InteractiveFly: GeneBrief

Na/Ca-exchange protein: Biological Overview | References

Phototransduction in Drosophila is mediated by phospholipase C (PLC) and Ca²⁺-permeable TRP channels, but the function of endoplasmic reticulum (ER) Ca²⁺ stores in this important model for Ca²⁺ signaling remains obscure. A low affinity Ca²⁺ indicator (ER-GCaMP6-150) was expressed in the ER, and its fluorescence was measured both in dissociated ommatidia and in vivo from intact flies of both sexes. Blue excitation light induced a rapid (tau approximately 0.8 s), PLC-dependent decrease in fluorescence, representing depletion of ER Ca²⁺ stores, followed by a slower decay, typically reaching approximately 50% of initial dark-adapted levels, with significant depletion occurring under natural levels of illumination. The ER stores refilled in the dark within 100-200 s. Both rapid and slow store depletion were largely unaffected in InsP₃ receptor mutants, but were much reduced in trp mutants. Strikingly, rapid (but not slow) depletion of ER stores was blocked by removing external Na⁺ and in mutants of the Na⁺/Ca²⁺ exchanger, CalX, which was immuno-localized to ER membranes in addition to its established localization in the plasma membrane. Conversely, overexpression of calx greatly enhanced rapid depletion. These results indicate that rapid store depletion is mediated by Na⁺/Ca²⁺ exchange across the ER membrane induced by Na⁺ influx via the light-sensitive channels. Although too slow to be involved in channel activation, this Na⁺/Ca²⁺ exchange-dependent release explains the decades-old observation of a light-induced rise in cytosolic Ca²⁺ in photoreceptors exposed to Ca²⁺-free solutions (Liu, 2020).

Phototransduction in microvillar photoreceptors is mediated by a G-protein-coupled phospholipase C (PLC), which hydrolyzes phosphatidyl inositol (4,5) bisphosphate (PIP₂) to generate diacylglycerol and inositol (1,4,5) trisphosphate (InsP₃). In Drosophila photoreceptors, activation of PLC leads to opening of two related Ca²⁺-permeable nonselective cation channels: TRP (transient receptor potential) and TRP-like (TRPL) in the microvillar membrane. TRP is the founding member of the TRP ion channel superfamily, so named because the light response in trp mutants is transient, decaying rapidly to baseline during maintained illumination. Because the most familiar product of PLC activity is InsP₃, it was initially thought that activation of the TRP/TRPL channels required release of Ca²⁺ from endoplasmic reticulum (ER) stores via InsP₃ receptors (InsP₃Rs) and that in the absence of Ca²⁺ influx via TRP channels the stores depleted leading to the response decay. However, it was subsequently found that phototransduction was intact in InsP₃R mutants, whereas response decay in trp mutants was associated with severe depletion of PIP₂. This suggested an alternative explanation of the trp decay phenotype, namely failure of Ca²⁺-dependent inhibition of PLC and the consequent runaway consumption of its substrate, PIP₂. Nevertheless, a role for InsP₃ and Ca²⁺ stores in Drosophila phototransduction remains debated. For example, a recent study reported that sensitivity to light was attenuated by RNAi knockdown of InsP₃R , although this study was unable to confirm this using either RNAi or null InsP₃R mutants (Bollepalli, 2017; Liu, 2020).

Relevant to this debate, Ca²⁺ imaging reveals a small, but significant light-induced rise in cytosolic Ca²⁺ in photoreceptors bathed in Ca²⁺-free solutions. Although some have attributed this to InsP₃-induced Ca²⁺ release from the ER, it was found that the rise was unaffected in InsP₃R mutants but was dependent on Na+/Ca²⁺ exchange (Hardie, 1996; Asteriti, 2017; Bollepalli, 2017). This suggested that the Ca²⁺ rise was due to Na+/Ca²⁺exchange following Na+ influx associated with the light response. However, it is difficult to understand how such a Ca²⁺ rise could be achieved by Na+/Ca²⁺ exchange across the plasma membrane when extracellular Ca²⁺ was buffered to low nanomolar levels. The source of the Ca²⁺ rise in Ca²⁺-free bath thus remains unresolved, and to date there have been no measurements of ER store Ca²⁺ levels in Drosophila photoreceptors. To address this, flies were generated expressing a low-affinity GCaMP6 variant in the ER lumen. Using this probe, a rapid light-induced depletion of ER Ca²⁺ was demonstrated and characterized, which, like the cytosolic Ca²⁺ signal in Ca²⁺-free bath, was unaffected by InsP₃R mutations, but dependent on Na+ influx and the CalX Na+/Ca²⁺ exchanger. These results indicate that the exchanger is also expressed on the ER membrane, that the Na+ influx associated with the light-induced current leads to Ca²⁺ extrusion from the ER by Na+/Ca²⁺exchange and that this accounts for the rise in cytosolic Ca²⁺ observed in Ca²⁺-free solutions (Liu, 2020).

This study measured ER Ca²⁺ levels using a low affinity GCaMP6 variant targeted to the photoreceptor ER lumen, where it generated bright fluorescence throughout the ER network. The probe (ER-GCaMP6-150), originally developed and expressed in mammalian neurons, has a 45-fold dynamic range, which was confirmed in situ, and allows measurements of ER luminal [Ca²⁺] with excellent signal-to-noise ratio. Not only could ER Ca²⁺ levels be monitored in dissociated ommatidia, it was also straightforward to make in vivo measurements from the eyes of completely intact flies. The results demonstrate rapid light-induced, PLC-dependent depletion of the ER Ca²⁺ stores, which refilled in the dark over a time course of 100-200 s (Liu, 2020).

Strikingly the results indicate that the rapid light-induced store depletion was mediated by Na+/Ca²⁺ exchange. Drosophila CalX belongs to the NCX family of Na+/Ca²⁺ exchangers, which are generally considered to act only at the plasma membrane. Although Drosophila CalX clearly does function at the plasma membrane, the results now provide compelling evidence that it also operates across the ER membrane. NCX activity has not previously been reported on the ER; however, Na+/Ca²⁺ exchange on internal membranes is not without precedent: for example NCX has been reported on the inner nuclear membrane providing a route for Ca²⁺ transfer between nucleoplasm and the nuclear envelope and hence ultimately the ER network with which it is continuous. In addition a dedicated mitochondrial Na+/Ca²⁺ exchanger (NCLX) plays important roles in uptake and release of mitochondrial Ca²⁺ (Liu, 2020).

