Bitter taste receptors (TAS2Rs or T2Rs) belong to the superfamily of seven-transmembrane G protein–coupled receptors, which are the targets of >50% of drugs currently on the market. Canonically, T2Rs are located in taste buds of the tongue, where they initiate bitter taste perception. However, accumulating evidence indicates that T2Rs are widely expressed throughout the body and mediate diverse nontasting roles through various specialized mechanisms. It has also become apparent that T2Rs and their polymorphisms are associated with human disorders. In this review, we summarize the physiological and pathophysiological roles that extraoral T2Rs play in processes as diverse as innate immunity and reproduction, and the major challenges in this emerging field.

Short-lived, localized Ca 2+ events mediate Ca 2+ signaling with high efficiency and great fidelity largely as a result of the close proximity between Ca 2+ -permeable ion channels and their molecular targets. However, in most cases, direct evidence of the spatial relationship between these two types of molecules is lacking, and, thus, mechanistic understanding of local Ca 2+ signaling is incomplete. In this study, we use an integrated approach to tackling this issue on a prototypical local Ca 2+ signaling system composed of Ca 2+ sparks resulting from the opening of ryanodine receptors (RYRs) and spontaneous transient outward currents (STOCs) caused by the opening of Ca 2+ -activated K + (BK) channels in airway smooth muscle. Biophysical analyses of STOCs and Ca 2+ sparks acquired at 333 Hz demonstrate that these two events are associated closely in time, and approximately eight RYRs open to give rise to a Ca 2+ spark, which activates ∼15 BK channels to generate a STOC at 0 mV. Dual immunocytochemistry and 3-D deconvolution at high spatial resolution reveal that both RYRs and BK channels form clusters and RYR1 and RYR2 (but not RYR3) localize near the membrane. Using the spatial relationship between RYRs and BK channels, the spatial-temporal profile of [Ca 2+ ] resulting from Ca 2+ sparks, and the kinetic model of BK channels, we estimate that an average Ca 2+ spark caused by the opening of a cluster of RYR1 or RYR2 acts on BK channels from two to three clusters that are randomly distributed within an ∼600-nm radius of RYRs. With this spatial organization of RYRs and BK channels, we are able to model BK channel currents with the same salient features as those observed in STOCs across a range of physiological membrane potentials. Thus, this study provides a mechanistic understanding of the activation of STOCs by Ca 2+ sparks using explicit knowledge of the spatial relationship between RYRs (the Ca 2+ source) and BK channels (the Ca 2+ target).

A central concept in the physiology of neurosecretion is that a rise in cytosolic [Ca 2+ ] in the vicinity of plasmalemmal Ca 2+ channels due to Ca 2+ influx elicits exocytosis. Here, we examine the effect on spontaneous exocytosis of a rise in focal cytosolic [Ca 2+ ] in the vicinity of ryanodine receptors (RYRs) due to release from internal stores in the form of Ca 2+ syntillas. Ca 2+ syntillas are focal cytosolic transients mediated by RYRs, which we first found in hypothalamic magnocellular neuronal terminals. ( scintilla , Latin for spark; found in nerve terminals, normally synaptic structures.) We have also observed Ca 2+ syntillas in mouse adrenal chromaffin cells. Here, we examine the effect of Ca 2+ syntillas on exocytosis in chromaffin cells. In such a study on elicited exocytosis, there are two sources of Ca 2+ : one due to influx from the cell exterior through voltage-gated Ca 2+ channels, and that due to release from intracellular stores. To eliminate complications arising from Ca 2+ influx, we have examined spontaneous exocytosis where influx is not activated. We report here that decreasing syntillas leads to an increase in spontaneous exocytosis measured amperometrically. Two independent lines of experimentation each lead to this conclusion. In one case, release from stores was blocked by ryanodine; in another, stores were partially emptied using thapsigargin plus caffeine, after which syntillas were decreased. We conclude that Ca 2+ syntillas act to inhibit spontaneous exocytosis, and we propose a simple model to account quantitatively for this action of syntillas.

