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The central nervous system (CNS) is constantly surveilled by microglia, highly motile and dynamic cells deputed to act as the first line of immune defense in the brain and spinal cord. Alterations in the homeostasis of the CNS are detected by microglia that respond by extending their processes or - following major injuries - by migrating toward the affected area. Understanding the mechanisms controlling directed cell migration of microglia is crucial to dissect their responses to neuroinflammation and injury. We used a combination of pharmacological and genetic approaches to explore the involvement of calcium (Ca 2+ ) signaling in the directed migration of human induced pluripotent stem cell (iPSC)-derived microglia challenged with a purinergic stimulus. This approach mimics cues originating from injury of the CNS. Unexpectedly, simultaneous imaging of microglia migration and intracellular Ca 2+ changes revealed that this phenomenon does not require Ca 2+ signals generated from the endoplasmic reticulum (ER) and store-operated Ca 2+ entry (SOCE) pathways. Instead, we find evidence that human microglial chemotaxis to purinergic signals is mediated by cyclic AMP in a Ca 2+ -independent manner. These results challenge prevailing notions, with important implications in neurological conditions characterized by perturbation in Ca 2+ homeostasis., Competing Interests: Declaration of competing interest M.B.-J. is a co-founder of NovoGlia Inc. I.F.S. is a consultant for, and holds stock options in NeuroQure, Inc. All other authors declare no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

KRas-induced actin-interacting protein (KRAP) has been identified as crucial for the appropriate localization and functioning of the inositol trisphosphate receptors (IP 3 Rs) that mediate Ca 2+ release from the endoplasmic reticulum. Here, we used siRNA knockdown of KRAP expression in HeLa and HEK293 cells to examine the roles of KRAP in the generation of IP 3 -mediated local Ca 2+ puffs and global, cell-wide Ca 2+ signals. High resolution Ca 2+ imaging revealed that the mean amplitude of puffs was strongly reduced by KRAP knockdown, whereas the Ca 2+ flux during openings of individual IP 3 R channels was little affected. In both control and KRAP knockdown cells the numbers of functional channels in the clusters underlying puff sites were stochastically distributed following a Poisson relationship, but the mean number of functional channels per site was reduced by about two thirds by KRAP knockdown. We conclude that KRAP is required for activity of IP 3 R channels at puff sites and stochastically 'licenses' the function of individual channels on a one-to-one basis, rather than determining the functioning of the puff site as a whole. In addition to puff activity ('punctate' Ca 2+ release), global, cell-wide Ca 2+ signals evoked by higher levels of IP 3 are further composed from a discrete 'diffuse' mode of Ca 2+ release. By applying fluctuation analysis to isolate the punctate component during global Ca 2+ signals, we find that KRAP knockdown suppresses to similar extents punctate and diffuse Ca 2+ release in wild-type cells and in HEK293 cells exclusively expressing type 1 and type 3 IP3Rs. Thus, KRAP appears essential for the functioning of the IP 3 Rs involved in diffuse Ca 2+ release as well as the clustered IP 3 Rs that generate local Ca 2+ puffs., Competing Interests: Declaration of Competing Interest None of the authors have any conflict of interest to declare., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Modulating cytoplasmic Ca 2+ concentration ([Ca 2+ ] i ) by endoplasmic reticulum (ER)-localized inositol 1,4,5-trisphosphate receptor (InsP 3 R) Ca 2+ -release channels is a universal signaling pathway that regulates numerous cell-physiological processes. Whereas much is known regarding regulation of InsP 3 R activity by cytoplasmic ligands and processes, its regulation by ER-luminal Ca 2+ concentration ([Ca 2+ ] ER ) is poorly understood and controversial. We discovered that the InsP 3 R is regulated by a peripheral membrane-associated ER-luminal protein that strongly inhibits the channel in the presence of high, physiological [Ca 2+ ] ER . The widely-expressed Ca 2+ -binding protein annexin A1 (ANXA1) is present in the nuclear envelope lumen and, through interaction with a luminal region of the channel, can modify high-[Ca 2+ ] ER inhibition of InsP 3 R activity. Genetic knockdown of ANXA1 expression enhanced global and local elementary InsP 3 -mediated Ca 2+ signaling events. Thus, [Ca 2+ ] ER is a major regulator of InsP 3 R channel activity and InsP 3 R-mediated [Ca 2+ ] i signaling in cells by controlling an interaction of the channel with a peripheral membrane-associated Ca 2+ -binding protein, likely ANXA1., Competing Interests: HV, MW, KM, RP, CM, JL, LS, CF, MC, IP, SS, JF, DM No competing interests declared, (© 2020, Vais et al.)

The 'building-block' model of inositol trisphosphate (IP 3 )-mediated Ca 2+ liberation posits that cell-wide cytosolic Ca 2+ signals arise through coordinated activation of localized Ca 2+ puffs generated by stationary clusters of IP 3 receptors (IP 3 Rs). Here, we revise this hypothesis, applying fluctuation analysis to resolve Ca 2+ signals otherwise obscured during large Ca 2+ elevations. We find the rising phase of global Ca 2+ signals is punctuated by a flurry of puffs, which terminate before the peak by a mechanism involving partial ER Ca 2+ depletion. The continuing rise in Ca 2+ , and persistence of global signals even when puffs are absent, reveal a second mode of spatiotemporally diffuse Ca 2+ signaling. Puffs make only small, transient contributions to global Ca 2+ signals, which are sustained by diffuse release of Ca 2+ through a functionally distinct process. These two modes of IP 3 -mediated Ca 2+ liberation have important implications for downstream signaling, imparting spatial and kinetic specificity to Ca 2+ -dependent effector functions and Ca 2+ transport., Competing Interests: JL, IP No competing interests declared, (© 2020, Lock and Parker.)

Cellular Ca 2+ signals are often constrained to cytosolic micro- or nano-domains where stochastic openings of Ca 2+ channels cause large fluctuations in local Ca 2+ concentration (Ca 2+ 'noise'). With the advent of TIRF microscopy to image the fluorescence of Ca 2+ -sensitive probes from attoliter volumes it has become possible to directly monitor these signals, which closely track the gating of plasmalemmal and ER Ca 2+ -permeable channels. Nevertheless, it is likely that many physiologically important Ca 2+ signals are too small to resolve as discrete events in fluorescence recordings. By analogy with noise analysis of electrophysiological data, we explore here the use of statistical approaches to detect and analyze such Ca 2+ noise in images obtained using Ca 2+ -sensitive indicator dyes. We describe two techniques - power spectrum analysis and spatio-temporal correlation - and demonstrate that both effectively identify discrete, spatially localized calcium release events (Ca 2+ puffs). Moreover, we show they are able to detect localized noise fluctuations in a case where discrete events cannot directly be resolved., Competing Interests: Declaration of Competing Interest The authors declare they have no competing financial interests., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The patterning of cytosolic Ca 2+ signals in space and time underlies their ubiquitous ability to specifically regulate numerous cellular processes. Signals mediated by liberation of Ca 2+ sequestered in the endoplasmic reticulum (ER) through inositol trisphosphate receptor (IP 3 R) channels constitute a hierarchy of events; ranging from openings of individual IP 3 channels, through the concerted openings of several clustered IP 3 Rs to generate local Ca 2+ puffs, to global Ca 2+ waves and oscillations that engulf the entire cell. Here, we review recent progress in elucidating how this hierarchy is shaped by an interplay between the functional gating properties of IP 3 Rs and their spatial distribution within the cell. We focus in particular on the subset of IP 3 Rs that are organized in stationary clusters and are endowed with the ability to preferentially liberate Ca 2+ ., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Piezo channels transduce mechanical stimuli into electrical and chemical signals to powerfully influence development, tissue homeostasis, and regeneration. Studies on Piezo1 have largely focused on transduction of "outside-in" mechanical forces, and its response to internal, cell-generated forces remains poorly understood. Here, using measurements of endogenous Piezo1 activity and traction forces in native cellular conditions, we show that cellular traction forces generate spatially-restricted Piezo1-mediated Ca 2+ flickers in the absence of externally-applied mechanical forces. Although Piezo1 channels diffuse readily in the plasma membrane and are widely distributed across the cell, their flicker activity is enriched near force-producing adhesions. The mechanical force that activates Piezo1 arises from Myosin II phosphorylation by Myosin Light Chain Kinase. We propose that Piezo1 Ca 2+ flickers allow spatial segregation of mechanotransduction events, and that mobility allows Piezo1 channels to explore a large number of mechanical microdomains and thus respond to a greater diversity of mechanical cues., Competing Interests: Competing interestsThe authors declare no competing interests.

