The mouse is proving to be an effective model for characterizing the roles of individual components because its relative simplicity can be reduced further by genetic engineering. There are two types of GC activating proteins (GCAPs) and two types of GCs in mouse rods, whereas cones express one type of GCAP and one type of GC. Mutant mouse rods and cones bereft of both GCAPs have large, long lasting photon responses. Thus, GCAPs normally mediate negative feedback tied to the light-induced decline in intracellular Ca2+ that accelerates GC activity to curtail the growth and duration of the photon response. Rods from other mutant mice that express a single GCAP type reveal how the two GCAPs normally work together as a team. Because of its lower Ca2+ affinity, GCAP1 is the first responder that senses the initial decrease in Ca2+ following photon absorption and acts to limit response amplitude. GCAP2, with a higher Ca2+ affinity, is recruited later during the course of the photon response as Ca2+ levels continue to decline further. The main role of GCAP2 is to provide for a timely response recovery and it is particularly important after exposure to very bright light. The multiplicity of GC isozymes and GCAP homologs in the retinas of other vertebrates confers greater flexibility in shaping the photon responses in order to tune visual sensitivity, dynamic range and frequency response.In vertebrate rods and cones, photon capture by rhodopsin leads to the destruction of cyclic GMP (cGMP) and the subsequent closure of cyclic nucleotide gated ion channels in the outer segment plasma membrane. Replenishment of cGMP and reopening of the channels limit the growth of the photon response and are requisite for its recovery. In different vertebrate retinas, there may be as many as four types of membrane guanylyl cyclases (GCs) for cGMP synthesis. Ten neuronal Ca When rods and cones receive light, photon capture by visual pigment within the outer segment is amplified by the subsequent activation of many transducin (G-protein) molecules, each of which stimulate phosphodiesterase (PDE) enzymatic activity. Cyclic GMP (cGMP) hydrolysis leads to closure of cyclic nucleotide-gated (CNG) channels thereby blocking the dark current. The ensuing hyperpolarization of membrane potential spreads through the photoreceptor to reduce neurotransmitter release at the synaptic terminal. To recover quickly from stimulation by light, cGMP, the second messenger that links photon capture to CNG channel opening, must be restored . Cation channels in the open state permit an influx of Na2+-binding, EF-hand bearing, GC activating protein (GCAP) subunits  catalyze the synthesis of cGMP. As opposed to other membrane GCs, those of photoreceptors do not respond to extracellular ligands, but instead are subject to regulation by Ca2+ sensing GCAP is likely to have already been in place in very ancient ciliary photoreceptors . The existence of multiple isoforms of GC and GCAP provided a substrate for natural selection to optimize the photon response for particular visual ecologies. Where an overly complex system of GCs and GCAPs was not needed, components were eventually lost. For example in mouse, three frame deletions degraded the GUCA1C gene for GCAP3 into a pseudogene , that inhibits GC at high Ca2+ but does not stimulate it at low Ca2+ (2+ (reviewed by Table 1). GCs and GCAPs have responsibilities in the inner segment . To understand how the Ca2+ feedback onto cGMP synthesis influences the cone photoresponse, it is useful to simplify the system further by genetic engineering. The tacit assumption is that the genetic manipulation “cleanly” removes a targeted protein(s) in an otherwise undisturbed system. Caution is necessary because full verification is impossible and there may be unpredictable consequences. As an illustration, knockout of GC1 in mice interferes with the expression of many cone phototransduction proteins, including transducin, PDE, GCAP1, and arrestin, and the cones eventually degenerate. Surprisingly, small cone ERG responses to light were detected at 1 month of age  may be a more specific experimental perturbation and cones retain good health. Figure 2A shows that the single photon response of a GCAPs-/- cone rises normally, but continues to do so for twice as long to finally reach a peak after 220 ms that is 2.7-fold higher than normal  = 6.2 times lower. There is such a shift for very weak responses, however, GCAPs-/- cones are about 20-fold more sensitive than wild type (WT) cones at the half maximal response level . As with mouse cones, a genetic approach is useful for trying to understand how the system works, but with rods, it is necessary to knock out components individually and in combinations to isolate the physiological function of each component . Negative feedback provided by GCAPs is a key factor. The feedback during the single photon response is somewhat more powerful in mouse rods than in mouse cones . In the absence of both GCAPs the rod response rises for three times longer than normal to reach an amplitude that is four times larger . When GCAP2 alone is missing (GCAP2-/-), the size of the single photon response in rods is normal and the only change is a slightly slower recovery (-/-) produces a single photon response roughly twice the normal size .It might seem that enlarged single photon responses should improve the ability of GCAPsrk noise , which wrk noise  and contrk noise . GCAPs crk noise . GCAP1-/2+ is driven down to lower and lower levels, one might expect GCAPs to extend the operating range of rods by keeping them out of saturation, just as GCAP1 does in cones . But that does not happen; the relation between fractional current and light intensity does not fall more gradually in WT rods compared to GCAPs-/- rods . One explanation might be that something happens at higher light intensities, such as the summation of long lasting, aberrant photon responses (see below), which undermines the efforts of GCAPs in their attempt to help rods evade saturation. Interestingly, knockout of GCAP1-/- does extend the operating range of rods, perhaps compensatory overexpression of GCAP2 boosts the maximal GC activity and the stimulation of GC activity is concentrated at higher intensities .Mammalian rods possess an odd characteristic: about once per several hundred events, rods generate an aberrant single photon response that climbs to an amplitude nearly twofold larger than normal and then remains at that amplitude for an average of 3–6 s, before recovering. In individual trials, the duration is unpredictable and some aberrant responses last for tens of seconds . Apparen2+ sensitivities. GCAP1 has a lower affinity for Ca2+ than GCAP2 . The K1/2 for Ca2+ of GCAP1 in the mouse rod is 130–140 nM, while that of GCAP2 is 50–60 nM (2+ inside a mouse rod range from 250 nM in darkness to 23 nM in saturating light (Table 3). So during the initial fall in Ca2+ incurred during the photon response, many more GCAP1 molecules release their Ca2+ than GCAP2. The majority of GCAP2 molecules only release their bound Ca2+ when intracellular Ca2+ plummets, during the saturating response to very bright light. A model illustrating the actions of GCAP1 and GCAP2 is shown in Figure 4B. A similar “relay” model was proposed earlier . Although it could have been the case that GCAP2 responds later because it has a slower release of Ca2+ than GCAP1 after a sudden drop in intracellular Ca2+, it will be argued below that both GCAPs are rapid Ca2+ sensors. Figure 5B shows the rising phases of the photon responses of mouse rods lacking GCAP1, GCAP2, or both. The GCAPs-/- response is the first to diverge at ~80 ms after the flash, while the GCAP2-/- response is the last to diverge. Based on this pattern, it may be inferred that GCAP1 senses the fall in Ca2+ and begins to stimulate GC by 80 ms post-flash. GCAP1-/- rods are highly variable in their response waveforms apparently because they upregulate GCAP2 expression and the degree of compensation differs from rod to rod. Rods with the least compensation show response divergence from the WT response nearly as early as the GCAPs-/- response. Accordingly, GCAP2 must respond rapidly to the initial fall in Ca2+. At normal expression levels, the paltry number of GCAP2 molecules responding to the initial fall in Ca2+ is unable to make its presence felt. But the mutant rods show that given enough