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Experimental records of single molecules or ion channels from fluorescence microscopy and patch-clamp electrophysiology often include high-frequency noise and baseline fluctuations that are not generated by the system under investigation and have to be removed. More-over, multiple channels or conductance levels can be present at a time in the data that need to be quantified to accurately understand the behavior of the system. Manual procedures for removing these fluctuations and extracting conducting states or multiple channels are laborious, prone to subjective bias, and hinder the processing of often very large data-sets. We introduce a maximum likelihood formalism for separating signal from a noisy and drifting background such as fluorescence traces from imaging of elementary Ca2+ release events called puffs arising from clusters of channels and patch-clamp recordings of ion channels. Parameters such as the number of open channels or conducting states, noise level, and back-ground signal can all be optimized using the expectation-maximization (EM) algorithm. We implement our algorithm following the Baum-Welch approach to EM in the portable java language with a user-friendly graphical interface and test the algorithm on both synthetic and experimental data from patch-clamp electrophysiology of Ca2+ channels and fluorescence microscopy of a cluster of Ca2+ channels and Ca2+ channels with multiple conductance levels. The resulting software is accurate, fast, and provides detailed information usually not available through manual analysis. Options for visual inspection of the raw and processed data with key parameters, and exporting a range of statistics such as the mean open probabilities, mean open times, mean close times, and dwell time distributions for different number of channels open or conductance levels, amplitude distribution of all opening events, and number of transitions between different number of open channels or conducting levels in asci format with a single click are provided.

Puffs are local Ca(2+) signals that arise by Ca(2+) liberation from the endoplasmic reticulum through the concerted opening of tightly clustered inositol trisphosphate receptors/channels (IP3Rs). The locations of puff sites observed by Ca(2+) imaging remain static over several minutes, whereas fluorescence recovery after photobleaching (FRAP) experiments employing overexpression of fluorescently tagged IP3Rs have shown that the majority of IP3Rs are freely motile. To address this discrepancy, we applied single-molecule imaging to locate and track type 1 IP3Rs tagged with a photoswitchable fluorescent protein and expressed in COS-7 cells. We found that ∼ 70% of the IP3R1 molecules were freely motile, undergoing random walk motility with an apparent diffusion coefficient of ∼ 0.095 μm s(-1), whereas the remaining molecules were essentially immotile. A fraction of the immotile IP3Rs were organized in clusters, with dimensions (a few hundred nanometers across) comparable to those previously estimated for the IP3R clusters underlying functional puff sites. No short-term (seconds) changes in overall motility or in clustering of immotile IP3Rs were apparent following activation of IP3/Ca(2+) signaling. We conclude that stable clusters of small numbers of immotile IP3Rs may underlie local Ca(2+) release sites, whereas the more numerous motile IP3Rs appear to be functionally silent.

Puffs are local Ca(2+) signals that arise by Ca(2+) liberation from the endoplasmic reticulum through concerted opening of tightly clustered inositol trisphosphate receptor/channels (IP(3)R). They serve both local signaling functions and trigger global Ca(2+) waves. The numbers of functional IP(3)R within clusters differ appreciably between different puff sites, and we investigated how the probability of puff occurrence varies with cluster size. We imaged puffs in SH-SY5Y cells using total internal fluorescence microscopy, and estimated cluster sizes from the magnitude of the largest puff observed at each site relative to the signal from a single channel. We find that the initial triggering rate of puffs following photorelease of IP(3), and the average frequency of subsequent repetitive puffs, vary about linearly with cluster size. These data accord well with stochastic simulations in which opening of any individual IP(3)R channel within a cluster triggers a puff via Ca(2+)-induced Ca(2+) release. An important consequence is that the signaling power of a puff site (average amount of Ca(2+) released per puff × puff frequency) varies about the square of cluster size, implying that large clusters contribute disproportionately to cellular signaling and, because of their higher puff frequency, preferentially act as pacemakers to initiate Ca(2+) waves.

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 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.

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.

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 from100 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.