Inositol 1,4,5-trisphosphate (IP 3 ) evokes Ca 2+ release through IP 3 receptors (IP 3 Rs) to generate both local Ca 2+ puffs arising from concerted openings of clustered IP 3 Rs and cell-wide Ca 2+ waves. Imaging Ca 2+ puffs with single-channel resolution yields information on the localization and properties of native IP 3 Rs in intact cells, but interpretation has been complicated because cells express varying proportions of three structurally and functionally distinct isoforms of IP 3 Rs. Here, we used TIRF and light-sheet microscopy to image Ca 2+ puffs in HEK-293 cell lines generated by CRISPR-Cas9 technology to express exclusively IP 3 R type 1, 2, or 3. Photorelease of the IP 3 analog i-IP 3 in all three cell lines evoked puffs with largely similar mean amplitudes, temporal characteristics, and spatial extents. Moreover, the single-channel Ca 2+ flux was similar among isoforms, indicating that clusters of different IP 3 R isoforms contain comparable numbers of active channels. Our results show that all three IP 3 R isoforms cluster to generate local Ca 2+ puffs and, contrary to findings of divergent properties from in vitro electrophysiological studies, display similar conductances and gating kinetics in intact cells., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

We describe the construction of a simplified, inexpensive lattice light-sheet microscope, and illustrate its use for imaging subcellular Ca 2+ puffs evoked by photoreleased i-IP 3 in cultured SH-SY5Y neuroblastoma cells loaded with the Ca 2+ probe Cal520. The microscope provides sub-micron spatial resolution and enables recording of local Ca 2+ transients in single-slice mode with a signal-to-noise ratio and temporal resolution (2ms) at least as good as confocal or total internal reflection microscopy. Signals arising from openings of individual IP 3 R channels are clearly resolved, as are stepwise changes in fluorescence reflecting openings and closings of individual channels during puffs. Moreover, by stepping the specimen through the light-sheet, the entire volume of a cell can be scanned within a few hundred ms. The ability to directly visualize a sideways (axial) section through cells directly reveals that IP 3 -evoked Ca 2+ puffs originate at sites in very close (≤a few hundred nm) to the plasma membrane, suggesting they play a specific role in signaling to the membrane., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Calcium is an essential cellular messenger that regulates numerous functions in living organisms. Here, we describe development and characterization of 'Salsa6f', a fusion of GCaMP6f and tdTomato optimized for cell tracking while monitoring cytosolic Ca 2+ , and a transgenic Ca 2+ reporter mouse with Salsa6f targeted to the Rosa26 locus for Cre-dependent expression in specific cell types. The development and function of T cells was unaffected in Cd4-Salsa6f mice. We describe Ca 2+ signals reported by Salsa6f during T cell receptor activation in naive T cells, helper Th17 T cells and regulatory T cells, and Ca 2+ signals mediated in T cells by an activator of mechanosensitive Piezo1 channels. Transgenic expression of Salsa6f enables ratiometric imaging of Ca 2+ signals in complex tissue environments found in vivo. Two-photon imaging of migrating T cells in the steady-state lymph node revealed both cell-wide and localized sub-cellular Ca 2+ transients ('sparkles') as cells migrate.

Ca 2+ influx through Orai1 channels is crucial for several T cell functions, but a role in regulating basal cellular motility has not been described. Here, we show that inhibition of Orai1 channel activity increases average cell velocities by reducing the frequency of pauses in human T cells migrating through confined spaces, even in the absence of extrinsic cell contacts or antigen recognition. Utilizing a novel ratiometric genetically encoded cytosolic Ca 2+ indicator, Salsa6f, which permits real-time monitoring of cytosolic Ca 2+ along with cell motility, we show that spontaneous pauses during T cell motility in vitro and in vivo coincide with episodes of cytosolic Ca 2+ signaling. Furthermore, lymph node T cells exhibited two types of spontaneous Ca 2+ transients: short-duration 'sparkles' and longer duration global signals. Our results demonstrate that spontaneous and self-peptide MHC-dependent activation of Orai1 ensures random walk behavior in T cells to optimize immune surveillance.

The inositol trisphosphate (IP 3 ) signaling pathway evokes local Ca 2+ signals (Ca 2+ puffs) that arise from the concerted openings of clustered IP 3 receptor/channels in the ER membrane. Physiological activation is triggered by binding of agonists to G-protein-coupled receptors (GPCRs) on the cell surface, leading to cleavage of phosphatidyl inositol bisphosphate and release of IP 3 into the cytosol. Photorelease of IP 3 from a caged precursor provides a convenient and widely employed means to study the final stage of IP 3 -mediated Ca 2+ liberation, bypassing upstream signaling events to enable more precise control of the timing and relative concentration of cytosolic IP 3 . Here, we address whether Ca 2+ puffs evoked by photoreleased IP 3 fully replicate those arising from physiological agonist stimulation. We imaged puffs in individual SH-SY5Y neuroblastoma cells that were sequentially stimulated by picospritzing extracellular agonist (carbachol, CCH or bradykinin, BK) followed by photorelease of a poorly-metabolized IP 3 analog, i-IP 3 . The centroid localizations of fluorescence signals during puffs evoked in the same cells by agonists and photorelease substantially overlapped (within ∼1μm), suggesting that IP 3 from both sources accesses the same, or closely co-localized clusters of IP 3 Rs. Moreover, the time course and spatial spread of puffs evoked by agonists and photorelease matched closely. Because photolysis generates IP 3 uniformly throughout the cytoplasm, our results imply that IP 3 generated in SH-SY5Y cells by activation of receptors to CCH and BK also exerts broadly distributed actions, rather than specifically activating a subpopulation of IP 3 Rs that are scaffolded in close proximity to cell surface receptors to form a signaling nanodomain., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The range of action of intracellular messengers is determined by their rates of diffusion and degradation. Previous measurements in oocyte cytoplasmic extracts indicated that the Ca 2+ -liberating second messenger inositol trisphosphate (IP 3 ) diffuses with a coefficient (~280 μm 2 s -1 ) similar to that in water, corresponding to a range of action of ~25 μm. Consequently, IP 3 is generally considered a "global" cellular messenger. We reexamined this issue by measuring local IP 3 -evoked Ca 2+ puffs to monitor IP 3 diffusing from spot photorelease in neuroblastoma cells. Fitting these data by numerical simulations yielded a diffusion coefficient (≤10 μm 2 s -1 ) about 30-fold slower than that previously reported. We propose that diffusion of IP 3 in mammalian cells is hindered by binding to immobile, functionally inactive receptors that were diluted in oocyte extracts. The predicted range of action of IP 3 (<5 μm) is thus smaller than the size of typical mammalian cells, indicating that IP 3 should better be considered as a local rather than a global cellular messenger., (Copyright © 2016, American Association for the Advancement of Science.)