The time course of the Na+/Ca²⁺-dependent rapid store depletion in Ca²⁺-free solutions appeared very similar to the rise in cytosolic Ca²⁺ reported from dissociated ommatidia in Ca²⁺-free bath, the source of which has been a subject of debate for over 20 years. It had recently been claimed that this 'Ca²⁺-free rise' was due to InsP₃-mediated release from ER Ca²⁺ stores; however, it was found that it was unaffected in null mutants of the InsP₃R (Asteriti, 2017; Bollepalli, 2017). Instead, it was found that the Ca²⁺-free cytosolic rise was dependent on Na+/Ca²⁺ exchange (Asteriti, 2017), but it was difficult to understand how this could be mediated by a plasma membrane exchanger when extracellular Ca²⁺ was buffered with EGTA to low nanomolar levels. The demonstration of rapid Na+/Ca²⁺-dependent release of Ca²⁺ from ER with a very similar time course now provides an obvious mechanism for this Ca²⁺-free rise and seems finally to have resolved this long-standing enigma. Interestingly the Na+/Ca²⁺-dependent rapid store depletion signal was most pronounced in very young flies around the time of eclosion. Also of note, it was found that trp mutants were very resistant to depletion, both in vivo and in dissociated ommatidia. This argues strongly and directly against the hypothesis that the trp decay phenotype reflects depletion of the ER Ca²⁺ stores (Liu, 2020).

Although up to ~80% rapid store depletion could be observed in newly eclosed adults, even in 1-d-old flies the rapid store depletion signal in vivo was much reduced (to ~10%). However, a much slower depletion was observed in mature adults in vivo, and in dissociated ommatidia after Na+/Ca²⁺ exchange was blocked. The origin of this slow phase depletion remains uncertain: in dissociated ommatidia from young flies this slower depletion was ~50% attenuated, but not blocked in null InsP₃R mutants (itpr), whereas in vivo measurements of the slow depletion phase in adult itpr mutants appeared similar to wild-type. This suggests that although Ca²⁺ release via InsP₃ receptors may contribute to the slow depletion in young flies, some other mechanism(s), such as Ca²⁺ release via ryanodine receptors, is largely responsible (Liu, 2020).

This evidence strongly suggests a novel role for NCX exchangers in mediating Na+/Ca²⁺ exchange across the ER membrane, but its physiological significance is unclear. Although rapid store depletion was routinely observed under experimental conditions used in this study, the Ca²⁺ released into the cytosol from the ER seems unlikely to play a direct role in phototransduction. First, it has a latency of ~100 ms (cf. ~10 ms for the light-induced current), and second it will in any case be swamped by the much more rapid Ca²⁺ influx via the light-sensitive channels. Thus measurements of cytosolic Ca²⁺ in 0 Ca²⁺ bath indicated a rise to only ~200-300 nm. This compares with much faster rises in the high micromolar range due to direct Ca²⁺ influx via the light-sensitive TRP channels. One possible role for an ER Na+/Ca²⁺ exchanger would be that it normally operates as a Ca²⁺ uptake mechanism and only briefly giving Ca²⁺ extrusion (and store depletion) following the extreme, and unnatural conditions of many of the current experiments. This the sudden onset of bright illumination from a dark-adapted state, which results in a massive transient surge of Na+ influx. Rapid Ca²⁺ uptake (store refilling), presumably via re-equilibration of the exchanger as the initial Na+ level subsided during the peak-to-plateau transition, was in fact routinely observed during maintained blue illumination. Furthermore, it is perhaps significant, that despite lacking the rapid depletion phase, the final level of store Ca²⁺ (i.e., after 30 s illumination) in calx^A mutants was if anything lower than that in wild-type backgrounds, although the cytosolic Ca²⁺ levels experienced in calx^A mutants are much higher because of the failure to extrude Ca²⁺ across the plasma membrane (Liu, 2020).

Although store depletion seems unlikely to contribute to activation of the phototransduction cascade, the possibility cannot be excluded that it may play some role in long-term light adaptation. Maintenance of ER Ca²⁺ levels is also important for many other cellular functions including protein folding and maturation in which Ca²⁺ is a cofactor for optimal chaperone activity. With conspicuously high cytosolic Ca²⁺ levels in the presence of light, photoreceptors face unusual homeostatic challenges and Na+/Ca²⁺ exchange across the ER may provide an important additional mechanism. In principle the balance between forward and reverse Na+/Ca²⁺ exchange (i.e., uptake vs release) by an ER Na+/Ca²⁺ exchanger will depend on the Na+ gradient across the ER membrane and whether this is actively regulated. There is no information on ER Na+ levels, although luminal Na+ in the nuclear envelope (which is continuous with the ER) has been reported to be concentrated (84 mm) in nuclei from hepatocytes by Na/K-ATPase expressed on nuclear membranes. The possibility that Na+/Ca²⁺ exchange across the ER might play only a minor physiological role cannot be excluded, but is an unavoidable consequence of the presence of functional CalX protein in ER membranes during protein synthesis and targeting. At least this may account for the enhanced depletion signal measured around the time of eclosion when there may be a rapid final phase of protein synthesis for the developing rhabdomere (Liu, 2020).

These results provide unique insight into ER Ca²⁺ stores in Drosophila photoreceptors. The ER-GCaMP6-150 probe lights up an extensive ER network and indicates a high luminal Ca²⁺ concentration probably in excess of 0.5 mm. The results reveal a rapid, and uniform light-induced depletion of the ER stores mediated by the CalX Na+/Ca²⁺ exchanger expressed on the ER membrane. The resulting extrusion of Ca²⁺ into the cytosol can readily account for the rise in cytosolic Ca²⁺ observed in dissociated ommatidia in Ca²⁺-free solutions), thus resolving this decades old mystery. In addition to the rapid depletion, a much slower depletion was also resolved that appears to be independent of Na+/Ca²⁺ exchange and also largely independent of InsP₃-induced Ca²⁺ release. The physiological significance of the ER Na+/Ca²⁺ exchange activity remains uncertain. It is perhaps more likely that it serves as a low affinity Ca²⁺ uptake mechanism supplementing the SERCA pump, and that rapid depletion is only seen during unnatural abrupt bright stimulation from dark-adapted backgrounds leading to massive Na+ influx and reverse exchange. Ultimately, to resolve the physiological significance of Na+/Ca²⁺ exchange across the ER membrane it will probably be necessary to selectively disrupt Na+/Ca²⁺ exchange on the ER without affecting the exchanger on the plasma membrane, which is known to play very important roles in Ca²⁺ homeostasis in the photoreceptors with direct consequences for channel activation and adaptation (Liu, 2020).