Ca 2+ sparks are highly localized, transient releases of Ca 2+ from sarcoplasmic reticulum through ryanodine receptors (RyRs). In smooth muscle, Ca 2+ sparks trigger spontaneous transient outward currents (STOCs) by opening nearby clusters of large-conductance Ca 2+ -activated K + channels, and also gate Ca 2+ -activated Cl − (Cl (Ca) ) channels to induce spontaneous transient inward currents (STICs). While the molecular mechanisms underlying the activation of STOCs by Ca 2+ sparks is well understood, little information is available on how Ca 2+ sparks activate STICs. In the present study, we investigated the spatial organization of RyRs and Cl (Ca) channels in spark sites in airway myocytes from mouse. Ca 2+ sparks and STICs were simultaneously recorded, respectively, with high-speed, widefield digital microscopy and whole-cell patch-clamp. An image-based approach was applied to measure the Ca 2+ current underlying a Ca 2+ spark (I Ca(spark) ), with an appropriate correction for endogenous fixed Ca 2+ buffer, which was characterized by flash photolysis of NPEGTA. We found that I Ca(spark) rises to a peak in 9 ms and decays with a single exponential with a time constant of 12 ms, suggesting that Ca 2+ sparks result from the nonsimultaneous opening and closure of multiple RyRs. The onset of the STIC lags the onset of the I Ca(spark) by less than 3 ms, and its rising phase matches the duration of the I Ca(spark) . We further determined that Cl (Ca) channels on average are exposed to a [Ca 2+ ] of 2.4 μM or greater during Ca 2+ sparks. The area of the plasma membrane reaching this level is <600 nm in radius, as revealed by the spatiotemporal profile of [Ca 2+ ] produced by a reaction-diffusion simulation with measured I Ca(spark) . Finally we estimated that the number of Cl (Ca) channels localized in Ca 2+ spark sites could account for all the Cl (Ca) channels in the entire cell. Taken together these results lead us to propose a model in which RyRs and Cl (Ca) channels in Ca 2+ spark sites localize near to each other, and, moreover, Cl (Ca) channels concentrate in an area with a radius of ∼600 nm, where their density reaches as high as 300 channels/μm 2 . This model reveals that Cl (Ca) channels are tightly controlled by Ca 2+ sparks via local Ca 2+ signaling.

Ca 2+ sparks are small, localized cytosolic Ca 2+ transients due to Ca 2+ release from sarcoplasmic reticulum through ryanodine receptors. In smooth muscle, Ca 2+ sparks activate large conductance Ca 2+ -activated K + channels (BK channels) in the spark microdomain, thus generating spontaneous transient outward currents (STOCs). The purpose of the present study is to determine experimentally the level of Ca 2+ to which the BK channels are exposed during a spark. Using tight seal, whole-cell recording, we have analyzed the voltage-dependence of the STOC conductance (g (STOC) ), and compared it to the voltage-dependence of BK channel activation in excised patches in the presence of different [Ca 2+ ]s. The Ca 2+ sparks did not change in amplitude over the range of potentials of interest. In contrast, the magnitude of g (STOC) remained roughly constant from 20 to −40 mV and then declined steeply at more negative potentials. From this and the voltage dependence of BK channel activation, we conclude that the BK channels underlying STOCs are exposed to a mean [Ca 2+ ] on the order of 10 μM during a Ca 2+ spark. The membrane area over which a concentration ≥10 μM is reached has an estimated radius of 150–300 nm, corresponding to an area which is a fraction of one square micron. Moreover, given the constraints imposed by the estimated channel density and the Ca 2+ current during a spark, the BK channels do not appear to be uniformly distributed over the membrane but instead are found at higher density at the spark site.