Tunneling membrane nanotubes (TNTs) are thin membrane projections linking cell bodies separated by many micrometers, which are proposed to mediate signaling and even transfer of cytosolic contents between distant cells. Several reports describe propagation of Ca(2+) signals between distant cells via TNTs, but the underlying mechanisms remain poorly understood. Utilizing a HeLa M-Sec cell line engineered to upregulate TNTs we replicated previous findings that mechanical stimulation elicits robust cytosolic Ca(2+) elevations that propagate to surrounding, physically separate cells. However, whereas this was previously interpreted to involve intercellular communication through TNTs, we found that Ca(2+) signal propagation was abolished - even in TNT-connected cells - after blocking ATP-mediated paracrine signaling with a cocktail of extracellular inhibitors. To then establish whether gap junctions may enable cell-cell signaling via TNTs under these conditions, we expressed sfGFP-tagged connexin-43 (Cx43) in HeLa M-Sec cells. We observed robust communication of mechanically-evoked Ca(2+) signals between distant but TNT-connected cells, but only when both cells expressed Cx43. Moreover, we also observed communication of Ca(2+) signals evoked in one cell by local photorelease of inositol 1,4,5-trisphosphate (IP3). Ca(2+) responses in connected cells began after long latencies at intracellular sites several microns from the TNT connection site, implicating intercellular transfer of IP3 and subsequent IP3-mediated Ca(2+) liberation, and not Ca(2+) itself, as the mediator between TNT-connected, Cx43-expressing cells. Our results emphasize the need to control for paracrine transmission in studies of cell-cell signaling via TNTs and indicate that, in this cell line, TNTs do not establish cytosolic continuity between connected cells but rather point to the crucial importance of connexins to enable communication of cytosolic Ca(2+) signals via TNTs., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Localized subcellular changes in Ca(2+) serve as important cellular signaling elements, regulating processes as diverse as neuronal excitability and gene expression. Studies of cellular Ca(2+) signaling have been greatly facilitated by the availability of fluorescent Ca(2+) indicators. The respective merits of different indicators to monitor bulk changes in cellular Ca(2+) levels have been widely evaluated, but a comprehensive comparison for their use in detecting and analyzing local, subcellular Ca(2+) signals is lacking. Here, we evaluated several fluorescent Ca(2+) indicators in the context of local Ca(2+) signals (puffs) evoked by inositol 1,4,5-trisphosphate (IP3) in cultured human neuroblastoma SH-SY5Y cells, using high-speed video-microscopy. Altogether, nine synthetic Ca(2+) dyes (Fluo-4, Fluo-8, Fluo-8 high affinity, Fluo-8 low affinity, Oregon Green BAPTA-1, Cal-520, Rhod-4, Asante Calcium Red, and X-Rhod-1) and three genetically-encoded Ca(2+)-indicators (GCaMP6-slow, -medium and -fast variants) were tested; criteria include the magnitude, kinetics, signal-to-noise ratio and detection efficiency of local Ca(2+) puffs. Among these, we conclude that Cal-520 is the optimal indicator for detecting and faithfully tracking local events; that Rhod-4 is the red-emitting indicator of choice; and that none of the GCaMP6 variants are well suited for imaging subcellular Ca(2+) signals., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Ca(2+) liberation from the endoplasmic reticulum mediated by inositol trisphosphate receptor/channels (IP3Rs) in response to production of the second messenger IP3 regulates numerous signaling pathways. However, estimates of resting and physiologically relevant cytosolic concentrations of IP3 vary appreciably. Here we directly address this question, taking advantage of the large size of Xenopus oocytes to image Ca(2+) liberation evoked by bolus intracellular injections of known concentrations of IP3. Our principal finding is that IP3 evokes both global and local Ca(2+) signals in freshly isolated oocytes at concentrations as low as a few pM. A corollary is that basal, resting [IP3] must be even lower, given the absence of detectable Ca(2+) signals before injection. The dose/response curve for IP3-activation of Ca(2+) liberation suggests that freshly isolated oocytes express two distinct functional populations of IP3 receptors with EC50 values around 200 pM and tens of nM, whereas the high-affinity receptors are not apparent in oocytes examined later than about 3 days after isolation from the ovary., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Amyloid beta (Aβ) oligomers associated with Alzheimer's disease (AD) form Ca2+-permeable plasma membrane pores, leading to a disruption of the otherwise well-controlled intracellular calcium (Ca2+) homeostasis. The resultant up-regulation of intracellular Ca2+ concentration has detrimental implications for memory formation and cell survival. The gating kinetics and Ca2+ permeability of Aβ pores are not well understood. We have used computational modeling in conjunction with the ability of optical patch-clamping for massively parallel imaging of Ca2+ flux through thousands of pores in the cell membrane of Xenopus oocytes to elucidate the kinetic properties of Aβ pores. The fluorescence time-series data from individual pores were idealized and used to develop data-driven Markov chain models for the kinetics of the Aβ pore at different stages of its evolution. Our study provides the first demonstration of developing Markov chain models for ion channel gating that are driven by optical-patch clamp data with the advantage of experiments being performed under close to physiological conditions. Towards the end, we demonstrate the up-regulation of gating of various Ca2+ release channels due to Aβ pores and show that the extent and spatial range of such up-regulation increases as Aβ pores with low open probability and Ca2+ permeability transition into those with high open probability and Ca2+ permeability.

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of >500 frames sec(-1) (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

The oscillating concentration of intracellular calcium is one of the most important examples for collective dynamics in cell biology. Localized releases of calcium through clusters of inositol 1,4,5-trisphosphate receptor channels constitute elementary signals called calcium puffs. Coupling by diffusing calcium leads to global releases and waves, but the exact mechanism of inter-cluster coupling and triggering of waves is unknown. To elucidate the relation of puffs and waves, we here model a cluster of IP3R channels using a gating scheme with variable non-equilibrium IP3 binding. Hybrid stochastic and deterministic simulations show that puffs are not stereotyped events of constant duration but are sensitive to stimulation strength and residual calcium. For increasing IP3 concentration, the release events become modulated at a timescale of minutes, with repetitive wave-like releases interspersed with several puffs. This modulation is consistent with experimental observations we present, including refractoriness and increase of puff frequency during the inter-wave interval. Our results suggest that waves are established by a random but time-modulated appearance of sustained release events, which have a high potential to trigger and synchronize activity throughout the cell.