Drosophila phototransduction is mediated by phospholipase C leading to activation of cation channels (TRP and TRPL) in the 30000 microvilli forming the light-absorbing rhabdomere. The channels mediate massive Ca²⁺ influx in response to light, but whether Ca²⁺ is released from internal stores remains controversial. This study generated flies expressing GCaMP6f in their photoreceptors and measured Ca²⁺ signals from dissociated cells, as well as in vivo by imaging rhabdomeres in intact flies. In response to brief flashes, GCaMP6f signals had latencies of 10-25ms, reached 50% Fmax with approximately 1200 effectively absorbed photons and saturated (DeltaF/F0 approximately 10-20) with 10000-30000 photons. In Ca²⁺ free bath, smaller (DeltaF/F0 approximately 4), long latency (~ 200ms) light-induced Ca²⁺ rises were still detectable. These were unaffected in InsP3 receptor mutants, but virtually eliminated when Na⁺ was also omitted from the bath, or in trpl;trp mutants lacking light-sensitive channels. Ca²⁺ free rises were also eliminated in Na⁺/Ca²⁺ exchanger mutants, but greatly accelerated in flies over-expressing the exchanger. These results show that Ca²⁺ free rises are strictly dependent on Na⁺ influx and activity of the exchanger, suggesting they reflect re-equilibration of Na⁺/Ca²⁺ exchange across plasma or intracellular membranes following massive Na⁺ influx. Any tiny Ca²⁺ free rise remaining without exchanger activity was equivalent to <10nM (DeltaF/F0 approximately 0.1), and unlikely to play any role in phototransduction (Asteriti, 2017).

Although Ca²⁺ signals in Drosophila photoreceptors were first studied over 20 years ago using Ca²⁺ indicator dyes, only one, recent study had used genetically encoded Ca²⁺ indicators. That study measured signals from dissociated ommatidia using the Gal4-UAS system, combining UAS-GCaMP6f with GMR-Gal4, which drives expression throughout the retina including all photoreceptor classes as well as accessory cells such as pigment and cone cells. GMR-Gal4 expression also causes significant abnormalities in photoreceptor structure and physiology. In the present study, flies were generated in which GCaMP6f expression was driven directly via the Rh1 (ninaE) promoter ensuring exclusive expression in R1-6 photoreceptors with wild-type morphology and physiology. The excellent signal-to-noise ratio of recordings in ninaE-GCaMP6f flies was distinctly superior to that in GMR-Gal4/UAS-GCaMP6f flies, and in many cases the maximum Δ/F0 ratio approached or exceeded 20 (cf ~3 using GMR-Gal4/UAS-GCaMP6f). This is close to the maximum value (23.5) determined by in situ calibrations or in vitro. Although the blue excitation light used for measuring GCaMP6f fluorescence is a super-saturating stimulus, 2-pulse paradigms allowed sensitive and accurate measurements of intensity and time dependence of signals in response to stimuli in the physiological range. Recordings in vivo from the deep pseudopupil (DPP) of intact flies are simple to perform and can be readily maintained over many hours, making this approach a valuable, and completely non-invasive tool for assessing in vivo photoreceptor performance. Even in the more vulnerable dissociated ommatidia preparation, multiple repeatable measurements could be made for up to at least an hour from the same ommatidium as long as metarhodopsin was reconverted to rhodopsin by long wavelength light after each measurement (Asteriti, 2017).

In vivo (DPP) or in dissociated ommatidia bathed in physiological solutions, the GCaMP6f signal reached 50% Fmax at intensities equivalent to ~1000-2500 effectively absorbed photons. It is believed that the elementary single photon response (quantum bump) is generated by activation of Ca²⁺ permeable channels (TRP and TRPL) within a single microvillus and that the consequent Ca²⁺ rise in the affected microvillus reaches near mM levels. Because such levels inevitably saturate GCaMP6f (Kd 290 nM, saturating at 1-2 μM), to a first approximation the Δ/F0 values under physiological conditions are probably best interpreted as the proportion of microvilli 'flooded' with Ca²⁺. In total, the rhabdomere contains ~30000 microvilli, meaning that 50% Fmax is reached when only ~3-8% of the microvilli have been activated by a photon. This implies that the Ca²⁺ influx into a single microvillus must spread to at least the immediately neighbouring microvilli within the timeframe of the response. In ninaE-calx flies over-expressing the Na+/Ca²⁺ exchanger, or in trp mutants lacking the major Ca²⁺ permeable channel, 50% Fmax was only obtained with flashes containing ~12000-15000 effective photons. This should activate ~50% of the microvilli, suggesting that in these flies Ca²⁺ is largely prevented from spreading to neighbouring microvilli under the same conditions (Asteriti, 2017).

The dark-adapted 'pedestal' level can be used to gain an estimate of the resting Ca²⁺ concentration in dissociated ommatidia (in physiological solutions) assuming in vitro calibration data. With reference to F0 measured in Ca²⁺ free solution in the same ommatidia, the mean dark-adapted value in normal bath was 0.77 ± 0.14 (mean ± S.E.M. n = 11). This would be equivalent to ~80 nM (assuming Kd = 290 nM and Fmax 23.5). This value was significantly lower in ninaE-calx flies over-expressing the exchanger (0.19 ± 0.04 n = 11 equivalent to ~50 nM) and higher in calx¹ mutants (1.94 ± 0.24 n = 14 equivalent to ~120 nM) (Asteriti, 2017).

The recovery of GCaMP6f fluorescence to baseline is likely to be a reasonably accurate reflection of the falling Ca²⁺ levels during response recovery, although the initial decrease (from initial ~mM levels to low μM levels) will still be subject to saturation effects. With relatively dim flashes (up to ~1000 effectively absorbed photons) the GCaMP6f signal in wild-type backgrounds fell to near baseline within ~2-3 s with a half time (t 1/2) of ~1 s. This is slower than the GCaMP6f off-rate (~200 ms), and thus likely to approximate the true time-course of Ca²⁺ recovery. The recovery was significantly accelerated in ninaE-calx flies (~500 ms), and slowed in calx¹ mutants (~2 s increasing to >10 s following brighter flashes), consistent with a dominant role of the Na+/Ca²⁺ exchanger in Ca²⁺ extrusion. Nevertheless, even after bright flashes, given sufficient dark-adaptation time (~30-60 s), resting [Ca²⁺] in calx¹ mutants fell to levels close to those in dark-adapted wild-type photoreceptors, reflecting either residual function of the exchanger in this hypomorphic mutant and/or alternative extrusion mechanism(s) (Asteriti, 2017).