Ca 2+ sparks are highly localized cytosolic Ca 2+ transients caused by a release of Ca 2+ from the sarcoplasmic reticulum via ryanodine receptors (RyRs); they are the elementary events underlying global changes in Ca 2+ in skeletal and cardiac muscle. In smooth muscle and some neurons, Ca 2+ sparks activate large conductance Ca 2+ -activated K + channels (BK channels) in the spark microdomain, causing spontaneous transient outward currents (STOCs) that regulate membrane potential and, hence, voltage-gated channels. Using the fluorescent Ca 2+ indicator fluo-3 and a high speed widefield digital imaging system, it was possible to capture the total increase in fluorescence (i.e., the signal mass) during a spark in smooth muscle cells, which is the first time such a direct approach has been used in any system. The signal mass is proportional to the total quantity of Ca 2+ released into the cytosol, and its rate of rise is proportional to the Ca 2+ current flowing through the RyRs during a spark (I Ca(spark) ). Thus, Ca 2+ currents through RyRs can be monitored inside the cell under physiological conditions. Since the magnitude of I Ca(spark) in different sparks varies more than fivefold, Ca 2+ sparks appear to be caused by the concerted opening of a number of RyRs. Sparks with the same underlying Ca 2+ current cause STOCs, whose amplitudes vary more than threefold, a finding that is best explained by variability in coupling ratio (i.e., the ratio of RyRs to BK channels in the spark microdomain). The time course of STOC decay is approximated by a single exponential that is independent of the magnitude of signal mass and has a time constant close to the value of the mean open time of the BK channels, suggesting that STOC decay reflects BK channel kinetics, rather than the time course of [Ca 2+ ] decline at the membrane. Computer simulations were carried out to determine the spatiotemporal distribution of the Ca 2+ concentration resulting from the measured range of I Ca(spark) . At the onset of a spark, the Ca 2+ concentration within 200 nm of the release site reaches a plateau or exceeds the [Ca 2+ ] EC50 for the BK channels rapidly in comparison to the rate of rise of STOCs. These findings suggest a model in which the BK channels lie close to the release site and are exposed to a saturating [Ca 2+ ] with the rise and fall of the STOCs determined by BK channel kinetics. The mechanism of signaling between RyRs and BK channels may provide a model for Ca 2+ action on a variety of molecular targets within cellular microdomains.

Localized, transient elevations in cytosolic Ca 2+ , known as Ca 2+ sparks, caused by Ca 2+ release from sarcoplasmic reticulum, are thought to trigger the opening of large conductance Ca 2+ -activated potassium channels in the plasma membrane resulting in spontaneous transient outward currents (STOCs) in smooth muscle cells. But the precise relationships between Ca 2+ concentration within the sarcoplasmic reticulum and a Ca 2+ spark and that between a Ca 2+ spark and a STOC are not well defined or fully understood. To address these problems, we have employed two approaches using single patch-clamped smooth muscle cells freshly dissociated from toad stomach: a high speed, wide-field imaging system to simultaneously record Ca 2+ sparks and STOCs, and a method to simultaneously measure free global Ca 2+ concentration in the sarcoplasmic reticulum ([Ca 2+ ] SR ) and in the cytosol ([Ca 2+ ] CYTO ) along with STOCs. At a holding potential of 0 mV, cells displayed Ca 2+ sparks and STOCs. Ca 2+ sparks were associated with STOCs; the onset of the sparks coincided with the upstroke of STOCs, and both had approximately the same decay time. The mean increase in [Ca 2+ ] CYTO at the time and location of the spark peak was ∼100 nM above a resting concentration of ∼100 nM. The frequency and amplitude of spontaneous Ca 2+ sparks recorded at −80 mV were unchanged for a period of 10 min after removal of extracellular Ca 2+ (nominally Ca 2+ -free solution with 50 μM EGTA), indicating that Ca 2+ influx is not necessary for Ca 2+ sparks. A brief pulse of caffeine (20 mM) elicited a rapid decrease in [Ca 2+ ] SR in association with a surge in [Ca 2+ ] CYTO and a fusion of STOCs, followed by a fast restoration of [Ca 2+ ] CYTO and a gradual recovery of [Ca 2+ ] SR and STOCs. The return of global [Ca 2+ ] CYTO to rest was an order of magnitude faster than the refilling of the sarcoplasmic reticulum with Ca 2+ . After the global [Ca 2+ ] CYTO was fully restored, recovery of STOC frequency and amplitude were correlated with the level of [Ca 2+ ] SR , even though the time for refilling varied greatly. STOC frequency did not recover substantially until the [Ca 2+ ] SR was restored to 60% or more of resting levels. At [Ca 2+ ] SR levels above 80% of rest, there was a steep relationship between [Ca 2+ ] SR and STOC frequency. In contrast, the relationship between [Ca 2+ ] SR and STOC amplitude was linear. The relationship between [Ca 2+ ] SR and the frequency and amplitude was the same for Ca 2+ sparks as it was for STOCs. The results of this study suggest that the regulation of [Ca 2+ ] SR might provide one mechanism whereby agents could govern Ca 2+ sparks and STOCs. The relationship between Ca 2+ sparks and STOCs also implies a close association between a sarcoplasmic reticulum Ca 2+ release site and the Ca 2+ -activated potassium channels responsible for a STOC.