Local Ca(2+) transients such as puffs and sparks form the building blocks of cellular Ca(2+) signaling in numerous cell types. They have traditionally been studied by linescan confocal microscopy, but advances in TIRF microscopy together with improved electron-multiplied CCD (EMCCD) cameras now enable rapid (>500 frames s(-1)) imaging of subcellular Ca(2+) signals with high spatial resolution in two dimensions. This approach yields vastly more information (ca. 1 Gb min(-1)) than linescan imaging, rendering visual identification and analysis of local events imaged both laborious and subject to user bias. Here we describe a routine to rapidly automate identification and analysis of local Ca(2+) events. This features an intuitive graphical user-interfaces and runs under Matlab and the open-source Python software. The underlying algorithm features spatial and temporal noise filtering to reliably detect even small events in the presence of noisy and fluctuating baselines; localizes sites of Ca(2+) release with sub-pixel resolution; facilitates user review and editing of data; and outputs time-sequences of fluorescence ratio signals for identified event sites along with Excel-compatible tables listing amplitudes and kinetics of events., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Calcium puffs are localized Ca(2+) signals mediated by Ca(2+) release from the endoplasmic reticulum (ER) through clusters of inositol trisphosphate receptor (IP3R) channels. The recruitment of IP3R channels during puffs depends on Ca(2+)-induced Ca(2+) release, a regenerative process that must be terminated to maintain control of cell signaling and prevent Ca(2+) cytotoxicity. Here, we studied puff termination using total internal reflection microscopy to resolve the gating of individual IP3R channels during puffs in intact SH-SY5Y neuroblastoma cells. We find that the kinetics of IP3R channel closing differ from that expected for independent, stochastic gating, in that multiple channels tend to remain open together longer than predicted from their individual open lifetimes and then close in near-synchrony. This behavior cannot readily be explained by previously proposed termination mechanisms, including Ca(2+)-inhibition of IP3Rs and local depletion of Ca(2+) in the ER lumen. Instead, we postulate that the gating of closely adjacent IP3Rs is coupled, possibly via allosteric interactions, suggesting an important mechanism to ensure robust puff termination in addition to Ca(2+)-inactivation., (Published by Elsevier Ltd.)

Oligomeric forms of β-amyloid (Aβ(42)) peptides associated with Alzheimer's disease (AD) disrupt cellular Ca(2+) regulation by liberating Ca(2+) into the cytosol from both extracellular and intracellular sources. We elucidated the actions of intracellular Aβ by imaging Ca(2+) responses to injections of Aβ oligomers into Xenopus oocytes. Two types of signal were observed: (1) local, "channel-like" transients dependent on extracellular Ca(2+) influx, which resembled signals from amlyoid pores formed by extracellular application of oligomers; and (2) local transients and global Ca(2+) waves, resembling Ca(2+) puffs and waves mediated by inositol trisphosphate (IP(3)). The latter responses were suppressed by antagonists of the IP(3) receptor (caffeine and heparin), pretreatment with the G(i)(o)-protein inhibitor pertussis toxin, and pretreatment with lithium to deplete membrane inositol lipids. We show that G-protein-mediated stimulation of IP(3) production and consequent liberation of Ca(2+) from the endoplasmic reticulum by intracellular Aβ oligomers is cytotoxic, potentially representing a novel pathological mechanism in AD which may be further exacerbated by AD-linked mutations in presenilins to promote opening of IP(3) receptor/channels.

InsP3-mediated puffs are fundamental building blocks of cellular Ca2+ signalling, and arise through the concerted opening of clustered InsP3Rs (InsP3 receptors) co-ordinated via Ca2+-induced Ca2+ release. Although the Ca2+ dependency of InsP3Rs has been extensively studied at the single channel level, little is known as to how changes in basal cytosolic [Ca2+] would alter the dynamics of InsP3-evoked Ca2+ signals in intact cells. To explore this question, we expressed Ca2+-permeable channels (nicotinic acetylcholine receptors) in the plasma membrane of voltage-clamped Xenopus oocytes to regulate cytosolic [Ca2+] by changing the electrochemical gradient for extracellular Ca2+ entry, and imaged Ca2+ liberation evoked by photolysis of caged InsP3. Elevation of basal cytosolic [Ca2+] strongly increased the amplitude and shortened the latency of global Ca2+ waves. In oocytes loaded with EGTA to localize Ca2+ signals, the number of sites at which puffs were observed and the frequency and latency of puffs were strongly dependent on cytosolic [Ca2+], whereas puff amplitudes were only weakly affected. The results of the present study indicate that basal cytosolic [Ca2+] strongly affects the triggering of puffs, but has less of an effect on puffs once they have been initiated.

The behavior of biological systems is determined by the properties of their component molecules, but the interactions are usually too complex to understand fully how molecular behavior generates cellular behavior. Ca(2+) signaling by inositol trisphosphate receptors (IP(3)R) offers an opportunity to understand this relationship because the cellular behavior is defined largely by Ca(2+)-mediated interactions between IP(3)R. Ca(2+) released by a cluster of IP(3)R (giving a local Ca(2+) puff) diffuses and ignites the behavior of neighboring clusters (to give repetitive global Ca(2+) spikes). We use total internal reflection fluorescence microscopy of two mammalian cell lines to define the temporal relationships between Ca(2+) puffs (interpuff intervals, IPI) and Ca(2+) spikes (interspike intervals) evoked by flash photolysis of caged IP(3). We find that IPI are much shorter than interspike intervals, that puff activity is stochastic with a recovery time that is much shorter than the refractory period of the cell, and that IPI are not periodic. We conclude that Ca(2+) spikes do not arise from oscillatory dynamics of IP(3)R clusters, but that repetitive Ca(2+) spiking with its longer timescales is an emergent property of the dynamics of the whole cluster array., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Liberation of Ca(2+) from the endoplasmic reticulum (ER) through inositol trisphosphate receptors (IP(3)R) is modulated by the ER Ca(2+) content, and overexpression of SERCA2b to accelerate Ca(2+) sequestration into the ER has been shown to potentiate the frequency and amplitude of IP(3)-evoked Ca(2+) waves in Xenopus oocytes. Here, we examined the effects of SERCA overexpression on the elementary IP(3)-evoked puffs to elucidate whether ER [Ca(2+)] may modulate IP(3)R function via luminal regulatory sites in addition to simply determining the size of the available store and electrochemical driving force for Ca(2+) release. SERCA2b and Ca(2+) permeable nicotinic plasmalemmal channels were expressed in oocytes, and hyperpolarizing pulses were delivered to induce Ca(2+) influx and thereby load ER stores. Puffs evoked by photoreleased IP(3) were significantly potentiated in terms of numbers of responding sites, frequency and amplitude following transient Ca(2+) influx in SERCA-overexpressing cells, whereas little change was evident with SERCA overexpression alone or following Ca(2+) influx in control cells not overexpressing SERCA. Intriguingly, we observed the appearance of a new population of puffs that arose after long latencies and had prolonged durations supporting the notion of luminal regulation of IP(3)R gating kinetics., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In addition to its well established function in activating Ca(2+) release from the endoplasmic reticulum (ER) through ryanodine receptors (RyR), the second messenger cyclic ADP-ribose (cADPR) also accelerates the activity of SERCA pumps, which sequester Ca(2+) into the ER. Here, we demonstrate a potential physiological role for cADPR in modulating cellular Ca(2+) signals via changes in ER Ca(2+) store content, by imaging Ca(2+) liberation through inositol trisphosphate receptors (IP(3)R) in Xenopus oocytes, which lack RyR. Oocytes were injected with the non-metabolizable analog 3-deaza-cADPR, and cytosolic [Ca(2+)] was transiently elevated by applying voltage-clamp pulses to induce Ca(2+) influx through expressed plasmalemmal nicotinic channels. We observed a subsequent potentiation of global Ca(2+) signals evoked by strong photorelease of IP(3), and increased numbers of local Ca(2+) puffs evoked by weaker photorelease. These effects were not evident with cADPR alone or following cytosolic Ca(2+) elevation alone, indicating that they did not arise through direct actions of cADPR or Ca(2+) on the IP(3)R, but likely resulted from enhanced ER store filling. Moreover, the appearance of a new population of puffs with longer latencies, prolonged durations, and attenuated amplitudes suggests that luminal ER Ca(2+) may modulate IP(3)R function, in addition to simply determining the size of the available store and the electrochemical driving force for release.