We applied single-molecule photobleaching to investigate the stoichiometry of human Orai1 and Orai3 channels tagged with eGFP and expressed in HEK cells. At low expression, GFP-tagged subunits were detected in TIRF microscopy as single fluorescent spots that decayed in a step-wise manner as individual GFP molecules bleached. By counting the number of photobleaching steps, the number of subunits per channel complex could be deduced. In fixed cells, Orai1 was detected primarily as dimers when expressed alone and as tetramers when co-expressed with the cytosolic STIM1 fragment, C-STIM1, to activate Ca2+ influx, as previously found for Drosophila Orai with and without activation by C-Stim (Penna et al., 2008, Nature 456:116-120). When co-expressed with full-length STIM1, Orai1 was also found to be predominantly dimeric in living cells under resting conditions with Ca2+ stores filled. We also investigated Orai3 alone and when activated by either C-STIM1 to form a Ca2+-selective channel in its store-operated mode, or by addition of 2-APB to form a Ca2+-permeable but relatively nonselective cation channel in its STIM1-independent mode. Similar to our observations with Orai1, eGFP-Orai3 alone was detected mostly as dimers under basal conditions, but predominantly as tetramers when co-expressed with C-STIM1. On the other hand, cells expressing only eGFP-Orai3 that were exposed to 2APB before fixation showed a distribution of bleaching steps closely similar to that observed without 2APB, with a predominance of dimers. These results indicate a predominantly dimeric state for Orai3 at rest or when activated by 2-APB, and a tetrameric channel when activated by C-STIM1. We conclude that the human Orai1 and Orai3 channels undergo a dimer-to-tetramer transition to form a Ca2+-selective pore during store-operated activation, and that Orai3 forms a dimeric non-selective cation pore upon activation by 2-APB.

A new mechanism of cell-cell communication was recently proposed after the discovery of tunneling nanotubes (TNTs) between cells. TNTs are membrane protrusions with lengths of tens of microns and diameters of a few hundred nanometers that permit the exchange of membrane and cytoplasmic constituents between neighboring cells. TNTs have been reported to mediate intercellular Ca(2+) signaling; however, our simulations indicate that passive diffusion of Ca(2+) ions alone would be inadequate for efficient transmission between cells. Instead, we observed spontaneous and inositol trisphosphate (IP(3))-evoked Ca(2+) signals within TNTs between cultured mammalian cells, which sometimes remained localized and in other instances propagated as saltatory waves to evoke Ca(2+) signals in a connected cell. Consistent with this, immunostaining showed the presence of both endoplasmic reticulum and IP(3) receptors along the TNT. We propose that IP(3) receptors may actively propagate intercellular Ca(2+) signals along TNTs via Ca(2+)-induced Ca(2+) release, acting as amplification sites to overcome the limitations of passive diffusion in a chemical analog of electrical transmission of action potentials.

The subcellular localization of membrane Ca2+ channels is crucial for their functioning, but is difficult to study because channels may be distributed more closely than the resolution of conventional microscopy is able to detect. We describe a technique, stochastic channel Ca2+ nanoscale resolution (SCCaNR), employing Ca2+-sensitive fluorescent dyes to localize stochastic openings and closings of single Ca2+-permeable channels within50 nm, and apply it to examine the clustered arrangement of inositol trisphosphate receptor (IP3R) channels underlying local Ca2+ puffs. Fluorescence signals (blips) arising from single functional IP3Rs are almost immotile (diffusion coefficient0.003 microm2 s(-1)), as are puff sites over prolonged periods, suggesting that the architecture of this signaling system is stable and not subject to rapid, dynamic rearrangement. However, rapid stepwise changes in centroid position of fluorescence are evident within the durations of individual puffs. These apparent movements likely result from asynchronous gating of IP3Rs distributed within clusters that have an overall diameter of approximately 400 nm, indicating that the nanoscale architecture of IP3R clusters is important in shaping local Ca2+ signals. We anticipate that SCCaNR will complement superresolution techniques such as PALM and STORM for studies of Ca2+ channels as it obviates the need for photoswitchable labels and provides functional as well as spatial information.