GCAP2, its feedback onto GC does restrain the early rising phase of the response.The timing of CaAs one might expect, knockout of both GCs in rods eliminates the dark current, sabotaging their capacity for phototransduction . DeletinIn summary, the functional significance to phototransduction of expressing two types of GCs in rods is not yet clear. For GCAPs, there is a division of labor. GCAP1 helps to achieve the proper balance of keeping noise to a minimum while restricting photon response size to optimize the range of flash strengths and light intensities over which the rod operates. Contrary to the situation in cones, the shutoffs of photoexcited visual pigment and activated transducin are slow in rods to allow the photon response to grow. The slow shutoffs pose a problem with bright flashes because the time that the rod stays in saturation gets prolonged. GCAP2 addresses that problem by boosting GC activity to bring the rod out of saturation sooner.2+ feedback onto cGMP synthesis reduces the size of the single photon response and quickens its kinetics in mouse rods and cones. The extent to which that occurs is dependent upon the selective expressions of GC(s) and GCAP(s). Therefore, variation that exists in the sensitivity and flash response kinetics of rods and cones from other vertebrates is likely to arise at least in part from differences in their cGMP synthetic machinery. This section will present a few examples of the variation between species and lay out three mechanisms by which cGMP production could be altered to tune the photon response: by augmenting the amounts of GC and GCAP expressed, by a change in intracellular Ca2+ dynamics or by a switch in the types of GCs or GCAPs expressed.While it is generally true that the photon response of rods is larger and slower than that of cones, the response can vary across rods and across cones of different species. The preceding sections described how CaTable 2). In general, the single photon response of cones is far smaller and peaks much sooner than the response of rods in the same animal. Across species, ground squirrel cones generate smaller, faster photon responses than mouse cones . At present, primate cones are something of an enigma. Their single photon response kinetics are faster than those of mouse yet the single photon response is larger and relative sensitivity is greater. In initial studies on isolated primate retina, cone flash responses exhibited a prominent and reproducible undershoot to the recovery  and kinetics inferred for cones in the intact human eye lack the undershoot  and the basal rate of cGMP synthesis in cones is an order of magnitude higher. The differential appears to be lower in salamander to account for a basal rate that is only ~3-fold higher in their cones compared to their rods . In salamander, a faster rate of Ca2+ extrusion in L cones than in S cones may contribute to faster response kinetics of L cones  expressed will need to be based in part on Ca2+ affinity.Dissimilarities in the operation of the same cGMP synthetic machinery may exist across photoreceptors because of how they handle Cafor Ca2+ . It is ns faster . As a re L cones . In bothd, 23 nM . For fee1/2 for Ca2+, cooperativity for Ca2+ binding, maximal and basal rates of cGMP synthetic activity. Further flexibility stems from having maximal stimulation of GC depend on the pairing of a GC with specific GCAP isoforms. The unstimulated rate of cGMP synthesis by mouse GC1 is nearly 10-fold less than that of GC2. But stimulation of GC1 activity by GCAP1 and GCAP2 is 28-fold and 13-fold, respectively, whereas for GC2 activity, it is only sixfold and fivefold, respectively , but stimulation of zebrafish GC3 by GCAPs 3–5 and 7 is more uniform at ~2-fold   , similarof (4:1) . It shouof (4:1) .1/2 for Ca2+ , but intracellular [Ca2+] in salamander cones falls to 5 nM upon exposure to bright light. So another GCAP with lower K1/2 for Ca2+ is needed if cGMP synthetic rate is to increase over the entire Ca2+ range. K1/2 values for zebrafish GCAPs 1–3 cluster at about 30 nM . It remains to be seen whether lower values are possible. Certainly GCAPs, e.g., zGCAPs 4,5, can have K1/2 values higher than those of mouse GCAPs , but while such GCAPs may be useful for photoreceptors of lizards and salamanders (Table 3), they cause degenerative retinal disease in mammals (reviewed in 2+-dependent activation of GC by GCAPs is cooperative (reviewed in 2+ (see above). Given the differences in the composition of their cGMP machineries (Table 1), it may be predicted that the physiological properties of zebrafish cones will vary with spectral type.GCAPs also vary in their Kfor Ca2+ , a prope50–60 nM , mouse Gin vitro (in vivo is not governed strictly by binding affinity observed in vitro. Thus while the Ca2+ sensitivity of the regulation is determined by the type of GCAP rather than isozyme of the cyclase (2+ sensitivity.The selectivity rules for forming GC complexes are not completely understood. The apparent affinity of mouse GC2 is slightly higher for GCAP2 than for GCAP1 in vitro , yet in in vitro . Apparen cyclase , the typ1/2 for Ca2+ that matches the value obtained by their mixture of GCAPs 1 and 2. This path was not chosen, perhaps because the flexibility of a mixture of components allowed for adjustments to be made on a shorter evolutionary time scale to accommodate other changes in the phototransduction cascade, as rods evolved as photoreceptors (It may be argued that Nature could have given mouse rods a single GCAP having a Keceptors .2+ (2+], which allows them to maintain their cyclase activating conformation in the light and affects the [Ca2+] at which they de-activate GC (2+] does not change in rods or in cones upon exposure to light and remains in the vicinity of 1 mM (On a much shorter time scale, relevant to the lifetime of an individual, the complexity of a mixture of components leaves open many options for plasticity. The absence of GCAP5 transcripts in zebrafish until 15 dpf , even th2+ . It is t2+  and PDEα2+ . In rat,ivate GC . However of 1 mM . So whil2+ affinity and GCs with different activities and preferences for the type of GCAP bound. By expressing selected components and regulating their levels of expression, vertebrates “design” the cGMP synthetic machinery in their photoreceptors in order to adjust their physiological responses to light stimuli. The possibility exists for plasticity during development or on a shorter time scale to meet changing visual demands.Evolution has favored the occurrence of multiple isoforms of GCAPs that vary in their CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The dynamics of [Ca2+]cyt elevation varied, reflecting the difference in the developmental stage of the eggs used. [Ca2+]cyt oscillation was exclusively observed in fertile, mature egg cells fused with the sperm cell. To determine how [Ca2+]cyt oscillation in mature egg cells is generated, egg cells were incubated in thapsigargin, which proved to be a specific inhibitor of the endoplasmic reticulum (ER) Ca2+-ATPase in wheat egg cells. In unfertilized egg cells, the addition of thapsigargin caused an abrupt transient increase in [Ca2+]cyt in the absence of extracellular Ca2+, suggesting that an influx pathway for Ca2+ is activated by thapsigargin. The [Ca2+]cyt oscillation seemed to require the filling of an intracellular calcium store for the onset of which, calcium influx through the plasma membrane appeared essential. This was demonstrated by omitting extracellular calcium from (or adding GdCl3 to) the fusion medium, which prevented [Ca2+]cyt oscillation in mature egg cells fused with the sperm. Combined, these data permit the hypothesis that the first sperm-induced transient increase in [Ca2+]cyt depletes an intracellular Ca2+ store, triggering an increase in plasma membrane Ca2+ permeability, and this enhanced Ca2+ influx results in [Ca2+]cyt oscillation.During  Fi et al.  and Fuji in vitro fertilization of gamete pairs, offer the possibility of studying the first events associated with gamete fusion , which was found to be induced by a fertilization-triggered increase in cytosolic Ca2+ levels cyt, was not possible, due to the failure of injecting the delicate egg cells, hence leaving important questions, such as the origin and the dynamics of the observed calcium signal, unanswered [Capitalizing on the Cae et al.  observedanswered .2+]cyt was