Postsynaptic release of Ca(2+) from intracellular stores is an important means of cellular signaling that mediates numerous forms of synaptic plasticity. Previous studies have identified a postsynaptic intracellular Ca(2+) requirement for a form of short-term plasticity, post-tetanic potentiation (PTP) at sensory neuron (SN)-motor neuron synapses in Aplysia. Here, we show that postsynaptic IP(3)-mediated Ca(2+) release in response to a presynaptic tetanus in an SN that induces PTP can confer transient plasticity onto a neighboring SN synapse receiving subthreshold activation. This heterosynaptic sharing of plasticity represents a dynamic, short-term synaptic enhancement of synaptic inputs onto a common postsynaptic target. Heterosynaptic sharing is blocked by postsynaptic disruption of Ca(2+)- and IP(3)-mediated signaling, and, conversely, it is mimicked by postsynaptic injection of nonhydrolyzable IP(3), and by photolysis of caged IP(3) in the MN. The molecular mechanism for heterosynaptic sharing involves metabotropic glutamate receptors and Homer-dependent interactions, indicating that Homer can facilitate the integration of Ca(2+)-dependent plasticity at neighboring postsynaptic sites and provides a postsynaptic mechanism for the spread of plasticity induced by presynaptic activation. Our results support a model in which postsynaptic summation of IP(3) signals from suprathreshold and subthreshold inputs results in molecular coincidence detection that gives rise to a novel form of heterosynaptic plasticity.

Intracellular calcium ion (Ca(2+)) signaling crucially depends on the clustered organization of inositol trisphosphate receptors (IP(3)Rs) in the endoplasmic reticulum (ER) membrane. These ligand-gated ion channels liberate Ca(2+) to generate local signals known as Ca(2+) puffs. We tested the hypothesis that IP(3) itself elicits rapid clustering of IP(3)Rs by using flash photolysis of caged IP(3) in conjunction with high-resolution Ca(2+) imaging to monitor the activity and localization of individual IP(3)Rs within intact mammalian cells. Our results indicate that Ca(2+) puffs arising with latencies as short as 100 to 200 ms after photorelease of IP(3) already involve at least four IP(3)R channels, and that this number does not subsequently grow. Moreover, single active IP(3)Rs show limited mobility, and stochastic simulations suggest that aggregation of IP(3)Rs at puff sites by a diffusional trapping mechanism would require many seconds. We thus conclude that puff sites represent preestablished, stable clusters of IP(3)Rs and that functional IP(3)Rs are not readily diffusible within the ER membrane.

Intracellular Ca(2+) release is a versatile second messenger system. It is modeled here by reaction-diffusion equations for the free Ca(2+) and Ca(2+) buffers, with spatially discrete clusters of stochastic IP(3) receptor channels (IP(3)Rs) controlling the release of Ca(2+) from the endoplasmic reticulum. IP(3)Rs are activated by a small rise of the cytosolic Ca(2+) concentration and inhibited by large concentrations. Buffering of cytosolic Ca(2+) shapes global Ca(2+) transients. Here we use a model to investigate the effect of buffers with slow and fast reaction rates on single release spikes. We find that, depending on their diffusion coefficient, fast buffers can either decouple clusters or delay inhibition. Slow buffers have little effect on Ca(2+) release, but affect the time course of the signals from the fluorescent Ca(2+) indicator mainly by competing for Ca(2+). At low [IP(3)], fast buffers suppress fluorescence signals, slow buffers increase the contrast between bulk signals and signals at open clusters, and large concentrations of buffers, either fast or slow, decouple clusters.

The spatiotemporal patterning of Ca(2+) signals regulates numerous cellular functions, and is determined by the functional properties and spatial clustering of inositol trisphosphate receptor (IP(3)R) Ca(2+) release channels in the endoplasmic reticulum membrane. However, studies at the single-channel level have been hampered because IP(3)Rs are inaccessible to patch-clamp recording in intact cells, and because excised organelle and bilayer reconstitution systems disrupt the Ca(2+)-induced Ca(2+) release (CICR) process that mediates channel-channel coordination. We introduce here the use of total internal reflection fluorescence microscopy to image single-channel Ca(2+) flux through individual and clustered IP(3)Rs in intact mammalian cells. This enables a quantal dissection of the local calcium puffs that constitute building blocks of cellular Ca(2+) signals, revealing stochastic recruitment of, on average, approximately 6 active IP(3)Rs clustered within <500 nm. Channel openings are rapidly ( approximately 10 ms) recruited by opening of an initial trigger channel, and a similarly rapid inhibitory process terminates puffs despite local [Ca(2+)] elevation that would otherwise sustain Ca(2+)-induced Ca(2+) release indefinitely. Minimally invasive, nano-scale Ca(2+) imaging provides a powerful tool for the functional study of intracellular Ca(2+) release channels while maintaining the native architecture and dynamic interactions essential for discrete and selective cell signaling.

The inositol 1,4,5-trisphosphate receptor/channel (IP(3)R) is a major regulator of intracellular Ca(2+) signaling, and liberates Ca(2+) ions from the endoplasmic reticulum in response to binding at cytosolic sites for both IP(3) and Ca(2+). Although the steady-state gating properties of the IP(3)R have been extensively studied and modeled under conditions of fixed [IP(3)] and [Ca(2+)], little is known about how Ca(2+) flux through a channel may modulate the gating of that same channel by feedback onto activating and inhibitory Ca(2+) binding sites. We thus simulated the dynamics of Ca(2+) self-feedback on monomeric and tetrameric IP(3)R models. A major conclusion is that self-activation depends crucially on stationary cytosolic Ca(2+) buffers that slow the collapse of the local [Ca(2+)] microdomain after closure. This promotes burst-like reopenings by the rebinding of Ca(2+) to the activating site; whereas inhibitory actions are substantially independent of stationary buffers but are strongly dependent on the location of the inhibitory Ca(2+) binding site on the IP(3)R in relation to the channel pore.

Elementary events such as puffs and sparks are cytosolic microdomains of Ca2+ from which cellular Ca2+ signals are constructed. Because of the tight localization and fast kinetics of elementary events, imaging studies have been hindered by instrumental limitations of confocal and deconvolution fluorescence microscopy which necessitate compromises between spatial and temporal resolution. Here, we describe a novel, yet simple 'multi-focal' fluorescence microscopy system that employs three high-speed cameras focused at different axial depths to enable 4-dimensional imaging with millisecond resolution. We demonstrate the utility of this system for studies of puffs in Xenopus oocytes by mapping the axial distribution of puff sites, by obtaining measurements of puff amplitudes undistorted by focus error, and by deriving deblurred images that reveal novel sub-micron jumps of Ca2+ release sites.