Advanced imaging techniques such as PALM and STORM have broken the diffraction limit of conventional optical microscopy through their ability to turn fluorescent molecules on and off at low enough densities such that the positions of single molecules can be determined, one at a time, with a precision of ∼10 nm (Gustafsson, 2008). However, these techniques involve the use of fluorescently tagged proteins or antibodies, which may alter protein properties and provide only positional, not functional information. Thus, we have developed a technique termed Single Channel Ca2+ Nanoscale Resolution (SCCaNR), based on similar principles except that it generates a super-resolution image by using Ca2+ sensitive fluorescent dyes to image the stochastic openings and closings of Ca2+ permeable ion channels. Subsequently, the point spread function resulting from the diffusion of calcium bound to the indicator dye can be fit to a 2-D Gaussian function, allowing the position of functional calcium channels to be localized with much higher precision (∼40 nm) than previously possible.The inositol triphosphate receptor (IP3R) is an ER Ca2+ channel that is both facilitated and inhibited by Ca2+ itself. This property enables a functional coupling between IP3Rs, which underlies the generation of localized Ca2+ events known as puffs (Yao, et al, 1995). This same property makes IP3Rs highly dependent on their spatial proximity to one another. Using our SCCaNR technique, we have found that the concerted opening of 4-10 IP3R channels likely underlies the generation of Ca2+ puffs in SH-SY5Y neuroblastoma cells. These puffs arise from clusters of IP3Rs approximately 300 nm in diameter, a dimension below the resolution limit of conventional optical microscopy.

We examined the effect of temperature (12–40°C) on local and global Ca2+ signals mediated by inositol trisphosphate receptor/channels (IP3R) in human neuroblastoma (SH-SY5Y) cells. The amplitudes and spatial spread of local signals arising from single IP3R (blips) and clusters of IP3R (puffs) showed little temperature dependence, whereas their kinetics (durations and latencies) were markedly accelerated by increasing temperature. In contrast, the amplitude of global Ca2+ waves increased appreciably at lower temperatures, probably as a result of the longer duration of IP3R channel opening. Several parameters, including puff and blip durations, puff latency and frequency, and frequency of repetitive Ca2+ waves, showed a biphasic temperature dependence on Arrhenius plots. In all cases the transition temperature occurred at ∼25°C, possibly reflecting a phase transition in the lipids of the endoplasmic reticulum membrane. Although the IP3-evoked Ca2+ signals were qualitatively similar at 25°C and 36°C, one should consider the temperature sensitivity of IP3-mediated signal amplitudes when extrapolating from room temperature to physiological temperature. Conversely, further cooling may be advantageous to improve the optical resolution of channel gating kinetics.

Upon endoplasmic reticulum Ca(2+) store depletion, Orai channels in the plasma membrane are activated directly by endoplasmic reticulum-resident STIM proteins to generate the Ca(2+)-selective, Ca(2+) release-activated Ca(2+) (CRAC) current. After the molecular identification of Orai, a plethora of functional and biochemical studies sought to compare Orai homologs, determine their stoichiometry, identify structural domains responsible for the biophysical fingerprint of the CRAC current, identify the physiological functions, and investigate Orai homologs as potential therapeutic targets. Subsequently, the solved crystal structure of Drosophila Orai (dOrai) substantiated many findings from structure-function studies, but also revealed an unexpected hexameric structure. In this review, we explore Orai channels as elucidated by functional and biochemical studies, analyze the dOrai crystal structure and its implications for Orai channel function, and present newly available information from molecular dynamics simulations that shed light on Orai channel gating and permeation.

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 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.