performed in order to investigate the characteristics of the calcium signal during fertilization in the wheat female gamete. Employing a microinjection technique elaborated by Pónya et al. cyt changes observed in the fertilized wheat egg, which permits quantitative comparisons to be made between the characteristics of the calcium signal ensuing upon fertilization in the animal egg and in the female gamete of wheat, a higher land plant. In view of the structural changes that the ER goes through during the in situ development of the wheat egg [ et al. [2+]cyt in wheat female gametes isolated at different maturational stages and fertilized in vitro were followed. Egg protoplasts were isolated at different developmental stages defined according to the time  elapsed from emasculation, carried out at a certain developmental window of the male gametophyte. Three maturational windows were defined for the female gametes to be isolated for the experiments: (1) three DAE, at which isolated eggs were considered immature; (2) six DAE, yielding mature, receptive eggs; and (3) 11 DAE, the isolation of overmature female gametes.In the present study, dual-ratio imaging of cytosolic calcium cyt elevation was also seen in egg cells incubated and fused in Ca2+ free medium , the ER was assumed to be the origin of the repetitive [Ca2+]cyt transients observed in mature, fertilized wheat  egg cells. To test this hypothesis, a pharmacological approach was employed to examine the origin of the fertilization-associated [Ca2+]cyt change in the egg cytoplasm. Wheat female gametes were treated with thapsigargin, an inhibitor of Ca2+-pumps cyt changes in the cytoplasm of the in vitro fertilized female gamete.The possibility of the injection of fura-2 dextran into egg cells isolated from wheat allowed for the dual-excitation-based ratio approach to be used to measure [Ca2+]cyt response of immature egg cells isolated three days after emasculation (DAE)  to sperm incorporation was investigated. During the recording period, [Ca2+]cyt did not rise above the basal level estimated by applying the GPT transformation  [n = 36). As shown in 2+]cyt rose only slightly above the basal level measured along an axis passing through the sperm entry site in immature egg cells isolated three DAE, whereas in 2+]cyt in a receptive egg cell ; whereas no [Ca2+]cyt elevation could be detected in overmature egg cells isolated 18 DAE (n = 17)   to the f(n = 17) c.2+]cyt images shown in 2+]cyt were observed to arise away from the sperm entry site. In the case of immature egg cells, this rise in [Ca2+]cyt was confined to a certain region of the cell away from the sperm entry site, whereas fully mature egg cells exhibited [Ca2+]cyt waves sweeping through the entire cell at the focal plane of sperm entry , which approximately 10 ± 2 min (n = 66) after gamete fusion rose to 1100 ± 21 nM (n = 66) as the highest [Ca2+]cyt peak reached its summit, which was followed by a global elevation in [Ca2+]cyt, as was observed at the focal plane corresponding to the Sperm Entry Site (SES) throughout the whole cell  egg cells, the first [Ca2+]cyt rise was typically followed by several [Ca2+]cyt pulses, the oscillatory maximum of which was estimated to be 1180 ± 40 nM (n = 66), as the resting [Ca2+]cyt level had increased about 13-fold when the [Ca2+]cyt spikes reached their peak (2+]cyt rise did not exceed 442 ± 15 nM (n = 66) and usually corresponded to about a 10-fold increase (1100 ± 21 nM) (n = 66) (n = 66). A typical measurement of [Ca2+]cyt in egg cells isolated at 11 DAE and fertilized with viable, mature sperm cells is depicted in 2+]cyt rise compared to that of receptive eggs. The last developmental stage at which changes in [Ca2+]cyt were elicited by the sperm cell in isolated egg cells was at 11 DAE. At this maturational stage, the [Ca2+]cyt rise occurred 10 ± 3 min (n = 45) later than in mature, fertilized egg cells (isolated six DAE) and presented a slow rise of [Ca2+]cyt, which reached a plateau at 424 ± 17 nM (n = 45) with a mean amplitude of 295 ± 22 nM (n = 45) 27 min post-fertilization  after fusion, the level began to decrease, until [Ca2+]cyt could not be distinguished from the basal level (2+]cyt elevations characteristic of mature egg cells could not be observed in any of the cells (n = 45) isolated at this maturational stage.The resting level of [Caole cell b. In receir peak b. The ma(n = 66) b. The calization c. The egal level c. Sperm-2+]cyt dynamics of calcium influx across the plasma membrane of the mature egg cell, IVF was carried out in fusion medium without calcium or in IVF medium containing 10 µM  of GdCl3, which had been previously demonstrated by Antoine et al. cyt oscillation could be detected (n = 18); instead, a single [Ca2+]cyt rise occurred  occurred .2+ permeability is necessary for the onset of the [Ca2+]cyt peaks  to chelate any external Ca2+ was not feasible, since it caused the loss of sperm membrane integrity within minutes after the introduction of the compound into the IVF medium, a finding corroborating that of Antoine et al. cyt do not reflect a putative stress response induced by the experimental procedures  in the egg. For this purpose, numerous control experiments were carried out, such as to demonstrate that impaling the fragile egg gametoplasts with the injection needle, injection itself and withdrawing the microcapillary did not elicit “artificial” changes in [Ca2+]cyt, nor did the electrofusion procedure (without the sperm cell) trigger events leading to [Ca2+]cyt rise . In unfertilized egg cells that were incubated in Ca2+-free IVF medium, the drug triggered a single Ca2+ peak with as high an amplitude as that caused by the sperm cell  compared to that triggered by incubating the cells in 10 μM thapsigargin, which suggests total depletion of the calcium pumps of the ER by thapsigargin added at 10 μM concentration to the cells  in the wheat egg , caused a single [Ca2+]cyt rise, the peak value  of which was not significantly different from that observed when the cells were incubated in calcium-free IVF medium   induced a [Ca2+]cyt rise, the peak value  of which was higher than that  produced by thapsigargin applied at a 10 μM concentration (compare 2+]cyt was sustained and in none of the egg cells (n = 15) analyzed returned to the basal [Ca2+]cyt level.However, the addition of 100 μM thapsigargin to unfertilized egg protoplasts incubated in IVF medium containing calcium  (DiIC16(3)) which had been previously shown by Pònya et al. [2+-ATPase pumps tethered on the membrane meshwork of the endoplasmic reticulum  when 2 mM CaCl2 was added to eggs previously incubated in thapsigargin in Ca2+-free IVF medium .To examine the effect of thapsigargin on the Ca2+]cyt transients (that were observed in mature egg cells fused with the sperm) was substantially reduced.As 2+]cyt comparable with that triggered by thapsigargin alone; [Ca2+]cyt transients continued for some time, and in all cases analyzed (n = 25), thapsigargin suppressed the sperm-induced [Ca2+]cyt transients observable during fertilization of the receptive wheat egg  used to induce fusion between the gamete pairs. The possibility that the dynamics of calcium signaling of fertilization has species-specific characteristics in angiosperms cannot be excluded. Contrary to the observation made by Digonnet et al. , who meat et al.  found thl et al. . Thus, t2+]cyt is generated in the fertilized wheat egg cell remains to be elucidated. In the thoroughly studied animal systems, several models were proposed to expound the generation of repetitive [Ca2+]cyt rises in non-excitable cells [2+ release and InsP3 production and as to whether both Ca2+ and InsP3 or only Ca2+ is thought to oscillate. Whether either of these models can be adopted in explaining the observed [Ca2+]cyt changes elicited by sperm incorporation into the wheat female gamete, or different mechanisms are involved, remains to be investigated. In the wheat egg cell, unlike many animal cells and maize, no [Ca2+]cyt wave arising at the site of sperm entry was apparent, neither could any local increase in [Ca2+]cyt be detected at the sperm entry site  egg cells .The truncated, spatially-confined [Cagg cells a might b was exploited in order to establish the origin of the [Ca2+]cyt rise and the contribution of extracellular calcium to the observed [Ca2+]cyt elevations. This fusion method combined with a pharmacological