Ca2+ liberation through inositol 1,4,5-trisphosphate receptor (IP3R) channels generates complex patterns of spatiotemporal cellular Ca2+ signals owing to the biphasic modulation of channel gating by Ca2+ itself. These processes have been extensively studied in Xenopus oocytes, where imaging studies have revealed local Ca2+ signals ("puffs") arising from clusters of IP3R, and patch-clamp studies on isolated oocyte nuclei have yielded extensive data on IP3R gating kinetics. To bridge these two levels of experimental data, we developed an IP3R model and applied stochastic simulation and transition matrix theory to predict the behavior of individual and clustered IP3R channels. The channel model consists of four identical, independent subunits, each of which has an IP3-binding site together with one activating and one inactivating Ca2+-binding site. The channel opens when at least three subunits undergo a conformational change to an "active" state after binding IP3 and Ca2+. The model successfully reproduces patch-clamp data; including the dependence of open probability, mean open duration, and mean closed duration on [IP3] and [Ca2+]. Notably, the biexponential distribution of open-time duration and the dependence of mean open time on [Ca2+] are explained by populations of openings involving either three or four active subunits. As a first step toward applying the single IP3R model to describe cellular responses, we then simulated measurements of puff latency after step increases of [IP3]. Assuming that stochastic opening of a single IP3R at basal cytosolic [Ca2+] and any given [IP3] has a high probability of rapidly triggering neighboring channels by calcium-induced calcium release to evoke a puff, optimal correspondence with experimental data of puff latencies after photorelease of IP3 was obtained when the cluster contained a total of 40-70 IP3Rs.

Calcium puffs are local Ca(2+) release events that arise from a cluster of inositol 1,4,5-trisphosphate receptor channels (IP(3)Rs) and serve as a basic "building block" from which global Ca(2+) waves are generated. Important questions remain as to the number of IP(3)Rs that open during a puff, their spatial distribution within a cluster, and how much Ca(2+) current flows through each channel. The recent discovery of "trigger" events-small Ca(2+) signals that immediately precede puffs and are interpreted to arise through opening of single IP(3)R channels-now provides a useful yardstick by which to calibrate the Ca(2+) flux underlying puffs. Here, we describe a deterministic numerical model to simulate puffs and trigger events. Based on confocal linescan imaging in Xenopus oocytes, we simulated Ca(2+) release in two sequential stages; representing the trigger by the opening of a single IP(3)R in the center of a cluster for 12 ms, followed by the concerted opening of some number of IP(3)Rs for 19 ms, representing the rising phase of the puff. The diffusion of Ca(2+) and Ca(2+)-bound indicator dye were modeled in a three-dimensional cytosolic volume in the presence of immobile and mobile Ca(2+) buffers, and were used to predict the observed fluorescence signal after blurring by the microscope point-spread function. Optimal correspondence with experimental measurements of puff spatial width and puff/trigger amplitude ratio was obtained assuming that puffs arise from the synchronous opening of 25-35 IP(3)Rs, each carrying a Ca(2+) current of approximately 0.4 pA, with the channels distributed through a cluster 300-800 nm in diameter.

The liberation of calcium ions sequestered in the endoplasmic reticulum through inositol 1,4,5-trisphosphate receptors/channels (IP(3)Rs) results in a spatiotemporal hierarchy of calcium signaling events that range from single-channel openings to local Ca(2+) puffs believed to arise from several to tens of clustered IP(3)Rs to global calcium waves. Using high-resolution confocal linescan imaging and a sensitive Ca(2+) indicator dye (fluo-4-dextran), we show that puffs are often preceded by small, transient Ca(2+) elevations that we christen "trigger events". The magnitude of triggers is consistent with their arising from the opening of a single IP(3) receptor/channel, and we propose that they initiate puffs by recruiting neighboring IP(3)Rs within the cluster by a regenerative process of Ca(2+)-induced Ca(2+) release. Puff amplitudes (fluorescence ratio change) are on average approximately 6 times greater than that of the triggers, suggesting that at least six IP(3)Rs may simultaneously be open during a puff. Trigger events have average durations of approximately 12 ms, as compared to 19 ms for the mean rise time of puffs, and their spatial extent is approximately 3 times smaller than puffs (respective widths at half peak amplitude 0.6 and 1.6 micro m). All these parameters were relatively independent of IP(3) concentration, although the proportion of puffs showing resolved triggers was greatest (approximately 80%) at low [IP(3)]. Because Ca(2+) puffs constitute the building blocks from which cellular IP(3)-mediated Ca(2+) signals are constructed, the events that initiate them are likely to be of fundamental importance for cell signaling. Moreover, the trigger events provide a useful yardstick by which to derive information regarding the number and spatial arrangement of IP(3)Rs within clusters.

Puffs are localized Ca(2+) signals that arise in oocytes in response to inositol 1,4,5-trisphosphate (IP(3)). They are analogous to the sparks of myocytes and are believed to be the result of the liberation of Ca(2+) from the endoplasmic reticulum through the coordinated opening of IP(3) receptor/channels clustered at a functional release site. In this article, we analyze sequences of puffs that occur at the same site to help elucidate the mechanisms underlying puff dynamics. In particular, we show a dependence of the interpuff time on the amplitude of the preceding puff, and of the amplitude of the following puff on the preceding interval. These relationships can be accounted for by an inhibitory role of the Ca(2+) that is liberated during puffs. We construct a stochastic model for a cluster of IP(3) receptor/channels that quantitatively replicates the observed behavior, and we determine that the characteristic time for a channel to escape from the inhibitory state is of the order of seconds.

Neuronal Ca2+ signaling through inositol triphosphate receptors (IP3R) and ryanodine receptors (RyRs) must be tightly regulated to maintain cell viability, both acutely and over a lifetime. Exaggerated intracellular Ca2+ levels have been associated with expression of Alzheimer's disease (AD) mutations in young mice, but little is known of Ca2+ dysregulations during normal and pathological aging processes. Here, we used electrophysiological recordings with two-photon imaging to study Ca2+ signaling in nontransgenic (NonTg) and several AD mouse models (PS1KI, 3xTg-AD, and APPSweTauP301L) at young (6 week), adult (6 months), and old (18 months) ages. At all ages, the PS1KI and 3xTg-AD mice displayed exaggerated endoplasmic reticulum (ER) Ca2+ signals relative to NonTg mice. The PS1 mutation was the predominant "calciopathic" factor, because responses in 3xTg-AD mice were similar to PS1KI mice, and APPSweTauP301L mice were not different from controls. In addition, we uncovered powerful signaling interactions and differences between IP3R- and RyR-mediated Ca2+ components in NonTg and AD mice. In NonTg mice, RyR contributed modestly to IP3-evoked Ca2+, whereas the exaggerated signals in 3xTg-AD and PS1KI mice resulted primarily from enhanced RyR-Ca2+ release and were associated with increased RyR expression across all ages. Moreover, IP3-evoked membrane hyperpolarizations in AD mice were even greater than expected from exaggerated Ca2+ signals, suggesting increased coupling efficiency between cytosolic [Ca2+] and K+ channel regulation. We conclude that lifelong ER Ca2+ disruptions in AD are related to a modulation of RyR signaling associated with PS1 mutations and represent a discrete "calciumopathy," not merely an acceleration of normal aging.