Puffs are localized, transient elevations in cytosolic Ca 2þ that serve both as the building blocks of global cellular Ca 2þ signals and as local signals in their own right. They arise from clustered inositol 1,4,5-trisphosphate receptor/channels (IP3Rs), whose openings are coordinated by Ca 2þ -induced Ca 2þ release (CICR). We utilized total internal reflection fluores- cence imaging of Ca 2þ signals in neuroblastoma cells with single-channel resolution to elucidate the mechanisms determining the triggering, amplitudes, kinetics, and spatial spread of puffs. We find that any given channel in a cluster has a mean probability of ~66% of opening following opening of an initial ''trigger'' channel, and the probability of puff triggering thus increases steeply with increasing number of channels in a cluster (cluster size). Mean puff amplitudes scale with cluster size, but individual amplitudes vary widely, even at sites of similar cluster size, displaying similar proportions of events involving any given number of the channels in the cluster. Stochastic variation in numbers of Ca 2þ -inhibited IP3Rs likely contributes to the variability of amplitudes of repeated puffs at a site but the amplitudes of successive puffs were uncorrelated, even though we observed statistical correlations between interpuff intervals and puff amplitudes. Initial puffs evoked following photorelease of IP3—which would not be subject to earlier Ca 2þ -inhibition—also showed wide variability, indicating that mechanisms such as stochastic vari- ation in IP3 binding and channel recruitment by CICR further determine puff amplitudes. The mean termination time of puffs lengthened with increasing puff amplitude size, consistent with independent closings of channels after a given mean open time, but we found no correlation of termination time with cluster size independent of puff amplitude. The spatial extent of puffs increased with their amplitude, and puffs of similar size were of similar width, independent of cluster size.

Recent RNAi screens have identified Stim and Orai as critical components of the Ca2+ release-activated Ca2+ (CRAC) channel. Stim senses depletion of the Endoplasmic Reticulum (ER) Ca2+ store, translocates from the ER to junctions adjacent to the plasma membrane (PM), and activates Orai pore-forming channel subunits in the PM to open the CRAC channel. The Orai oligomerization interface was investigated by co-immunoprecipitation of N-and/or C-terminal Orai deletion mutants and expressed Orai N- and C-terminal fragments. The transmembrane core domain plays a predominant role in subunit assembly; a weaker interaction interface was identified at the N-terminal region. We analyzed the quaternary structure of the Orai subunit and showed by cross-linking, and by non-denaturing gel electrophoresis that Orai is predominantly a dimer under resting conditions with or without co-expression of Stim. Single-molecule imaging of GFP-tagged Orai expressed in Xenopus oocytes revealed predominantly two-step photo-bleaching, consistent with a dimeric basal state. In contrast, co-expression of GFP-tagged Orai with the C-terminus of Stim as a cytosolic protein to activate the Orai channel without inducing Ca2+ store depletion or clustering of Orai into punctae yielded predominantly four-step photobleaching, consistent with a tetrameric Orai stoichiometry of the active CRAC channel. Interaction of the Orai C-terminal coiled-coil domain (as shown by structure-disruptive mutations) with the C-terminus of Stim thus induces Orai dimers to dimerize, forming a tetramer that constitutes the Ca2+-selective pore. This represents a novel mechanism in which assembly and activation of the functional ion channel are mediated by the same triggering molecule and may reveal a new channel gating mechanism. New data will be presented on the Stim-Orai stoichiometry and activation mechanism.

Cytosolic Ca2+ is a universal intracellular messenger with a central role in a diverse array of physiological processes. Ca2+ signals can emanate from the endoplasmic reticulum (ER), through inositol trisphosphate receptor/channels (IP3R), as either highly localized signals arising from the opening of one channel (blip) or several clustered channels (puff) or as global Ca2+ waves capable of engulfing the whole cell. We previously examined the temperature dependence of global Ca2+signals in Xenopus oocytes, finding that upon cooling the frequency of repetitive Ca2+ waves slowed while their amplitudes increased markedly. Since then, advances in imaging technology have made it possible to resolve cytosolic Ca2+ signals down to the single channel level. Here, we utilized total internal reflection fluorescence (TIRF) microscopy for high-resolution Ca2+ imaging in mammalian SH-SY5Y neuroblastoma cells from 12-40oC, in conjunction with cytosolic loading of the slow Ca2+ buffer EGTA to inhibit cluster-cluster interactions to record local blips and puffs, or omitting EGTA to record global Ca2+ waves. We found that the amplitudes and spatial spread of blips and puffs showed little temperature dependence, whereas their kinetics (durations and latencies) were markedly accelerated by increasing temperature. In contrast, the amplitudes of Ca2+ waves increased appreciably at lower temperatures, probably resulting from longer durations of IP3R channel openings. Several parameters, including puff and blip durations, puff latency and frequency, and frequency of repetitive Ca2+ waves showed a biphasic temperature dependence on Arrhenius plots. In all cases the transition temperature occurred at about 25oC, possibly reflecting a phase transition in the lipids of the ER. As IP3-evoked Ca2+ signals were qualitatively similar at 25oC and 36oC, room temperature experiments should reflect physiological responses at body temperature, but cooling to 12oC, or lower, may be advantageous to improving optical resolution of channel gating kinetics.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder affecting up to 2% of children and characterized by impaired social skills, delayed or disordered language and communication skills, and repetitive, stereotypic behavior. Growing evidence supports a role of Ca2+ signaling in the pathogenesis of ASD. Inositol trisphosphate (IP3)-mediated Ca2+ release from intracellular stores participates in a variety of functions, from synaptic plasticity and memory, to long-term gene transcription changes and immune response. IP3 is produced upon stimulation of G-protein coupled receptors (GPCR) and binds to IP3 receptor/channel (IP3R) in the membrane of the endoplasmic reticulum (ER), liberating Ca2+ sequestered in the ER lumen into the cytoplasm. Here, we report that human fibroblasts from three genetically distinct monogenic models of ASD – fragile X and tuberous sclerosis TSC1 and TSC2 – uniformly display depressed Ca2+ release through IP3 receptors. We observed defects in whole-cell Ca2+ signals evoked by G-protein-coupled cell surface receptors and by photoreleased IP3, and at the level of local elementary Ca2+ events, suggesting fundamental defects in IP3R channel activity in ASD. Given its ubiquitous functions in the body, malfunctioning of IP3-mediated signaling may account for the heterogeneity of non-neuronal symptoms seen in ASD, such as gastrointestinal tract problems and immunological complications.