approach allowed for addressing these questions. First, unfertilized wheat egg cells incubated in calcium-free IVF medium were treated with thapsigargin. Thapsigargin was mainly used in animal systems and shown to block ER/SR Ca-ATPases without interfering with the plasma membrane Ca2+-ATPases [2+]cyt in a variety of cell types without stimulating the production of inositol polyphosphates cyt which per se had to originate from an intracellular calcium store.In the unfertilized egg incubated in IVF medium without extracellular Ca(2)2+]cyt rise . It may be reasoned, however, that other intracellular Ca2+ pumps, such as those located, e.g., in the Golgi apparatus membrane or on vacuole membranes, may have remained unaffected by thapsigargin, which could still facilitate sequestering Ca2+ into the Golgi apparatus or into vacuoles. Indeed, Ordenes et al. cyt transient was not significantly higher compared to that measured when cells were incubated in calcium-free medium  “Ca2+ pressure” on the cell membrane. In concert with this assumption, when applied to unfertilized egg cells incubated in IVF medium containing 2 mM CaCl2, thapsigargin at a high concentration (100 µM) caused a rapidly rising increase in [Ca2+]cyt, the peak value of which was higher than that observed in wheat egg cells treated with thapsigargin at 10 µM (compare 2+]cyt was sustained during [Ca2+]cyt measurement (n = 17) and only slightly diminished due to photobleaching of the calcium-sensitive dye. This finding lends credit to the hypothesis that at this concentration, Ca2+ overload occurs in the cell, most probably due to the inhibitory effect exerted by thapsigargin on the plasma membrane calcium pumps.The observation that 10 µM thapsigargin treatment caused Cadium see a suggest compare a,b. The 2+-ATPase to thapsigargin could, therefore, be attributed to differences in hydrophobicity of the two types of membranes. This notion is corroborated by our finding that fluorescent thapsigargin at a 100 µM concentration stained both the ER and the plasma membrane  by the proposed mode of action of the compound: thapsigargin, being highly hydrophobic, is believed to partition selectively into the phospholipid component of membranes, where it interacts with the hydrophobic domains of membrane proteins, hence affecting lipid-protein interactions and, consequently, general ATPase activity. The differing degree of sensitivity of the ER and the PM Carane see a,b.2+]cyt transient acts as a signal for an increase in plasma membrane Ca2+ permeability. We examined the hypothesis that the [Ca2+]cyt oscillation observed in mature egg cells following fertilization is due to an increased Ca2+ permeability and subsequent filling and periodic emptying of an intracellular Ca2+ store. The depletion of intracellular Ca2+ by thapsigargin in the analyzed egg cells increased Ca2+ influx through the cell’s plasma membrane, since an immediate elevation in [Ca2+]cyt could be observed when Ca2+ was added to unfertilized eggs previously treated with thapsigargin and incubated in IVF medium without extracellular calcium  ensue after a relatively longer “lag period” , rendering it doubtful that [Ca2+]cyt in these angiosperm species have (direct) relevance in mechanisms ensuring the avoidance of polyspermy. However, it appears to be clear that in a number of described systems, the intracellular calcium waves induce cortical vesicle fusion with the plasma membrane, leading to the elevation of the so-called “fertilization envelope”, which acts as a mechanical barrier to further sperm penetration, hence indirectly implying changes in [Ca2+]cyt. This is thought to be a slow block to polyspermy. Whether elevations observed in [Ca2+]cyt in the wheat egg have relevance in mechanisms ensuring the avoidance of polyspermy remains to be elucidated. In any case, the difference observed in the two species between the time lapse measured from plasmogamy to the onset of the elevation in [Ca2+]cyt may be explained by the different in vitro fusion systems used .The present study demonstrates that repetitive Cand Reger , suppliecyt measurements. In this manner, the incremental positioning of the micromanipulator arm by the step motor could reproducibly drive the microcapillary into the cytoplasm of the firmly immobilized egg cell. The signal for injection was triggered by pushing a pedal, which generated a “start”-signal recorded by a computer.The microinjection procedure employed to introduce the calcium-sensitive dye, fura-2 dextran  delivered by a cell fusion instrument  following the dielectric alignment of the gamete pairs on one of the electrodes by using an AC field . The fusion droplets were composed of 600 mOsmol mannitol containing 2 mM CaCl2 (pH 6.0). Thapsigargin and calcium-containing solutions were directly added by a microsyringe controlled by a micromanipulator to the fusion droplet in an equal volume of medium to ensure rapid and thorough mixing.The electrofusion of selected pairs of isolated gametes was implemented following the method of Kranz et al. . ElectroFor cell wall visualization egg cells fused with the sperm were stained for 5 min in the dark in the IVF medium containing 0.001% Triton X-100 and calcofluor white M2R , then washed twice in a 600 mOsmol/kg mannitol solution before observing them on an inverted microscope  equipped with epifluorescence.Fura-2 dextran-loaded egg cells were observed with a Zeiss Axiovert 35M inverted microscope equipped with a 75-W xenon epifluorescence burner . The images were obtained using a Zeiss Plan-Neofluar 63× oil immersion objective ; Carl Zeiss Microscopy, GmbH, Jena, Germany). A rotating filter wheel  and a shutter apparatus were used to alternate excitation wavelengths between 340 and 380 nm. A 400-nm dichroic mirror was positioned after the shutter assembly. The emitted light was collected using a 510-nm emission filter with a 10-nm half-bandwidth. Confocal images were produced by using a laser scanning confocal system  coupled with a BX50F4 research microscope . The cells were excited at 488 nm, and the emitted fluorescence was detected at 500–530 nm.2+ binding/free-acid forms, respectively. Image processing and ratio calculation were performed with the Lucida 3.53 image processing software system . The resulting ratio images were color-coded to represent different calcium concentrations determined after calibration. An in vitro (extracellular) calibration of the ratio versus free [Ca2+] was performed using Ca2+-ethyleneglycol-bis(aminoethyl ether)-N,N'-tetraacetic acid (EGTA) buffers. For the in vitro calibration, the buffer contained 0.5 µM fura-2 dextran , 120 mM KCl, 0.5 mM MgCl2, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.2) and 0.2 mM EGTA (with or without CaCl2). To account for the spectral changes of fura-2 fluorescence in cells and in salt solutions, due to viscosity differences  = Kd × [((R − Rmin)/(Rmax − R))Sf2/Sb2], where: [Ca2+] = the concentration of calcium ions (nM); Kd = the dissociation constant of fura-2; R = the ratio recorded under appropriate physiological conditions; Rmin = the ratio recorded at zero external calcium; Rmax = the ratio recorded in the presence of excess calcium; Sf2 = the signal at 380 nm in zero calcium; Sb2 = the signal at 380 nm in excess calcium.The fluorescent ratio-values were converted into Ca formula , [Ca2+] 16(3) obtained from Molecular Probes  into the egg cells was described in detail by Pònya et al. cyt elevations in the mature angiosperm female gamete activated by the sperm cell appears to be comparable to the dynamics of [Ca2+]cyt oscillations triggered by an oscillation-inducing sperm protein (“oscillogen”) demonstrated to induce a characteristic series of Ca2+ oscillations in the mammalian egg at fertilization [2+ signatures identified in widely differing systems transcend the specific system that they are found in [2+ wave signatures appear conspicuously similar [2+]cyt may represent the initial steps in egg cell activation during fertilization in higher plants . Furthermore, the repetitive [Calization . It seemfound in . For ins similar . Calcium similar . The finiew, see ).Our observations may have ramifications for enhancing our understanding of the mechanisms of double fertilization in angiosperms with particular regard to the presumable interaction between the synergids and the egg cell during pollen tube penetration and discharge .