The smaller signals recorded in Ca²⁺ free bath fall within the dynamic range of GCaMP6f and allow estimates of the absolute Ca²⁺ levels reached under these conditions (e.g., Δ/F0 of 6 corresponds to ~200 nM). These signals were used to investigate the long disputed origin of the light-induced rise in cytosolic Ca²⁺ in Ca²⁺ free solutions. Originally, using INDO-1, it was found that this Ca²⁺ free rise was dependent upon extracellular Na+ and suggested that the rise might be due to re-equilibration of Na+/Ca²⁺ exchange in response to the massive light-induced Na+ influx that persists under these conditions. This was challenged by by a study that confirmed the requirement of external Na+ for a significant Ca²⁺ rise in Ca²⁺ free solutions, Na²⁺, but reported that a rise still occurred in Ca²⁺ free bath in the presence of Na+ when the photoreceptors were voltage clamped at the Na+ equilibrium potential to prevent Na+ influx. It was concluded that a Na+ gradient − but not influx − was required, that the Ca²⁺ free rise reflected release from internal stores, and that the requirement of extracellular Na+ reflected involvement of some other Na+ dependent process, such as Na/H transport. But how this might affect release of Ca²⁺ from intracellular stores is far from clear. A more recent study reported that the Ca²⁺ free rise was attenuated following RNAi knockdown of the IP3R. However, this is difficult to reconcile with an earlier study using INDO-1, where the rise was found to be unaffected in null IP3R mosaic eyes and confirmed again in this study using GCaMP6f (Asteriti, 2017).

This study used a variety of approaches to investigate the source of this Ca²⁺ free signal further. It was first confirmed that it was all but abolished in the absence of external Na+, whether substituted for Li+, Cs+, K+ or NMDG+. Importantly, it was found that the rise was also effectively eliminated in trpl;trp double mutants both in vivo and in dissociated ommatidia despite the presence of normal extracellular solutions containing both Na+ and Ca²⁺. Although it might be argued that, for some reason, PLC activity (and hence InsP3 generation) was compromised in trpl;trp mutants, convincing evidence indicates that net PLC activity is in fact greatly enhanced in trpl;trp due to the lack of Ca²⁺ and PKC dependent inhibition of PLC. Thus the rate and intensity dependence of PIP2 hydrolysis, measured using GFP-tagged PIP2 binding probes are greatly enhanced in trpl;trp mutants, as are the PLC-induced photomechanical contractions, and the acidification due to the protons released by the PLC reaction. Overall, therefore these results strongly suggest that Na+ influx is indeed required for the Ca²⁺ free rise. Crucially, the involvement of the Na+/Ca²⁺ exchanger in this rise was confirmed by finding that it was essentially eliminated in an exchanger mutant (calx¹), but greatly accelerated in ninaE-calx photoreceptors over-expressing the exchanger (Asteriti, 2017).

The question remains, how Na+/Ca²⁺ exchanger activity could generate such a sizeable Ca²⁺ signal (~100-200 nM) in cells perfused with EGTA buffered solutions, when free Ca²⁺ in the bath should be reduced to low nM levels. There is no unequivocal answer to this, and assuming the standard equation for the Na+/Ca²⁺ exchange equilibrium it would seem difficult for reverse Na+/Ca²⁺ exchange to raise Ca²⁺ into the range that was observed. However, at least three, not mutually exclusive factors might result in higher cytosolic Cai levels than predicted. Firstly, external Ca²⁺ might be relatively resistant to buffering in the intra-ommatidial space, and specifically the extremely narrow spaces between the microvilli or their bases, where the exchanger is believed to be localised (Wang, 2005). For example, with 500 nM Ca_o remaining, it is predicted that 130 nM Cai would be reached with 70 mM Na_i, 110 mM Na_o and the cell depolarised to 0 mV (values that could realistically be reached with the huge inward Na+ currents flowing under these conditions). Although one might also expect Ca²⁺ influx via the light-sensitive channels at such Ca_o concentrations, experiments buffering external Ca²⁺ at different concentrations with EGTA showed that direct Ca²⁺ influx signals could only be detected once external Ca²⁺ was raised above ~400 nM. Secondly, resting cytosolic Ca²⁺ concentration is determined not only by the exchanger, but also by any other Ca²⁺ fluxes, which might include tonic leakage from intracellular compartments such as endoplasmic reticulum (ER) or mitochondria. Massive Na+ influx would compromise the ability of the exchanger to counter any such fluxes. A third possibility is that, contrary to conventional dogma, the exchanger might also be expressed on intracellular membranes of endoplasmic reticulum or other Ca²⁺ containing compartments and that Na+ influx leads to re-equilibration of Na+/Ca²⁺ exchange across these (Asteriti, 2017).

Whatever the exact mechanism, the results indicate that the Ca²⁺ rise in Ca²⁺ free bath is strictly dependent upon both Na+ influx and the activity level of the Na+/Ca²⁺ exchanger, but unaffected in null IP3R mutants. Its time-course, with no detectable rise for ~200 ms, also appears much too slow to play any role in initiating the light response, which has a latency of ~10 ms and peaks within ~100-200 ms in response to bright illumination even under Ca²⁺ free conditions. The residual GCaMP6f signal remaining in the absence of Na+ influx and/or in the absence of Na+/Ca²⁺ exchanger activity − whether achieved by Na+ substitution, trpl;trp or calx mutants − was also still observed in IP3R mutants and was so small that it is questionable whether it reflects a Ca²⁺ signal. Because of the rapid inhibition of PLC by Ca²⁺ influx under physiological conditions any presumptive PLC-mediated Ca²⁺ release under physiological conditions would be even less. Together with a study in which no phototransduction defects were found in null IP3R mutants, these results suggest that InsP3-induced Ca²⁺ release plays no significant role in Drosophila phototransduction (Asteriti, 2017).

The Drosophila melanogaster Na⁺/Ca²⁺ exchanger CALX controls the Ca²⁺ level in olfactory sensory neurons at rest and after odorant receptor activation

CALX, the Na⁺/Ca²⁺ exchanger in Drosophila, is highly expressed in the outer dendrites of olfactory sensory neurons (OSNs) which are equipped with the odorant receptors (ORs). Insect OR/Orco dimers are nonselective cation channels that pass also calcium which leads to elevated calcium levels after OR activation. CALX exhibits an anomalous regulation in comparison to its homolog in mammals sodium/calcium exchanger, NCX: it is inhibited by increasing intracellular calcium concentration [Ca²⁺]i. Thus, CALX mediates only Ca²⁺ efflux, not influx. The main goal of this study was to elucidate a possible role of this protein in the olfactory response. It was first asked whether already described NCX inhibitors were capable of blocking CALX. By means of calcium imaging techniques in ex-vivo preparations and heterologous expression systems, it was determined ORM-10962 as a potent CALX inhibitor. CALX inhibition did not affect the odor response but it affected the recovery of the calcium level after this response. In addition, CALX controls the calcium level of OSNs at rest (Halty-deLeon, 2018).