Inositol trisphosphate receptors (IP3Rs) are calcium-permeable channels in the membrane of the endoplasmic reticulum (ER) that liberate calcium to generate cytosolic calcium signals that control diverse cellular functions including gene expression, secretion and synaptic plasticity. The spatial distribution of these channels is crucial in determining the patterning of intracellular calcium signals. The mechanisms underlying the aggregation and maintenance of IP3Rs into clusters are controversial. Local calcium puffs reflecting concerted openings of clustered IP3Rs arise at just a few, fixed locations within a cell, suggesting clusters are stable entities; and calcium blips generated by ‘lone’ IP3Rs are also immotile. In contrast, GFP-tagged or immunostained IP3Rs show a dense distribution throughout a cell. Moreover, the majority IP3Rs can diffuse freely within the ER membrane, and aggregate into clusters following activation of IP3 signaling and/or cytosolic calcium elevation. These apparently different behaviors may be explained because calcium imaging detects only functional IP3Rs, whereas immunostained or GFP-tagged IP3Rs report the entire population of IP3R proteins. We therefore hypothesize that most IP3Rs are motile, but functionally unresponsive. Local calcium signals arise, instead, from a small subset of IP3Rs that are anchored, individually or in clusters, by association with static cytoskeletal structures and possibly as a consequence of this anchoring, display high sensitivity to IP3 to produce calcium signals. To test this hypothesis we expressed type 1 IP3R tagged with a photoactivatable genetically encoded protein to track the motility of thousands of individual IP3R molecules with nanoscale spatial and millisecond temporal resolution. We find that IP3Rs can be distinguished into two groups with relatively high or low motility, and that the apparently immotile IP3Rs are preferentially grouped within tight clusters.Support: NIH GM40871 (IP) and GM100201 (IFS).

Cellular signaling mediated by the inositol trisphosphate (IP3) messenger pathway involves hierarchical Ca2+ liberation from the endoplasmic reticulum (ER), whereby local ‘elementary’ Ca2+ transients (Ca2+ puffs) serve autonomous signaling functions and as well as constituting the building blocks from which global cellular Ca2+ waves are constructed. These channels are inaccessible to single-channel study by patch-clamp in intact cells, and excised organelle and bilayer reconstitution systems disrupt the Ca2+ induced Ca2+ release (CICR) process that mediates channel-channel coordination. We report here the use of total internal reflection fluorescence (TIRF) microscopy to image single-channel Ca2+ flux through individual and clustered IP3R's in intact mammalian cells. This enables a quantal dissection of calcium puffs involving stochastic recruitment of an average of 6 active IP3Rs clustered within