The balance among AC, PDE1 and CaN might determine the occurrence of opposite Ca2+-induced forms of synaptic plasticity, long-term potentiation (LTP) and long-term depression (LTD). CaN is essential for LTD. AC and PDE1 regulate, indirectly, protein kinase A, which counteracts CaN during LTP. Stimulations of AC, PDE1 and CaN with artificial and physiological Ca2+ signals demonstrated that AC and CaN have Ca2+ requirements modulated dynamically by different properties of the signals used to stimulate them, because their interactions with Ca2+ often occur under kinetic control. Contrarily, PDE1 responds to the immediate amplitude of different Ca2+ transients and usually with the same Ca2+ requirements observed under steady state. Therefore, AC, PDE1 and CaN decode different dynamic properties of Ca2+ signals.Frequently, a common chemical entity triggers opposite cellular processes, which implies that the components of signalling networks must detect signals not only through their chemical natures, but also through their dynamic properties. To gain insights on the mechanisms of discrimination of the dynamic properties of cellular signals, we developed a computational stochastic model and investigated how three calcium ion (Ca With the expansion of our knowledge on cell signalling, it became evident that signal transduction emerges from the combination of highly interconnected networks often formed by signalling pathways with opposing actions, but activated by the same signal22+) to the cytosol12+ concentration ([Ca2+]), which suggests the participation of molecules with high Ca2+ affinities42+] rises and, supposedly, requires the activation of molecules with moderate to weak Ca2+ affinities42+ affinities of the molecules implicated with each type of plasticity and the magnitude of the [Ca2+] rise.Long-term forms of synaptic plasticity are persistent alterations in the efficacy of synaptic transmission, which can be depressed or potentiated2+ in its free form and complexed with calmodulin (Ca2+/CaM)Among the molecules involved with LTP are several protein kinases, in contrast, LTD requires protein phosphatases12+, but indirectly82+/CaM2+/CaMIn the hippocampus, PKA is regulated by Ca2+ and the subsequent elevation of cAMP activates PKA during LTP, but also activates Epac, a molecule involved with LTD2+] elevations activate, among other targets, CaN, a phosphatase essential for LTD, AC and PDE1, both of which regulate the level of cAMP, a second messenger implicated with LTD and LTP. In such a scenario, it is very difficult to predict how the Ca2+ signals are decoded to ensure the occurrence of specific forms of synaptic plasticity. AC, CaN, and PDE1 have Ca2+ requirements below 1 μmol.L−1 682+ signals. We developed a stochastic computational model of the detailed mechanisms of activation of each one of these enzymes placed inside a hippocampal dendritic spine containing comprehensive mechanisms of Ca2+ dynamics. This model was used to investigate how CaN, AC and PDE1 decode simple and complex Ca2+ transients to promote specificity among their activations. Our results demonstrated that these enzymes have different modes of Ca2+ signals decoding that arise from thermodynamic and kinetic factors.The rise of Ca2+ transients, which are highly variable2+ dynamics formed by its influx through NMDARs channels, buffering and extrusion .Structurally, NMDARs are assemblies of two GluN1 and two GluN2 subunits192018192+], two buffers were simulated, an unspecific buffer (UB) and CaM. We simulated CaM according to its structure composed by two distinct domains, each one containing a pair of Ca2+-binding sites2+ to CaM by fitting a dose-response curve with the Hill equation:To reproduce physiological changes of  is the concentration of its activator, nHill is the Hill coefficient, and K1/2 is the concentration of activator required to activate the half maximum concentration of [P]. The nHill and K1/2 obtained by fitting the dose-response curve of activation of CaM as a function of free [Ca2+] ([Ca2+]free)   were in 2+ dynamics implemented in the computational model through its ability to reproduce synaptically evoked Ca2+ transients observed experimentally. NMDARs have a very characteristic current (I)-membrane potential (Vm) relationship caused by the voltage-dependent blockage of their channels by magnesium ions (Mg2+)2+ signals, first we verified the number of open NMDAR channels and the NMDAR-mediated current evoked by a glutamate pulse (amplitude of ~700 μmol.L−1 and duration of 1 ms) as a function of Vm. The number of open NMDAR varied with Vm  obtained for our simulations varied from 77 to 177 ms. Experimental works have reported values ranging from 89 msWe verified the accuracy of the Ca with Vm , and theblockage 2324. We  current 2425. Typ2+-binding sites that, when filled, promote a conformational change that exposes sites for target interactions2+, but the binding of all four ions stabilizes its open conformation2+ can interact with AC, PDE1, and CaN11292+ and CaM: (Ca2+)4CaM denotes CaM loaded with four Ca2+, and Ca2+/CaM refers to all complexes of CaM with four or less Ca2+.As mentioned previously, CaM has four Ca2+] systematically. We performed the simulations until the system had reached equilibrium. After that, we annotated the remaining [Ca2+]free and the concentration of the activated molecule under analysis to plot its dose-response curve of activation. To validate AC, we verified its activation as a function of [Ca2+] in the presence of saturating CaM (−1 of AC to 10 μmol.L−1 of CaM). The parameters obtained by fitting the 1/2 of ~0.2 μmol.L−1 and nHill of 2)2+ not only stimulated AC, but also inhibited its activity when present in high concentrations (50 ~80 μmol.L−1)To validate the reactions and parameters used to simulate PDE, AC, and CaN, we simulated each one of them isolated from the other components of the model and, in the presence of saturating concentrations of CaM, we varied  with saturating CaM 8We validated the model of PDE1 through its dose-response curve of activation as a function of  to become fully saturated (~20 μmol.L−1), the maximum activations of its targets involve lower [Ca2+] rises (~0.5–1 μmol.L−1)682+ 11302+ dependency of CaM in its native environment reflects the relative concentrations of all of its potential competing targetsThe dose-response curves obtained with the models of AC, PDE1 and CaN demonstrated that, while CaM requires a high elevation of [Ca2+ requirements under steady state indicated by their K1/2s  and high (>10 μmol.L−1) rises of [Ca2+]2+ signals in the intracellular environment are continuously changing over timeA common aspect of AC, PDE1 and CaN activations is their low Cair K1/2s . ConsequThe activation and inactivation of proteins can occur under thermodynamic or kinetic control, which can lead to striking different outcomes. When a system is under thermodynamic control (steady state), the concentrations of the competing species are determined by the stability difference among them. The most stable species are present in larger amounts. If a system is not in steady state, the formation of competing species is governed by competing rates (kinetic control), and the species formed faster are present in larger concentrations.2+] rise considering exclusively their Ca2+ requirements obtained under steady state. Based on this aspect, the next step in our work focused on the analysis of the activations of AC, PDE1, and CaN when stimulated by Ca2+ transients.Most biological systems are incessantly changing over time and operate far from steady state. Thus, it is unlikely that the activations of AC, PDE1 and CaN in their native environment are determined exclusively by thermodynamic factors. Consequently, it is impossible to predict their levels of activation for a given . As the three enzymes are activated by Ca2+ by binding to Ca2+/CaM, we also included the formation of (Ca2+)4CaM in our analysis. We used the peak activations of the molecules analysed obtained for each Ca2+ signal to plot curves of activation versus the peak amplitude of [Ca2+]. For that, we annotated the maximum activation of the molecules analysed during the occurrence of a Ca2+ pulse and the corresponding peak [Ca2+]. The dose-responses curves of maximum activations as functions of the peak [Ca2+] were fitted using 2+ requirements obtained for dynamic patterns of Ca2+ stimulation with the values observed under steady state.To verify how Ca2+)4CaM demonstrated that the activity of CaM was independent of the durations of the Ca2+ pulses tested and of the number of pulses used to stimulate the model , but had no effect on its activation when longer transients were used as input signals  were used to stimulate the model of the hippocampal spine and promote the Ca2+ influx through NMDAR channels. As the opening of NMDARs requires both the binding of glutamate and the depolarization of the postsynaptic cellular membranem to produce physiological Ca2+ signals with different amplitudes.LTP and LTD are typically induced by glutamate pulses released at high and low frequencies, respectively2+ signals 4CaM (indicated as CaM activity in the panels) remained unchanged during the trains of stimulation. Consequently, all the curves were superposed, and their corresponding K1/2s  than the K1/2 observed under steady state  and dephosphorylated by CaN. We used the same rate constants for the reactions of phosphorylation and dephosphorylation to facilitate the comparisons between PKA and CaN. Under basal conditions, PKA has a higher activity than CaN2+], but promotes low levels of PKA activation  for the protocols tested. Thus, we observed stronger levels of dephosphorylation of subsP for stimulations at 1 Hz in comparison to 10 Hz . The results demonstrated the binding of CaM partially loaded with Ca2+ to AC was important to determine its pattern of activation, but had smaller effects on the activations of CaN and PDE1 4CaM with a constant Ca2+ requirement independent of the dynamic properties of these signals. Thus, in the situations tested, the interactions of Ca2+ and CaM were in fast equilibrium, which is consistent with experimental dataStimulations of our model with simple and complex patterns of Ca2+ transients because their interactions with Ca2+ occurred under kinetic control for most of the simulated protocols. Consistently with experimental observations2+ signals induced by glutamate pulses, but had poor sensitivity to the frequencies of stimulation tested. In our work, the ability of CaN to count pulses was caused from a combination of its slow interaction with Ca2+ and high affinity for Ca2+/CaM, which allowed it to respond gradually and consistently to the signals used. Previous models of CaN have indicated that the high affinity of CaN for Ca2+/CaM is important for the integration of different patterns of signals4350Our results demonstrated that AC and CaN decode the dynamic features of the Cam. Thus, AC is probably less activated during protocols of LTD than during protocols of LTP induction.The role of AC during LTP and LTD is not completely clear2+ transients independently of their dynamic properties for the large majority of the situations simulated. The regulation of PDE1 by the amplitude of the Ca2+ signals for the protocols tested indicates that it can have lower Ca2+ requirements than AC when both enzymes are stimulated by brief Ca2+ transients, which is contrary to what would be predicted from dose-response curves obtained under steady state.In contrast to AC, PDE1 responded to the amplitude of the Ca2+-dependent proteins can respond differently to the dynamic features of the Ca2+ signals to ensure the occurrence of the appropriate cellular response. The ability to decode the dynamic properties of the Ca2+ signals are caused by a combinations of factors, including differences in affinities and competing rates of reactions and mechanisms of activations, which allows some molecules to interact with Ca2+ under thermodynamic control, while others interact with it under kinetic control. Therefore, the balance between thermodynamic and kinetic control can be an important tool for decoding physiological Ca2+ signals.In conclusion, our results demonstrated that Ca−15 L was set to simulate a single hippocampal spine2+ dynamics  that were used to simulate physiological signals of Ca2+. In addition, in some simulations of the model, we included the production and degradation of cAMP by AC and PDE1, respectively, and the subsequent activation of PKA. All components of the model were validated based on comparisons with published experimental data, as described in the Results. The detailed descriptions of the reactions used to simulate each component of the model with their respective parameters and references are described in the The computational model described in this paper was constructed using BioNetGenHow to cite this article: Antunes, G. et al. Modelling intracellular competition for calcium: kinetic and thermodynamic control of different molecular modes of signal decoding. Sci. Rep.6, 23730; doi: 10.1038/srep23730 (2016).