We describe an optical technique using total internal reflection fluorescence (TIRF) microscopy to obtain simultaneous and independent recordings from numerous ion channels via imaging of single-channel Ca2+ flux. Muscle nicotinic acetylcholine (ACh) receptors made up of alphabetagammadelta subunits were expressed in Xenopus oocytes, and single channel Ca2+ fluorescence transients (SCCaFTs) were imaged using a fast (500 fps) electron-multiplied c.c.d. camera with fluo-4 as the indicator. Consistent with their arising through openings of individual nicotinic channels, SCCaFTs were seen only when a nicotinic agonist was present in the bathing solution, were blocked by curare, and increased in frequency as roughly the second power of [ACh]. Their fluorescence amplitudes varied linearly with membrane potential and extrapolated to zero at about +60 mV. The rise and fall times of fluorescence were as fast as 2 ms, providing a kinetic resolution adequate to characterize channel gating kinetics; which showed mean open times of 7.9 and 15.8 ms when activated, respectively, by ACh or suberyldicholine. Simultaneous records were obtained from >400 channels in the imaging field, and we devised a novel "channel chip" representation to depict the resultant large dataset as a single image. The positions of SCCaFTs remained fixed (<100 nm displacement) over tens of seconds, indicating that the nicotinic receptor/channels are anchored in the oocyte membrane; and the spatial distribution of channels appeared random without evidence of clustering. Our results extend single-channel TIRFM imaging to ligand-gated channels that display only partial permeability to Ca2+, and demonstrate an order-of-magnitude improvement in kinetic resolution. We believe that functional single-channel imaging opens a new approach to ion channel study, having particular advantages over patch-clamp recording in that it is massively parallel, and provides high-resolution spatial information that is inaccessible by electrophysiological techniques.

Increasing evidence suggests that amyloid peptides associated with a variety of degenerative diseases induce neurotoxicity in their intermediate oligomeric state, rather than as monomers or fibrils. To test this hypothesis and investigate the possible involvement of Ca2+ signaling disruptions in amyloid-induced cytotoxicity, we made homogeneous preparations of disease-related amyloids (Abeta, prion, islet amyloid polypeptide, polyglutamine, and lysozyme) in various aggregation states and tested their actions on fluo-3-loaded SH-SY5Y cells. Application of oligomeric forms of all amyloids tested (0.6-6 microg ml-1) rapidly (approximately 5 s) elevated intracellular Ca2+, whereas equivalent amounts of monomers and fibrils did not. Ca2+ signals evoked by Abeta42 oligomers persisted after depletion of intracellular Ca2+ stores, and small signals remained in Ca2+-free medium, indicating contributions from both extracellular and intracellular Ca2+ sources. The increased membrane permeability to Ca2+ cannot be attributed to activation of endogenous Ca2+ channels, because responses were unaffected by the potent Ca2+-channel blocker cobalt (20 microm). Instead, observations that Abeta42 and other oligomers caused rapid cellular leakage of anionic fluorescent dyes point to a generalized increase in membrane permeability. The resulting unregulated flux of ions and molecules may provide a common mechanism for oligomer-mediated toxicity in many amyloidogenic diseases, with dysregulation of Ca2+ ions playing a crucial role because of their strong trans-membrane concentration gradient and involvement in cell dysfunction and death.

Local intracellular Ca(2+) signals result from Ca(2+) flux into the cytosol through individual channels or clusters of channels. To gain a mechanistic understanding of these events we need to know the magnitude and spatial distribution of the underlying Ca(2+) flux. However, this is difficult to infer from fluorescence Ca(2+) images because the distribution of Ca(2+)-bound dye is affected by poorly characterized processes including diffusion of Ca(2+) ions, their binding to mobile and immobile buffers, and sequestration by Ca(2+) pumps. Several methods have previously been proposed to derive Ca(2+) flux from fluorescence images, but all require explicit knowledge or assumptions regarding these processes. We now present a novel algorithm that requires few assumptions and is largely model-independent. By testing the algorithm with both numerically generated image data and experimental images of sparklets resulting from Ca(2+) flux through individual voltage-gated channels, we show that it satisfactorily reconstructs the magnitude and time course of the underlying Ca(2+) currents.

Recent developments in microscopy and fluorescent indicators now make it possible to monitor the activity and localization of membrane ion channels by imaging Ca(2+) flux through individual channels. Such optical approaches have advantages over electrophysiological single-channel techniques in that they are less invasive, provide spatial information and can simultaneously and independently monitor hundreds of channels. However, their kinetic resolution does not yet approach that of patch-clamp recordings. To help understand the processes that determine the temporal resolution and noise level of single-channel Ca(2+) fluorescence signals (SCCaFTs), we simulated the microdomains of Ca(2+) ions and Ca(2+)-bound indicator dye that exist around the mouth of an open channel. Further, as an aid to development of improved optical techniques, we modeled the dependence of the amplitude and kinetics of SCCaFTs on parameters such as the imaging volume, the indicator concentration, affinity and mobility, and the presence of endogenous and exogenous Ca(2+) buffers. The results indicate that under optimal conditions, including the use of confocal or total-internal reflection microscopy to image from sub-femtolitre volumes, SCCaFTs should resolve channel openings as brief as 1ms with a signal-to-noise ratio >10.

Developments in imaging technology now enable visualization of the functioning of individual ion channels in living cells: something previously possible only by the electrophysiological patch-clamp technique. We review techniques that track channel gating via changes in intracellular [Ca2+] resulting from openings of Ca(2+)-permeable channels. Spatial and temporal resolution are optimized by monitoring Ca2+ close to the channel mouth, and we describe the use of two imaging modalities: confocal laser scan microscopy (linescan CLSM) and total internal reflection fluorescence microscopy (TIRFM). Both currently achieve a kinetic resolution of <10 ms, provide a simultaneous and independent readout from many channels, and enable their locations to be mapped with submicrometer resolution. TIRFM provides 2-D images from a very thin (approximately 100 nm) optical section, but it is restricted to channels in the plasma membrane of cells adhering close to a cover glass. In contrast, CLSM can image channels in intracellular membranes but, to achieve good temporal resolution, has been utilized only in a linescan mode with limited spatial information. We anticipate that imaging techniques will develop as a useful adjunct to patch-clamping for single-channel studies, with capabilities including simultaneous readout from multiple channels, high-resolution mapping of channel location, and mobility that is inaccessible by electrophysiological means. Optical single-channel recording is applicable to diverse voltage- and ligand-gated Ca(2+)-permeable channels and has potential for high-throughput functional analysis., (Copyright 2005 Society of Photo-Optical Instrumentation Engineers)

The patch-clamp technique has enabled functional studies of single ion channels, but suffers limitations including lack of spatial information and inability to independently monitor currents from more than one channel. Here, we describe the use of total internal reflection fluorescence microscopy as an alternative, noninvasive approach to optically monitor the activity and localization of multiple Ca(2+)-permeable channels in the plasma membrane. Images of near-membrane Ca(2+) signals were obtained from >100 N-type channels expressed within restricted areas (80 x 80 micro m) of Xenopus oocytes, thereby permitting simultaneous resolution of their gating kinetics, voltage dependence, and localization. Moreover, this technique provided information inaccessible by electrophysiological means, demonstrating that N-type channels are immobile in the membrane, show a patchy distribution, and display diverse gating kinetics even among closely adjacent channels. Total internal reflection fluorescence microscopy holds great promise for single-channel recording of diverse voltage- and ligand-gated Ca(2+)-permeable channels in the membrane of neurons and other isolated or cultured cells, and has potential for high-throughput functional analysis of single channels.

