79 and L-type VGCCs function as components of a biosynthetic mechanism that favors membrane incorporation of organized molecular complexes in a manner that is independent of PKA phosphorylation events.     

Local Ca2+ signals in cellular signalling

Local Ca2+ rises and propagated Ca2+ signals represent different patterns that are differentially decoded for fine tuning cellular signalling. This Ca2+ concentration plasticity is absolutely required to allow adaptation to different needs of the cells ranging from contraction or increased learning to proliferation and cell death. A wide diversity of molecular structures and specific location of Ca2+ signalling molecules confer spatial and temporal versatility to the Ca2+ changes allowing specific cellular responses to be elicited. Various types of local Ca2+ signals have been described. Ca2+ spikes correspond to Ca2+ signals spanning several micrometers but displaying limited propagation into a cell leading to regulation of cellular functions in one particular zone of this cell. This is of particular relevance in cells presenting distinct morphological specializations, i.e. apical versus basal sites or dendritic versus somatic/axonal sites. More stereotyped elementary Ca2+ events (denominated Ca2+ sparks or Ca2+ puffs depending on the type of endoplasmic reticulum Ca2+ release channel involved) are highly confined and non-propagated Ca2+ rises which are observed in the close neighbouring of the Ca2+ channels. These elementary Ca2+ events play a major role in controlling cellular excitability. Elementary Ca2+ events involve Ca2+ release channels such as the ryanodine receptors (RyRs) and the inositol 1,4,5-trisphosphate receptors (InsP3Rs). The molecular bases underlying the various local Ca2+ release events will be discussed by reviewing the channels and particularly the different isoforms of RyRs and InsP3Rs and their role in inducing localized Ca2+ responses. These calcium release events are controlled by various second messengers and are regulated by Ca2+ channel-associated proteins, intra-luminal Ca2+ content of the endoplasmic reticulum (ER) and other Ca2+ organelles. We will discuss on how the control of local cellular Ca2+ content may account for cellular functions in physiological and physiopathological conditions.     

The cellular and molecular basis of store-operated calcium entry

The impact of calcium signalling on so many areas of cell biology reflects the crucial role of calcium signals in the control of diverse cellular functions. Despite the precision with which spatial and temporal details of calcium signals have been resolved, a fundamental aspect of the generation of calcium signals -- the activation of 'store-operated channels' (SOCs) -- remains a molecular and mechanistic mystery. Here we review new insights into the exchange of signals between the endoplasmic reticulum (ER) and plasma membrane that result in activation of calcium entry channels mediating crucial long-term calcium signals.     

Opioid Receptor Regulation of Neuronal Voltage-Gated Calcium Channels

Neuronal voltage-gated calcium channels play a pivotal role in the conversion of electrical signals into calcium entry into nerve endings that is required for the release of neurotransmitters. They are under the control of a number of cellular signaling pathways that serve to fine tune synaptic activities, including G-protein coupled receptors (GPCRs) and the opioid system. Besides modulating channel activity via activation of second messengers, GPCRs also physically associate with calcium channels to regulate their function and expression at the plasma membrane. In this mini review, we discuss the mechanisms by which calcium channels are regulated by classical opioid and nociceptin receptors. We highlight the importance of this regulation in the control of neuronal functions and their implication in the development of disease conditions. Finally, we present recent literature concerning the use of novel μ-opioid receptor/nociceptin receptor modulators and discuss their use as potential drug candidates for the treatment of pain.

     

Delay of intracellular calcium transients using a calcium chelator: application to high-throughput screening of the capsaicin receptor ion channel and G-protein-coupled receptors.

Whole-cell functional assays are often used for high-throughput screening (HTS) of molecular targets such as ion channels and G-protein-coupled receptors. A common method for assaying the activity of these membrane proteins is to measure the change in intracellular calcium concentration upon receptor stimulation. These changes in calcium concentration are typically transient and therefore not readily adapted to high-density plate formats used in HTS instruments. We have demonstrated that an intracellular calcium chelator, BAPTA, was able to delay by 5- to 20-fold and extend for several minutes the observed calcium signals initiated by extracellular calcium influx or release of calcium from intracellular stores. As examples, we used cells expressing a calcium-permeable ion channel, vanilloid receptor type 1 (the capsaicin receptor), and two G-protein-coupled receptors. These receptor-mediated increases in intracellular calcium concentration were measured by both fluorescence-based and luminescence-based detection methods. The use of an intracellular calcium chelator to delay calcium signaling should have wide application since it allows the measurement of the functional activity of any cellular receptor that signals through calcium. With this procedure, calcium fluorescence and luminescence whole-cell functional assays may be performed with standard laboratory pipetting and detection systems.     