Ca2+ signals in the cytosol are initiated by the activation of Ca2+ channels in the plasma membrane and/or in endomembranes. Yeast (Saccharomyces cerevisiae) contains a Ca2+-permeable channel of the TRP family, TRPY1, which is localized in the vacuolar membrane and contributes to cytosolic free Ca2+ ([Ca2+]cyt) elevations, for example in response to osmotic upshock. A TRPY1 homologue in the rice blast fungus is known to be important for growth and pathogenicity. To determine the role of the TRP channel family in the maize pathogen Colletotrichum graminicola, proteins homologous to TRPY1 were searched. This identified not one, but four genes in the C. graminicola genome, which had putative orthologs in other fungi, and which we named CgTRPF1 through 4. The topology of the CgTRPF proteins resembled that of TRPY1, albeit with a variable number of transmembrane (TM) domains additional to the six-TM-domain core and a diverse arrangement of putatively Ca2+-binding acidic motifs. All CgTRPF genes were expressed in axenic culture and throughout the infection of maize. Like TRPY1, all TRPF proteins of C. graminicola were localized intracellularly, albeit three of them were found not in large vacuoles, but co-localized in vesicular structures. Deletion strains for the CgTRPF genes were not altered in processes thought to involve Ca2+ release from internal stores, i.e. spore germination, the utilization of complex carbon sources, and the generation of tip-focussed [Ca2+]cyt spikes. Heterologous expression of CgTRPF1 through 4 in a tryp1Δ yeast mutant revealed that none of the channels mediated the release of Ca2+ in response to osmotic upshock. Accordingly, aequorin-based [Ca2+]cyt measurements of C. graminicola showed that in this fungus, osmotic upshock-triggered [Ca2+]cyt elevations were generated entirely by influx of Ca2+ from the extracellular space. Cgtrpf mutants did not show pathogenicity defects in leaf infection assays. In summary, our study reveals major differences between different fungi in the contribution of TRP channels to Ca2+-mediated signal transduction.Calcium (Ca Colletotrichum graminicola, is generated by the accumulation of osmotically active compounds cyt) is terminated by the activity of Ca2+/H+ antiporters and Ca2+-ATPases which transport Ca2+ out of the cytosol cyt. Ca2+-permeable channels may be activated by a number of ligands, such as inositol phosphates, cyclic nucleotides or amino acids, and by physical parameters, such as voltage or stretch of the membrane cyt signal generation, germination, growth rates, tolerance to osmotic stress and Ca2+ starvation, as well as virulence. Surprisingly, our results differed considerably from data obtained previously on TRPY1 homologues in other fungal species, indicating that fungi vary largely in their employment of Ca2+ channel types.Despite the obvious importance of TRPY1-like channels in filamentous fungi, their functioning has been rarely analysed, with the notable exception of TRPGz from minearum . We therC. graminicola whole genome sequence hosted at the Broad Institute was carried out using default parameters. This identified four fragments with similarity to the TRPY1 channel  of Saccharomyces cerevisiae. The corresponding genes were denominated CgTRPF1 through CgTRPF4. Full-length sequences of the CgTRPF genes were obtained by RACE-PCR as described below. Membrane topology of the CgTRPF proteins was analysed with TOPCONS (accessed at http://topcons.net/) using default settings. The putative pore loop was inferred from previous analyses cyt, yeast cultures were grown on a rotating shaker in liquid SC-Leu-Ura medium containing 2 μM coelenterazine (Carl Roth) at 30°C to a final density of 1 to 5 x 107 cells per mL and diluted to 1 x 107 cells per mL with fresh coelenterazine-containing medium. For [Ca2+]cyt measurements in a tube luminometer , 20 μl of the cell suspension were used per experiment. After 1 min baseline recording, cells were treated with 200 μl of a solution (pH 7.0) containing 1.5 M NaCl, 50 mM MES, and 25 mM EGTA. Total aequorin luminescence was discharged at the end of the experiment by injecting 220 μl of a solution containing 2 M CaCl2 and 20% ethanol. [Ca2+]cyt was calculated as described by Allen and co-workers (1977) cyt values obtained by this procedure may be offset from the calculated values due to the cytosolic aequorin environment, this effect should be similar in all yeast studies employing this formula.The aequorin  as descraequorin . Full-le plasmid . The pEVs (1977) , which n2+]cyt responses of C. graminicola wild type and ΔCgtrpf deletion strains to osmotic upshock, the strains were transformed with the pAEQS1-G418 plasmid cyt measurements, 1 mL mLCM agar supplemented with 10 μM coelenterazine was poured into sterile polystyrene cylinders with slip lid . 300 washed macroconidia were inoculated onto the solidified medium, and the polystyrene cylinders were covered loosely to allow for gas exchange. The fungal cultures were incubated in darkness for 3 d at 23°C and 65% relative humidity. Prior to [Ca2+]cyt measurements, cultures were overlaid with 3 mL of a solution (pH 7.0) containing 50 mM MES-KOH and 0 or 25 mM EGTA. Cylinders were covered with parafilm and incubated for 30 min in the chamber of the Sirius-1 luminometer. After 1 min baseline recording, 4 mL of a solution (pH 7.0) containing 50 mM MES-KOH and 0 or 3 M NaCl plus 0 or 25 mM EGTA were injected through the parafilm cover. Aequorin luminescence was recorded for 30 min. To discharge total aequorin at the end of the experiment, 8 mL discharge solution containing 2 M CaCl2 and 100 μM digitonin  were injected, and recording continued for 60 min. Data shown represent three biological repetitions performed on different days. [Ca2+]cyt was calculated as described above.To test [Ca plasmid  which en plasmid . Transfo2+]cyt analyses in individual hyphae, wild type and ΔCgtrpf deletion strains were transformed with a pGEM-T-PtrpC-nptII-TtrpC-PtoxB-YC3.6-TtrpC plasmid [2+ reporter protein Yellow Cameleon 3.6 (YC3.6) [2+]cyt spikes during hyphal growth were performed as described before . Collectively, an important role of these genes in Ca2+-related processes, in particular those involving Ca2+ release from internal stores, seemed likely, but could not be unveiled in our