Calcium entry following receptor activation in OSNs needs to be balanced to restore resting calcium levels in preparation for new stimuli. Calcium can be taken up by intracellular stores such as mitochondria and endoplasmic reticulum or extruded from the cell by Ca2+ pumps or exchangers. Sensory cascades operating through rapid Ca2+-mediated signaling seem to rely on Na+/Ca2+ exchange mechanisms. For example, Drosophila photoreceptor cells are very sensitive to perturbations in the Na+/Ca2+ exchange activity mediated by CALX (Wang, 2005). Furthermore, NCX was reported to be responsible for returning the concentration of intracellular Ca2+ to its basal level after odor stimulation in frog olfactory neurons. However, the involvement of CALX in the Drosophila odor response was so far unknown. The aim of the present study was to investigate this process (Halty-deLeon, 2018).

These immunohistochemistry results are in good agreement with previous studies where NCX was observed to be expressed in olfactory cilia and dendrites. By measuring the change in calcium within the different neuronal compartments in Xenopus, an increase in calcium was observed first in the dendritic compartments, whereas the increase in the soma and dendritic knob was delayed and less pronounced. Due to the fact that ATPase has a lower transport capacity for calcium than CALX, it seems plausible that CALX in the dendrites would be as a sink for calcium under conditions of elevated intracellular calcium concentration, such as after a receptor activation event, transporting calcium from the dendritic cytosol into the sensillum lymph (Halty-deLeon, 2018).

In mammals, NCX is particularly important in cardiac myocytes. It has a key role in removing Ca2+ after excitation and contraction under normal conditions. However, it is also known to play an important role under pathological situations. In the case of arrhythmias, the reversed mode of NCX could lead to a Ca2+ overload. The development of NCX inhibitors has therefore been targeted as a strategy to study regulatory calcium mechanisms. In contrast to KB-R7943 and SEA 0400, where both compounds preferentially block the reverse mode of NCX, ORM-10962 acts on the two opposite NCX operational modes. Yet, no inhibitor of the Drosophila Na+/Ca2+ exchanger CALX had been described. To understand a possible role of CALX in Drosophila olfactory transduction, it was crucial to selectively block it independently of other elements in the transduction cascade. Given evidence of three NCX inhibitors, these compounds were studied as potential blockers of CALX (Halty-deLeon, 2018).

The first two compounds, KB-R7943 and SEA 0400, are amiloride derivatives. Besides mainly blocking the reverse mode of NCX, amiloride derivatives have been shown to block odorant-evoked activity in lobster olfactory receptor neurons. Specifically, KB-R7943 blocked the olfactory response in lobster and mosquito. In both studies, inhibition of the olfactory response was almost total between 50 μM and 100 μM of KB-R7943. Accordingly, the experiments confirmed that KB-R7943 attenuated the activation of Drosophila ORs significantly. In addition, these results in HEK cells strongly suggest that KB-R7943 acts on the co-receptor Orco directly. This is further supported by the fact that-when testing the other putative inhibitors, namely SEA 0400 or ORM-10962-no attenuation was seen in the Orco response. These data indicate that KB-R7943 blocks the co-receptor Orco, and hence cannot be used to study the role of CALX in olfaction (Halty-deLeon, 2018).

In contrast to KB-R7943, SEA 0400 appear to have no side effect on Orco. The minor, insignificant inhibition could be due to a weak specificity for NCX. This result, together with the fact a state-dependent inhibition of NCX by SEA 0400 has been postulated, made this compound a putative CALX inhibitor. However, although SEA had been reported to be more selective for NCX and being 30 times more potent than KB-R7943, the current results indicate that it is not potently acting on CALX. Even at a high concentration of 10 μM, SEA 0400 failed to inhibit the forward mode of the exchanger. It has been shown that SEA 0400 preferentially inhibits the reverse mode of mainly NCX1 but not the other NCX isoforms (NCX2 and NCX3) at concentrations between 10 nM and 1 μM. Such isoform specificity could be the reason for the lack of effect on CALX. Nonetheless, the calcium binding domain (CBD1) in CALX and NCX share 60% sequence identity. Therefore, the lack of effect on CALX could be attributable to the absence of a reverse mode in CALX or the reduced specificity mentioned before (Halty-deLeon, 2018).

The experiments with the last candidate for inhibition of CALX, ORM-10962, indicated that there was no negative effect in the Orco-response. By contrast, the decay of the Ca2+ signal back to baseline was significantly altered. This indicates that the restitution of the Ca2+ levels in the presence of ORM-10962 was impaired, which is confirmed by experiments in resting conditions. The importance of CALX in restoring calcium levels is also highlighted by results under Na+ free conditions. Under this circumstance, CALX function is impaired and thus the decay of the first and the second response is comparable to the decay in presence of ORM-10926. Elevated calcium levels could be reduced by efflux through the plasma membrane by Na+/Ca2+ exchange and/or the plasma membrane Ca2+ ATPase (PMCA). Previous studies reported that NCX acts as the major Ca2+ extrusion mechanism in frogs and mouse olfactory response. However, it has been suggested that PMCA could also play an important role in restoring calcium basal levels in rat (Sprague–Dawley) and toad (Caudiverbera caudiverbera) olfactory neurons. They argued that because of its lower affinity to calcium and its voltage dependent properties, NCX's efficiency will decline with depolarization of the neurons during an odor response. Their evidence suggests that both Ca2+ transporters contribute to re-establish resting Ca2+ levels in the cilia following olfactory responses. However, the current results suggest that in Drosophila, CALX plays a more important role in maintaining calcium homeostasis. Calmodulin, a Ca2+ binding protein, modulates Drosophila odorant receptor function through Orco and is able to potentiate the action of PMCA in olfactory cilia. Hence, the slower decay observed in the presence of ORM after stimulation of Orco could be due to the action of PMCA. Further experiments to investigate these processes will be important to shed more light into Ca2+ regulatory mechanisms in Drosophila olfactory transduction (Halty-deLeon, 2018).

In conclusion, the current study identified ORM-10962 as potent CALX inhibitor. As in other organisms, where Na+/Ca2+ exchangers are important for the dynamics of the olfactory response, CALX appears to function as the major calcium extrusion mechanisms in Drosophila olfactory neurons both under resting conditions and after enhanced activity (Halty-deLeon, 2018).