The TRP family of channels: a complex gallery of characters

Calcium influxes are of fundamental importance in eukaryotic cell functions. These calcium influxes are carried by different classes of membrane proteins that allow regulated calcium entry. If in excitable cells, such as neurones or muscle, voltage-dependent calcium channels represent the main source of calcium influx, other proteins are needed to assume such a function in non-excitable cells. In these, a sustained calcium influx is observed, secondary to phospholipase C activation, IP3 synthesis and internal calcium release. The identity of proteins implicated in this second messenger calcium-driven influx, as well as the mechanisms of activation of these channels have long been debated. In recent years, genes encoding a new kind of cationic channels called TRP channels have been identified. This molecular work has set the basis for further functional studies and helped to gain crucial information on the mechanisms by which extracellular calcium can penetrate into non-excitable cells. This review will present the most recent advances obtained on the molecular diversity of TRP channels and their mode of gating.     

Integrin-mediated signal transduction pathways.

Integrins serve as adhesion receptors for extracellular matrix proteins and also transduce biochemical signals into the cell. They regulate a variety of cellular functions, including spreading, migration, proliferation and apoptosis. Many signaling pathways downstream of integrins have been identified and characterized and are discussed here. In particular, integrins regulate many protein tyrosine kinases and phosphatases, such as FAK and Src, to coordinate many of the cell processes mentioned above. The regulation of MAP kinases by integrins is important for cell growth or other functions, and the putative roles of Ras and FAK in these pathways are discussed. Phosphatidylinositol lipids and their modifying enzymes, particularly PI 3-kinase, are strongly implicated as mediators of integrin-regulated cytoskeletal changes and cell migration. Similarly, actin cytoskeleton regulation by the Rho family of GTPases is coordinated with integrin signaling to regulate cell spreading and migration, although the exact relationship between these pathways is not clear. Finally, intracellular pH and calcium fluxes by integrins are suggested to affect a variety of cellular proteins and functions.     

Advances of calcium signals involved in plant anti-drought

Considerable progresses have taken place, both in the methodology available to study changes in intracellular cytosolic calcium and in our understanding of calcium signaling cascades, but how calcium signals function in plant drought resistance is questionable. In plant cells, calcium plays roles as a second messenger coupling a wide range of extracellular stimuli with intracellular responses. Different extracellular stimuli trigger specific calcium signatures: dynamics, amplitude and duration of calcium transients specify the nature, implication and intensity of stimuli. Calcium-binding proteins (sensors) play a critical role in decoding calcium signatures and transducing signals by activating specific targets and corresponding metabolic pathways. Calmodulin is a calcium sensor known to regulate the activity of many mammalian proteins, whose targets in plants are now being identified. Higher plants possess a rapidly growing list of calmodulin targets with a variety of cellular functions. Nevertheless, many targets appear to be unique to higher plants and remain characterized, calling for a concerted effort to elucidate their functions. To date, three major classes of plant calcium signals, including calcium permeable ion channels, Ca(2+)/H(+) antiporters and Ca(2+)-ATPases, have been responsible for drought-stress signal transduction. This review summarizes the current knowledge of calcium signals involved in plant anti-drought and plant water use efficiency (WUE) and presents suggestions for future focus of study.     

Dynamic action of neurometals at the synapse

There are synaptic vesicles that are labeled by Timm's sulfide-silver staining method in the brain, suggesting that synaptic vesicles contain metals such as zinc and copper. Zinc is co-released with glutamate and the importance of zinc signaling in the intracellular compartment, in addition to extracellular compartment, is becoming recognized. Zinc can pass through calcium channels, while blocking them. Calcium signaling plays a critical role for synaptic activity and crosstalk between zinc signaling with calcium signaling through calcium channels may participate in synaptic neurotransmission including synaptic plasticity such as long-term potentiation. Copper released into the synaptic cleft during synaptic excitation may also participate in synaptic neurotransmission. Other metals including copper potentially serve as calcium channel blockers and also influence calcium signaling and zinc signaling via the interaction with metal-binding proteins such as metallothioneins. Homeostasis of metals needs to be controlled spatiotemporally for proper brain function, and their dyshomeostasis is associated with neurological diseases. However, the data on the dynamic action of metals at synapses is limited and their significance poorly understood. This paper summarizes the action of metals in synaptic neurotransmission focused on calcium signaling at glutamatergic synapses.