experiments.A number of pharmacological studies have suggested a role of Camination  and hyphmination , as wellmination , 32, 56. signals . In suppy growth . We therus fungi . The memof TRPY1 . These m1 (TPC1) , a vacuo1 (TPC1) . CgTRPF1S. cerevisiae, the only TRP member, TRPY1 (Yvc1), forms a stretch-activated Ca2+-permeable cation channel in the vacuolar membrane [2+]cyt signal cyt response of the S. cerevisiae trpy1Δ mutant to osmotic upshock. This might indicate that technical problems of the heterologous expression system  have prevented yeast complementation. However, we consider this as not very likely because other TRP channels are functional in this system. Alternatively, the failure to complement the trpy1Δ response to osmotic upshock may indicate that not all TRPF proteins act in the perception of osmotic stress. To further test this presumption, the response of C. graminicola to hyperosmotic shock was analysed. In C. graminicola, 1.5 M NaCl triggered a massive Ca2+ influx via the plasma membrane, but, unlike in yeast, a negligible release of Ca2+ from internal stores. Since TRPF channels of C. graminicola were localized to intracellular membranes, these results further substantiate the idea that in C. graminicola TRP proteins are not involved in Ca2+ release into the cytosol upon hyperosmotic stress.In membrane , 33 thatt signal . By compalbicans , as wellminearum . In cont2+ release from the fusing vesicles cyt dependence of enzyme secretion, the Cgtrpf deletion strains did not differ from the wild type in their growth on complex carbon sources.A further process that we considered likely to be regulated by TRPF proteins is the utilization of complex carbon sources, which relies on the exocytosis of hydrolytic enzymes. In yeast, membrane fusion, a prerequisite of exocytosis, is dependent on the activation of calmodulin by Cavesicles . Furtherin yeast . HoweverC. graminicola colonies is very sensitive to the inhibitors 2-APB and capsazepine, which block a range of TRP channels in animals cyt pulses that can be visualized by fluorescence ratio imaging microscopy of the Yellow Cameleon (YC) reporter protein [2+]cyt pulses are apparently not related to growth kinetics, but may rather be involved in environmental sensing [2+]cyt spikes of short duration are also detectable on whole-colony level as luminescence of the Ca2+ reporter aequorin cyt spike generation was affected neither on single-hypha level nor on whole-colony level. This corresponds well to the fact that on whole-colony level, spike occurrence was nearly completely abolished by chelation of extracellular Ca2+ [2+]cyt spikes depend on Ca2+ influx rather than Ca2+ release from internal stores.During undisturbed growth, filamentous fungi, including  protein , 65, 66. protein , these [ sensing . [Ca2+]caequorin . In N. cvesicles . Howeverlar Ca2+ , suggestCgTRPF genes were expressed throughout the infection process of C. graminicola on maize plants. Interestingly, there was a transcriptional regulation of the genes by up to 13-fold during the course of infection. This is unexpected for ion channels, which are primarily regulated on post-translational level. However, regulation of TRP channels on mRNA level is also known for a number of mammalian family members, such as TRPC1, TRPC3, TRPV4, and TRPV6 cyt elevation after osmotic upshock, as shown for homologues from other fungal species. This indicates a functional diversification of this ion channel family in fungi. A change in functionality can be similarly observed in an animal TRP family member, TRPV1, the receptor for capsaicin (the spicy component of hot chilli pepper): Rabbits are about 100 times less sensitive to capsaicin than humans and rats, and birds are completely insensitive. A molecular basis for these drastic differences lies in just two point mutations in the TRPV1 gene cyt-dependent luminescence was detected for 20 min. Data are the means ± SE (N = 4).Colonies of (TIF)Click here for additional data file.S4 Fig2+, colonies were pre-treated with a solution (pH 7.0) containing 50 mM MES-KOH and 25 mM EGTA for 30 min prior to measurement, followed by treatment with a solution (pH 7.0) containing 50 mM MES-KOH, 25 mM EGTA, and no NaCl (green line) or 1.5 M NaCl . Treatment solutions were added after 1 min of measurement. Traces show single measurements in order to demonstrate [Ca2+]cyt spikes in the MES-KOH control treatment.Whole colonies were pre-treated with 50 mM MES-KOH (pH 7.0) for 30 min prior to recording, followed by treatment with a solution (pH 7.0) containing 50 mM MES-KOH and no NaCl (grey line) or 1.5 M NaCl . To abolish the influx of extracellular Ca(TIF)Click here for additional data file.S5 FigTRPF genes were amplified from RNA extracted from log-phase cultures of S. cerevisiae trpy1Δ transformed with pFL61-CgTRPF1 through pFL61-CgTRPF4. Products were expected at 2098, 2152, 3522, and 2101 bp for CgTRPF1, CgTRPF2, CgTRPF3, and CgTRPF4, respectively. RT: + reverse transcriptase added in cDNA synthesis, − reverse transcriptase omitted in cDNA synthesis.Full-length cDNAs of the (TIF)Click here for additional data file.S1 File(TXT)Click here for additional data file.S2 FileExons are indicated in uppercase letters, introns are indicated in lowercase letters.(TXT)Click here for additional data file.S3 FileCytosolic amino acid residues are highlighted in yellow, luminal amino acid residues are highlighted in light blue, amino acid residues in the putative pore loop are highlighted in dark blue. Acidic amino acid residues are indicated in red.(PDF)Click here for additional data file.S4 File(XLSX)Click here for additional data file.S5 File(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Tablehttp://topcons.net/) using standard settings and the full-length protein sequences of S. cerevisiae TRPY1, and CgTRPF1, CgTRPF2, CgTRPF3, and CgTRPF4.Topology prediction was performed with TOPCONS ((PDF)Click here for additional data file.S3 Tablephylum and class. The sequenced strain and the NCBI Taxid of each species are given in the indicated columns. To link the proteins of this table with the tree, see column TRPY1 Homologue No. For the distinct identification of the proteins the locus tag may be used. E-values were calculated on NCBI.Organisms were ordered by (PDF)Click here for additional data file.