Model for the allosteric regulation of the Na+/Ca²⁺ exchanger NCX

The Na⁺ /Ca²⁺ exchanger provides a major Ca²⁺ extrusion pathway in excitable cells and plays a key role in the control of intracellular Ca²⁺ concentrations. In Canis familiaris, Na⁺ /Ca²⁺ exchanger (NCX) activity is regulated by the binding of Ca²⁺ to two cytosolic Ca²⁺ -binding domains, CBD1 and CBD2, such that Ca²⁺ -binding activates the exchanger. Despite its physiological importance, little is known about the exchanger's global structure, and the mechanism of allosteric Ca²⁺ -regulation remains unclear. It was found previously that for NCX in the absence of Ca²⁺ the two domains CBD1 and CBD2 of the cytosolic loop are flexibly linked, while after Ca²⁺ -binding they adopt a rigid arrangement that is slightly tilted. A realistic model for the mechanism of the exchanger's allosteric regulation should not only address this property, but also it should explain the distinctive behavior of Drosophila melanogaster's sodium/calcium exchanger, CALX, for which Ca²⁺ -binding to CBD1 inhibits Ca²⁺ exchange. In this study, NMR spin relaxation and residual dipolar couplings were used to show that Ca²⁺ modulates CBD1 and CBD2 interdomain flexibility of CALX in an analogous way as for NCX. A mechanistic model for the allosteric Ca²⁺ regulation of the Na⁺ /Ca²⁺ exchanger is proposed. In this model, the intracellular loop acts as an entropic spring whose strength is modulated by Ca²⁺ -binding to CBD1 controlling ion transport across the plasma membrane (Abiko, 2016).

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

The Na⁺-Ca²⁺ exchanger (NCX) mediated Ca²⁺ fluxes are essential for handling Ca²⁺ homeostasis in many cell-types. Eukaryotic NCX variants contain regulatory CBD1 and CBD2 domains, whereas in distinct variants the Ca²⁺ binding to Ca3-Ca4 sites of CBD1 results either in sustained activation, inhibition or no effect. CBD2 contains an alternatively spliced segment, which is expressed in a tissue-specific manner although its impact on allosteric regulation remains unclear. Recent studies revealed that the Ca²⁺ binding to Ca3-Ca4 sites results in interdomain tethering of CBDs, which rigidifies CBDs movements with accompanied slow dissociation of "occluded" Ca²⁺. This study investigate the effects of CBD2 variants on Ca²⁺ occlusion in the two-domain construct (CBD12). Mutational studies revealed that both sites (Ca3 and Ca4) contribute to Ca²⁺ occlusion, whereas after dissociation of the first Ca²⁺ ion the second Ca²⁺ ion becomes occluded. This mechanism is common for the brain, kidney and cardiac splice variants of CBD12, although the occluded Ca²⁺ exhibits 20-50-fold difference in off-rates among the tested variants. Therefore, the spliced exons on CBD2 affect the rate-limiting step of the occluded Ca²⁺ dissociation at the primary regulatory sensor to shape dynamic features of allosteric regulation in NCX variants (Giladi, 2012a).

A common Ca²⁺-driven interdomain module governs eukaryotic NCX regulation

Na⁺/Ca²⁺ exchanger (NCX) proteins mediate Ca²⁺-fluxes across the cell membrane to maintain Ca²⁺ homeostasis in many cell types. Eukaryotic NCX contains Ca²⁺-binding regulatory domains, CBD1 and CBD2. Ca²⁺ binding to a primary sensor (Ca3-Ca4 sites) on CBD1 activates mammalian NCXs, whereas CALX, a Drosophila NCX ortholog, displays an inhibitory response to regulatory Ca²⁺. To further elucidate the underlying regulatory mechanisms, this study determined the 2.7 Å crystal structure of mammalian CBD12-E454K, a two-domain construct that retains wild-type properties. In conjunction with stopped-flow kinetics and SAXS (small-angle X-ray scattering) analyses of CBD12 mutants, this study shows that Ca²⁺ binding to Ca3-Ca4 sites tethers the domains via a network of interdomain salt-bridges. This Ca²⁺-driven interdomain switch controls slow dissociation of 'occluded' Ca²⁺ from the primary sensor and thus dictates Ca²⁺ sensing dynamics. In the Ca²⁺-bound conformation, the interdomain angle of CBD12 is very similar in NCX and CALX, meaning that the interdomain distances cannot account for regulatory diversity in NCX and CALX. Since the two-domain interface is nearly identical among eukaryotic NCXs, including CALX, it is suggested that the Ca²⁺-driven interdomain switch described in this study represents a general mechanism for initial conduction of regulatory signals in NCX variants (Giladi, 2012b).

Crystal structures of progressive Ca²⁺ binding states of the Ca²⁺ sensor Ca²⁺ binding domain 1 (CBD1) from the CALX Na+/Ca²⁺ exchanger reveal incremental conformational transitions

Na⁺/Ca²⁺ exchangers (NCX) constitute a major Ca²⁺ export system that facilitates the re-establishment of cytosolic Ca²⁺ levels in many tissues. Ca²⁺ interactions at its Ca²⁺ binding domains (CBD1 and CBD2) are essential for the allosteric regulation of Na⁺/Ca²⁺ exchange activity. The structure of the Ca²⁺-bound form of CBD1, the primary Ca²⁺ sensor from canine NCX1, but not the Ca²⁺-free form, has been reported, although the molecular mechanism of Ca²⁺ regulation remains unclear. This study reporta crystal structures for three distinct Ca²⁺ binding states of CBD1 from CALX, a Na⁺/Ca²⁺ exchanger found in Drosophila sensory neurons. The fully Ca²⁺-bound CALX-CBD1 structure shows that four Ca²⁺ atoms bind at identical Ca²⁺ binding sites as those found in NCX1 and that the partial Ca²⁺ occupancy and apoform structures exhibit progressive conformational transitions, indicating incremental regulation of CALX exchange by successive Ca²⁺ binding at CBD1. The structures also predict that the primary Ca²⁺ pair plays the main role in triggering functional conformational changes. Confirming this prediction, mutagenesis of Glu(455), which coordinates the primary Ca²⁺ pair, produces dramatic reductions of the regulatory Ca²⁺ affinity for exchange current, whereas mutagenesis of Glu(520), which coordinates the secondary Ca²⁺ pair, has much smaller effects. Furthermore, the structures indicate that Ca²⁺ binding only enhances the stability of the Ca²⁺ binding site of CBD1 near the hinge region while the overall structure of CBD1 remains largely unaffected, implying that the Ca²⁺ regulatory function of CBD1, and possibly that for the entire NCX family, is mediated through domain interactions between CBD1 and the adjacent CBD2 at this hinge (Wu, 2010).

Light activation, adaptation, and cell survival functions of the Na+/Ca2+ exchanger CalX

In sensory neurons, Ca(2+) entry is crucial for both activation and subsequent attenuation of signaling. Influx of Ca(2+) is counterbalanced by Ca(2+) extrusion, and Na⁺/Ca(2+) exchange is the primary mode for rapid Ca(2+) removal during and after sensory stimulation. However, the consequences on sensory signaling resulting from mutations in Na⁺/Ca(2+) exchangers have not been described. This paper reports that mutations in the Drosophila Na⁺/Ca(2+) exchanger calx have a profound effect on activity-dependent survival of photoreceptor cells. Loss of CalX activity resulted in a transient response to light, a dramatic decrease in signal amplification, and unusually rapid adaptation. Conversely, overexpression of CalX had reciprocal effects and greatly suppressed the retinal degeneration caused by constitutive activity of the TRP channel. These results illustrate the critical role of Ca(2+) for proper signaling and provide genetic evidence that Ca(2+) overload is responsible for a form of retinal degeneration resulting from defects in the TRP channel (Wang, 2005).