Ca2+ liberation through inositol 1,4,5-trisphosphate receptors (IP3Rs) plays a universal role in cell regulation, and specificity of cell signalling is achieved through the spatiotemporal patterning of Ca2+ signals. IP3Rs display Ca2+-induced Ca2+ release (CICR), but are grouped in clusters so that regenerative Ca2+ signals may remain localized to individual clusters, or propagate globally between clusters by successive cycles of Ca2+ diffusion and CICR. We used confocal microscopy and photoreleased IP3 in Xenopus oocytes to study how these properties are modulated by mobile cytosolic Ca2+ buffers. EGTA (a buffer with slow 'on-rate') speeded Ca2+ signals and 'balkanized' Ca2+ waves by dissociating them into local signals. In contrast, BAPTA (a fast buffer with similar affinity) slowed Ca2+ responses and promoted 'globalization' of spatially uniform Ca2+ signals. These actions are likely to arise through differential effects on Ca2+ feedback within and between IP3R clusters, because Ca2+ signals evoked by influx through voltage-gated channels were little affected. We propose that cell-specific expression of Ca2+-binding proteins with distinct kinetics may shape the time course and spatial distribution of IP3-evoked Ca2+ signals for specific physiological roles.

Functional studies of single membrane ion channels were made possible by the introduction of the patch-clamp technique, which allows single-channel currents to be measured with unprecedented resolution. Nevertheless, patch clamping has some limitations: including the need for physical access of the patch pipette, possible disruption of local cellular architecture, inability to monitor multiple channels, and lack of spatial information. Here, we demonstrate the use of confocal fluorescence microscopy as a non-invasive technique to optically monitor the gating of individual Ca2+ channels. Near-membrane fluorescence signals track the gating of N-type Ca2+ channels with a kinetic resolution of about 10ms, provide a simultaneous and independent readout from several channels, and allow their locations to be mapped with sub-micrometer spatial resolution. Optical single-channel recording should be applicable to diverse voltage- and ligand-gated Ca2+-permeable channels, and has the potential for high-throughput functional analysis of single channels.

We describe the construction of a video-rate two-photon laser scanning microscope, compare its performance to a similar confocal microscope, and illustrate its use for imaging local Ca(2+) transients from cortical neurons in brain slices. Key features include the use of a Ti-sapphire femtosecond laser allowing continuous tuning over a wide (700-1000 nm) wavelength range, a resonant scanning mirror to permit frame acquisition at 30 Hz, and efficient wide-field fluorescence detection. Two-photon imaging provides compelling advantages over confocal microscopy in terms of improved imaging depth and reduced phototoxicity and photobleaching, but the high cost of commercial instruments has limited their widespread adoption. By constructing one's own system the expense is greatly reduced without sacrifice of performance, and the microscope can be more readily tailored to specific applications., (Copyright 2001 Harcourt Publishers Ltd.)

1. The purpose of this study was to compare oocytes from the pipid frogs Xenopus tropicalis and Xenopus laevis, with respect to their utility for studying Ca(2+) signalling mechanisms and for expression of heterologous proteins. 2. We show that X. tropicalis oocytes possess an intracellular Ca(2+) store that is mobilized by inositol (1,4,5) trisphosphate (IP(3)). Ca(2+) signalling is activated by endogenous lysophosphatidic acid receptors and cytosolic Ca(2+) activates a plasma membrane chloride conductance. The spatiotemporal organization of cytosolic Ca(2+) signals, from the microscopic architecture of elementary Ca(2+) 'puffs' to the macroscopic patterns of Ca(2+) spiking are closely similar to the local and global patterns of Ca(2+) release previously characterized in oocytes from X. laevis. 3. By injecting X. tropicalis oocytes with cDNA encoding an ER-targeted fluorescent protein construct, we demonstrate the capacity of the X. tropicalis oocyte to readily express heterologous proteins. The organization of ER is polarized across the oocyte, with the IP(3)-releaseable store targeted within an approximately 8 microm wide band that circumscribes the cell. 4. We conclude that the X. tropicalis oocyte shares many of the characteristics that have made oocytes of X. laevis a favoured system for studying Ca(2+) signalling mechanisms. Moreover, X. tropicalis oocytes display further practical advantages in terms of imaging depth, Ca(2+) signal magnitude and electrical properties. These further enhance the appeal of X. tropicalis as an experimental system, in addition to its greater amenability to transgenic approaches.

Inositol (1,4,5)-trisphosphate (IP(3)) liberates intracellular Ca(2+) both as localized 'puffs' and as repetitive waves that encode information in a frequency-dependent manner. Using video-rate confocal imaging, together with photorelease of IP(3) in Xenopus oocytes, we investigated the roles of puffs in determining the periodicity of global Ca(2+) waves. Wave frequency is not delimited solely by cyclical recovery of the cell's ability to support wave propagation, but further involves sensitization of Ca(2+)-induced Ca(2+) release by progressive increases in puff frequency and amplitude at numerous sites during the interwave period, and accumulation of pacemaker Ca(2+), allowing a puff at a 'focal' site to trigger a subsequent wave. These specific 'focal' sites, distinguished by their higher sensitivity to IP(3) and close apposition to neighboring puff sites, preferentially entrain both the temporal frequency and spatial directionality of Ca(2+) waves. Although summation of activity from many stochastic puff sites promotes the generation of regularly periodic global Ca(2+) signals, the properties of individual Ca(2+) puffs control the kinetics of Ca(2+) spiking and the (higher) frequency of subcellular spikes in their local microdomain.

Ca(2+) liberation by inositol 1,4,5-trisphosphate (IP(3)) is 'quantal', in that low [IP(3)] causes only partial Ca(2+) release, but further increasing [IP(3)] evokes more release. This characteristic allows cells to generate graded Ca(2+) signals, but is unexpected, given the regenerative nature of Ca(2+)-induced Ca(2+) release through IP(3) receptors. Two models have been proposed to resolve this paradox: (i) all-or-none Ca(2+) release from heterogeneous stores that empty at varying [IP(3)]; and (ii) phasic liberation from homogeneously sensitive stores. To discriminate between these hypotheses, we imaged subcellular Ca(2+) puffs evoked by IP(3) in Xenopus oocytes where release sites were functionally uncoupled using EGTA. Puffs were little changed by 300 microM intracellular EGTA, but sites operated autonomously and did not propagate waves. Photoreleased IP(3) generated flurries of puffs-different to the prolonged Ca(2+) elevation following waves in control cells-and individual sites responded repeatedly to successive increments of [IP(3)]. These data support the second hypothesis while refuting the first, and suggest that local Ca(2+) signals exhibit rapid adaptation, different to the slower inhibition following global Ca(2+) waves.

Ca(2+)-activated Cl(-) currents (I(Cl,Ca)) were examined using fluorescence confocal microscopy to monitor intracellular Ca(2+) liberation evoked by flash photolysis of caged inositol 1,4, 5-trisphosphate (InsP(3)) in voltage-clamped Xenopus oocytes. Currents at +40 mV exhibited a steep dependence on InsP(3) concentration ([InsP(3)]), whereas currents at -140 mV exhibited a higher threshold and more graded relationship with [InsP(3)]. Ca(2+) levels required to half-maximally activate I(Cl,Ca) were about 50% larger at -140 mV than at +40 mV, and currents evoked by small Ca(2+) elevations were reduced >25-fold. The half-decay time of Ca(2+) signals shortened at increasingly positive potentials, whereas the decay of I(Cl,Ca) lengthened. The steady-state current-voltage (I-V) relationship for I(Cl,Ca) exhibited outward rectification with weak photolysis flashes but became more linear with stronger stimuli. Instantaneous I-V relationships were linear with both strong and weak stimuli. Current relaxations following voltage steps during activation of I(Cl,Ca) decayed with half-times that shortened from about 100 ms at +10 mV to 20 ms at -160 mV. We conclude that InsP(3)-mediated Ca(2+) liberation activates a single population of Cl(-) channels, which exhibit voltage-dependent Ca(2+) activation and voltage-independent instantaneous conductance.