Structure-function analysis of calx¹.1, a Na+-Ca²⁺ exchanger from Drosophila. Mutagenesis of ionic regulatory sites

Cytoplasmic Na⁺ and Ca²⁺ regulate the activity of Na+-Ca²⁺ exchange proteins, in addition to serving as the transported ions, and protein regions involved in these processes have been identified for the canine cardiac Na+-Ca²⁺ exchanger, NCX1.1. Although protein regions associated with Na+_i- and Ca²⁺_i-dependent regulation are highly conserved among cloned Na+-Ca²⁺ exchangers, it is unknown whether or not the structure-function relationships characteristic of NCX1.1 apply to any other exchangers. Therefore, structure-function relationships were studied in a Na+-Ca²⁺ exchanger from Drosophila, CALX1.1, which is unique among characterized members of this family of proteins in that microM levels of Ca²⁺i_i- inhibit exchange current. Wild-type and mutant CALX1.1 exchangers were expressed in Xenopus oocytes and characterized electrophysiologically using the giant excised patch technique. Mutations within the putative regulatory Ca²⁺_i binding site of CALX1.1, like corresponding alterations in NCX1.1, led to reduced ability (i.e. D516V and D550I) or inability (i.e. G555P) of Ca²⁺_i to inhibit Na+-Ca²⁺ exchange activity. Similarly, mutations within the putative XIP region of CALX1.1, as in NCX1.1, led to two distinct phenotypes: acceleration (i.e. K306Q) and elimination (i.e. Delta310-313) of Na+_i-dependent inactivation. These results indicate that the respective regulatory roles of the Ca²⁺_i binding site and XIP region are conserved between CALX1.1 and NCX1.1, despite opposite responses to Ca²⁺_i. These findings were extended using chimeric constructs of CALX1.1 and NCX1.1 to determine whether or not functional interconversion of Ca²⁺i_i- regulatory phenotypes was feasible. With one chimera (i.e., CALX:NCX:CALX), substitution of a 193-amino acid segment, from the large intracellular loop of NCX1.1, for the corresponding 177-amino acid segment of CALX1.1 led to an exchanger that was stimulated by Ca²⁺_i. This result indicates that the regulatory Ca²⁺_i binding site of NCX1.1 retains function in a CALX1.1 parent transporter and that the substituted segment contains some of the amino acid sequence(s) required for transduction of the Ca²⁺i_i- binding signal (Dyck, 1998).

INDO-1 measurements of absolute resting and light-induced Ca²⁺ concentration in Drosophila photoreceptors

Absolute Ca²⁺ levels in dissociated Drosophila photoreceptors were measured using the ratiometric indicator dye INDO-1 loaded via patch pipettes, which simultaneously recorded whole-cell currents. In wild-type photoreceptors, the ultraviolet (UV) excitation light used to measure fluorescence elicited a massive Ca²⁺ influx that saturated the dye (>10 microM Ca²⁺), but lagged the electrical response by 2.8 msec. Resting Ca²⁺ levels in the dark, measured during the latent period before the response, averaged 160 nM in normal Ringer's (1.5 mM Ca²⁺). Ca²⁺ increases in response to weak illumination were estimated (1) by using a weak adapting stimulus before the UV excitation light and measuring Ca²⁺ during the latent period; and (2) by using ninaE mutants with greatly reduced rhodopsin levels. Ca²⁺ rose linearly as a function of the time integral of the light-sensitive current with a slope of 2.7 nM/pC. In the transient receptor potential (trp) mutant, which lacks a putative light-sensitive channel subunit, the slope was only 1.1 nM/pC, indicating a 2.5-fold reduction in the fractional Ca²⁺ current. From these data, it can also be estimated that >99% of the Ca²⁺ influx is effectively buffered by the cell. In Ca²⁺-free Ringer's, resting cytosolic Ca²⁺ was reduced (to 30-70 nM), but contrary to previous reports, significant light-induced increases (approximately 250 nM) could be elicited. This rise was reduced to <20 nM when extracellular Na+ was replaced with N-methyl-D-glucamine, suggesting that it could be attributed to Na+ influx altering the Na/Ca exchanger equilibrium. It is concluded that any light-induced release from internal stores amounts to <20 nM (Hardie, 1996).

Na⁺/Ca²⁺ exchanger mediates cold Ca²⁺ signaling conserved for temperature-compensated circadian rhythms

Historically, before the discovery of the clock genes, a feedback system involving ions and ion regulators in plasma membranes was proposed as the oscillation mechanism of the circadian clock. This 'membrane model' is based on the observation that the circadian rhythms are notably affected by manipulating ion concentrations or ion regulator activities in various eukaryotes. To date, several ions, especially Ca²⁺, have been shown to play an essential role for oscillation of the TTFLs in mammals, insects (Harrisingh, 2007), and plants. In mice and Drosophila, intracellular Ca²⁺ levels were shown to exhibit robust circadian oscillations (Guo, 2016), which elicit rhythmic activation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) (Kon, 2014). CaMKII phosphorylates CLOCK to activate CLOCK-BMAL1 heterodimer, a key transcriptional activator in the animal TTFLs. The upstream regulator of the Ca²⁺-dependent phosphorylation signaling has been a missing link between the TTFL and the membrane model (Kon, 2021).

Circadian TTFLs are an elaborate system that drives a wide range of overt rhythms with various phase angles and amplitudes. The oscillation speed of the TTFLs is temperature compensated, although many of the biochemical reactions in TTFLs are slowed down by decreasing temperature. This study demonstrates that the temperature compensation of the TTFL in mammalian cells was compromised when Ca²⁺-dependent phosphorylation signaling was inhibited. An important role was found of NCX-CaMKII activity as the state variable of the circadian oscillator. This present study and a series of preceding works demonstrate that the Ca²⁺ oscillator plays essential roles in the circadian oscillation mechanism. Functional studies clearly demonstrated essential roles of NCX-dependent Ca²⁺ signaling in the three important properties of the circadian clock, i.e., cell-autonomous oscillation, temperature compensation, and entrainment. The circadian Ca²⁺ oscillation is observed in mice lacking Bmal1 or Cry1/Cry2, implicating that the Ca²⁺ oscillator is an upstream regulator of the TTFL in mammals (Kon, 2021).

The effects of NCX2 and NCX3 deficiencies on the regulation of mouse behavioral rhythms (Fig. 7, A to C) suggest involvement of Na+/Ca²⁺ exchanging activity in the Ca²⁺ dynamics of the SCN. Previous studies showed that L-type Ca²⁺ channel (LTCC) and voltage-gated Na+ channel (VGSC) are required for high-amplitude Ca²⁺ rhythms in the SCN. Because NCX activities are regulated by local concentrations of Na+/Ca²⁺ and the membrane potential, cooperative actions of LTCC, VGSC, and NCX seem to play important roles in generation mechanism of the robust Ca²⁺ oscillations in the SCN (Kon, 2021).

It should be emphasized that the role of Ca²⁺/calmodulin-dependent protein kinases is conserved among clockworks in insects, fungi, and plants, suggesting that the Ca²⁺ oscillator might be a core timekeeping mechanism in their common ancestor (see Involvement of ancient Ca²⁺ signaling for temperature-compensated circadian rhythms). After divergence of each lineage, a subset of clock genes should have independently evolved in association with the Ca²⁺ oscillator. It is noteworthy that NCX is also required for temperature compensation of PTO-based cyanobacterial clock. Because intracellular Ca²⁺ in cyanobacteria is elevated in response to temperature decrease, YrbG-mediated Ca²⁺ signaling may regulate the PTO in vivo. Conservation of NCX among eukaryotes, eubacteria, and archaea suggests that NCX-dependent temperature signaling is essential for adaptation of a wide variety of organisms to environment. Further studies on NCX-regulated Ca²⁺ flux will provide evolutionary insights into the origin of the circadian clocks (Kon, 2021).

Search PubMed for articles about Drosophila Calx

Abiko, L. A., Vitale, P. M., Favaro, D. C., Hauk, P., Li, D. W., Yuan, J., Bruschweiler-Li, L., Salinas, R. K. and Bruschweiler, R. (2016). Model for the allosteric regulation of the Na+/Ca²⁺ exchanger NCX. Proteins 84(5): 580-590. PubMed ID: 26850381

Asteriti, S., Liu, C. H. and Hardie, R. C. (2017). Calcium signalling in Drosophila photoreceptors measured with GCaMP6f. Cell Calcium 65: 40-51. PubMed ID: 28238353

Bollepalli, M. K., Kuipers, M. E., Liu, C. H., Asteriti, S. and Hardie, R. C. (2017). Phototransduction in Drosophila is compromised by Gal4 expression but not by InsP3 receptor knockdown or mutation. eNeuro 4(3). PubMed ID: 28660247

Dyck, C., Maxwell, K., Buchko, J., Trac, M., Omelchenko, A., Hnatowich, M. and Hryshko, L. V. (1998). Structure-function analysis of calx¹.1, a Na+-Ca²⁺ exchanger from Drosophila. Mutagenesis of ionic regulatory sites. J Biol Chem 273(21): 12981-12987. PubMed ID: 9582332

Giladi, M., Bohbot, H., Buki, T., Schulze, D. H., Hiller, R. and Khananshvili, D. (2012a). Dynamic features of allosteric Ca²⁺ sensor in tissue-specific NCX variants. Cell Calcium 51(6): 478-485. PubMed ID: 22571864

Giladi, M., Sasson, Y., Fang, X., Hiller, R., Buki, T., Wang, Y. X., Hirsch, J. A. and Khananshvili, D. (2012b). A common Ca²⁺-driven interdomain module governs eukaryotic NCX regulation. PLoS One 7(6): e39985. PubMed ID: 22768191

Guo, F., Yu, J., Jung, H. J., Abruzzi, K. C., Luo, W., Griffith, L. C. and Rosbash, M. (2016). Circadian neuron feedback controls the Drosophila sleep--activity profile. Nature 536(7616): 292-297. PubMed ID: 27479324

Halty-deLeon, L., Hansson, B. S. and Wicher, D. (2018). The Drosophila melanogaster Na⁺/Ca²⁺ exchanger CALX controls the Ca²⁺ level in olfactory sensory neurons at rest and after odorant receptor activation. Front Cell Neurosci 12: 186. PubMed ID: 30018538

Hardie, R. C. (1996). INDO-1 measurements of absolute resting and light-induced Ca²⁺ concentration in Drosophila photoreceptors. J Neurosci 16(9): 2924-2933. PubMed ID: 8622123

Harrisingh, M. C., Wu, Y., Lnenicka, G. A. and Nitabach, M. N. (2007). Intracellular Ca²⁺ regulates free-running circadian clock oscillation in vivo. J Neurosci 27(46): 12489-12499. PubMed ID: 18003827

Kon, N., Yoshikawa, T., Honma, S., Yamagata, Y., Yoshitane, H., Shimizu, K., Sugiyama, Y., Hara, C., Kameshita, I., Honma, K. and Fukada, Y. (2014). CaMKII is essential for the cellular clock and coupling between morning and evening behavioral rhythms. Genes Dev 28(10): 1101-1110. PubMed ID: 24831701

Kon, N., Wang, H. T., Kato, Y. S., Uemoto, K., Kawamoto, N., Kawasaki, K., Enoki, R., Kurosawa, G., Nakane, T., Sugiyama, Y., Tagashira, H., Endo, M., Iwasaki, H., Iwamoto, T., Kume, K. and Fukada, Y. (2021). Na⁺/Ca²⁺ exchanger mediates cold Ca²⁺ signaling conserved for temperature-compensated circadian rhythms. Sci Adv 7(18). PubMed ID: 33931447

Liu, C. H., Chen, Z., Oliva, M. K., Luo, J., Collier, S., Montell, C. and Hardie, R. C. (2020). Rapid release of Ca²⁺ from endoplasmic reticulum mediated by Na⁺/Ca²⁺ exchange. J Neurosci 40(16): 3152-3164. PubMed ID: 32156830

Wang, T., Xu, H., Oberwinkler, J., Gu, Y., Hardie, R. C. and Montell, C. (2005). Light activation, adaptation, and cell survival functions of the Na+/Ca2+ exchanger CalX. Neuron 45(3): 367-378. PubMed ID: 15694324

Wu, M., Le, H. D., Wang, M., Yurkov, V., Omelchenko, A., Hnatowich, M., Nix, J., Hryshko, L. V. and Zheng, L. (2010). Crystal structures of progressive Ca²⁺ binding states of the Ca²⁺ sensor Ca²⁺ binding domain 1 (CBD1) from the CALX Na+/Ca²⁺ exchanger reveal incremental conformational transitions. J Biol Chem 285(4): 2554-2561. PubMed ID: 19815561

date revised: 14 October 2021

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