Here, we discuss the implications of differences in EC coupling when considering the potential applications of human iPSC-CMs in a number of areas as well as detailing the current understanding of this fundamental process in these cells.Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) hold enormous potential in many fields of cardiovascular research. Overcoming many of the limitations of their embryonic counterparts, the application of iPSC-CMs ranges from facilitating investigation of familial cardiac disease and pharmacological toxicity screening to personalized medicine and autologous cardiac cell therapies. The main factor preventing the full realization of this potential is the limited maturity of iPSC-CMs, which display a number of substantial differences in comparison to adult cardiomyocytes. Excitation–contraction (EC) coupling, a fundamental property of cardiomyocytes, is often described in iPSC-CMs as being more analogous to neonatal than adult cardiomyocytes. With Ca Studies of EC coupling in human iPSC-CMs (hiPSC-CMs) have described substantial differences between their properties and those of adult cardiomyocytes. While understanding these differences will be crucial to fully realizing the potential of hiPSC-CMs, research to date is limited. Furthermore, electrophysiological study of hiPSC-CMs is confounded by the variability of these cells in culture. We have previously shown the markedly increased variability in action potential morphology when hiPSC-CMs are cultured as single cells as compared to in a monolayer. As patch clamping studies necessitate the use of individual cells, these findings raise questions over the usefulness of investigations performed in this manner  is the key link between electrophysiological stimulation and the production of mechanical force as well as a feature that distinguishes cardiomyocytes from other types of muscle cells. Cardiomyocyte depolarization causes Ca2+ entry via voltage-sensitive L-type Ca2+ channels, triggering the release of Ca2+ from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyR), raising [Ca2+]i and causing contraction. Relaxation is achieved by removing the Ca2+ from the cytosol, either by re-sequestering it in the SR via the sarco-endoplasmic reticulum ATP-ase (SERCA), or extruding it into the extracellular space through the Na+-Ca2+ exchanger (NCX) or Ca2+ ATPase pump  through patch clamp techniques have measured substantial peak current densities, ranging from −8 pA/pF  are the Ca2+ release mechanism of the SR, and are closely apposed to L-type Ca2+ channels. RYR2, the cardiac isoform of RyRs, appear diffuse throughout the cytosol on immunostaining with reports of both intense, punctate perinuclear . Due to this stoichiometry, NCX generates a substantial inward current (INCX) that contributes to cell depolarization, pace maker activity and action potential duration  and as such need to be interpreted with caution. In summary, hiPSC-CMs display significantly impaired diastolic Ca2+ removal.Regarding the relative contribution of the two main mechanisms, SR Ca2+ handling in these cells.A key feature of hiPSC-CMs is that they beat spontaneously, making for a convenient means to confirm successful differentiation of stem cells into cardiomyocytes. Unexpectedly, study of this phenomenon has given us a number of insights into the regulation of Ca2+ release identified small Ca2+ wavelets propagating within the cell, the majority of which were too small to trigger a global Ca2+ release (/ΔFCaff)(, averaging at ~0.3 vs. 0.6–0.9 in adult human cells (Bassani et al., 2+ release and limited association with sarcolemmal Ca2+ channels contributes to slow and inefficient rise of [Ca2+]i. hiPSC-CMs are devoid of t-tubules (Gherghiceanu et al., 2+ transients, low fractional release and increased dependence on sarcolemmal Ca2+ flux (Figure The lack of the sophisticated adult myocyte structure in hiPSC-CMs may explain some functional observations. From studies discussed here it is clear that SR Cax Figure . This st2+ extrusion rate, high PLN:SERCA ratio and a negative force-frequency response point to decreased SR function and regulation, associated with a greater dependence on sarcolemmal flux to source and extrude Ca2+. As such, greater structural co-ordination may yield limited benefits without improvements in the underlying function of EC coupling components, and vice-versa.Equally so the opposite can be argued. T-tubule formation and maintenance is an inherently energy-intensive process. Low SERCA expression and Ca2+ handling, and as such it could be argued that it is more efficient to rely on sarcolemmal flux driven by electrochemical gradients than ATP-intensive processes. We and others have shown how using culture techniques to manipulate myocyte structure can result in substantial changes in Ca2+ handling properties (Kaji et al., Standard culture environments place little demand on hiPSC-CMs in terms of Ca2+ handling properties of these cells a number of questions remain, particularly with regards to how well Ca2+ channels and RyRs are coupled and what the dominant sources of Ca2+ are in EC coupling.hiPSC-CMs hold enormous promise in many fields of cardiovascular research. While we have a reasonable understanding of the CaWith many of the end applications of hiPSC-CMs dependent on the ability of these cells to recapitulate adult human cardiomyocyte properties to a satisfactory degree, differences in channel pharmacology, mechanisms of automaticity and structural organization between hiPSC-CMs and adult cardiomyocytes pose challenges to fully realizing the potential of this technology. It is important however to remember that there is no truly definitive understanding of human cardiac physiology. Difficulties in obtaining healthy adult tissue have meant that the field has come to rely on failing hearts, and in particular on rabbit physiology, as a basis of comparison. While this by no means invalidates the comparative studies above nor the concerns raised about hiPSC-CM function, it is essential to consider this limitation when discussing hiPSC-CM properties.From a more practical perspective, the minimal variability shown between different cells lines and different differentiation protocols is reassuring, and gives us greater confidence when comparing and collating the work of multiple groups. Consideration must be given however to culture conditions, with substantial variability in hiPSC-CM electrophysiology apparently dependent on cell density (Du et al., Overall, a more solid understanding of the electrophysiological properties and limits of hiPSC-CMs will be extremely valuable in the interpretation of the effects of novel drugs and making judgments on the ability of these cells to successfully recapitulate human disease.We confirm that CK, LC, and CT meet the criteria for authorship of this review based on the recommendations of the ICMJE.CK is funded by a British Heart Foundation MBBS/PhD studentship FS/13/46/30282.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Moreover, recent observations showed that modifying the cellular Ca2+ signalling might be beneficial in AP. The inhibition of Ca2+ release from the endoplasmic reticulum or the activity of plasma membrane Ca2+ influx channels decreased the severity of AP in experimental models. Similarly, inhibition of mitochondrial permeability transition pore (MPTP) opening also seems to improve the outcome of AP in in vivo animal models. At the moment MPTP blockers are under detailed clinical investigation to test whether interventions in MPTP openings and/or Ca2+ homeostasis of the cells can be specific targets in prevention or treatment of cell damage in AP.Acute pancreatitis (AP) is a leading cause of hospitalization among non-malignant gastrointestinal disorders. The mortality of severe AP can reach 30–50%, which is most probably owing to the lack of specific treatment. Therefore, AP is a major healthcare problem, which urges researchers to identify novel drug targets. Studies from the last decades highlighted that the toxic cellular Ca2+ and ATP together to control life and death’.This article is part of the themed issue ‘Evolution brings Ca It is a well-known fact that in the exocrine pancreas acinar cells secrete digestive enzymes and pancreatic ductal epithelial cells secrete 2+ has a central role in both pancreatic physiology and pathophysiology. Recent studies suggest that these two cell types cannot be handled separately since they are more likely integrated into a functional unit i in pancreatic acinar cells even in extremely high concentrations [2+]i elevation and a drop of cellular ATP leading to necrosis i, elevation and augments fluid secretion, whereas high concentration moderately inhibits the stimulated fluid secretion in secretin-stimulated guinea pig pancreatic ducts [2+, inhibited adenosine 3’,5’-cyclic monophosphate and ATP production and depolarization of ΔΨm. We also showed that ethanol reduced expression of CFTR via multiple pathways, which in turn augmented the severity of experimental alcohol-induced AP in mice.The second most frequent form of pancreatitis is alcohol-induced AP . Whereastrations . Whereasnecrosis –30. Impo [et al. . They shic ducts . Our groic ducts . We showtrations ; howeverobserved . The rem(c)2+ toxicity and mitochondrial injury. Notably, these cellular changes seem to be the key of AP pathogenesis since a considerable number of studies showed that other stress factors provoke the same alterations in Ca2+ signalling and energy metabolism. Intrapancreatic trypsinogen activation is a hallmark of AP pathogenesis and we showed earlier that trypsin acting via PAR2 on the luminal membrane induces intracellular Ca2+ elevation and inhibits the luminal acid/base transporters in PDEC [et al. investigated the pathomechanism of an iatrogen form of AP, the post-ERCP pancreatitis [in vitro and in vivo models, they showed that exposure of pancreatic acinar cells to iohexol (a radiocontrast agent) triggered sustained intracellular Ca2+ elevation. The downstream activation of NF-κB and NFAT is completely abolished by the suppression of the Ca2+ signals. Moreover, they proved that the downstream effects of Ca2+ were mediated by calcineurin since genetic, or pharmacological inhibition of calcineurin prevented the radiocontrast-induced damage. This interesting study further underlines the central role of pathophysiological Ca2+ signalling in the pathogenesis of AP regardless of the etiological factor.As demonstrated above, the two most common pathogenic factors of AP—BA and ethanol—damage the exocrine pancreas via Ca in PDEC . Moreovereatitis . Using s3.(a)3) by hydrolysing phosphatidylinositol 4,5-bisphosphate (PIP2) [2+ signals have a strict spatio-temporal localization [2+ signals to the apical pole of the cells. As in other non-excitable cell types, this is ensured by two ATP-dependent pumps that clear the cytosol from the free Ca2+. The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pumps and the plasma membrane Ca2+-ATPase (PMCA) pumps move Ca2+ from the cytosol to the ER and the extracellular space, respectively. This activity restores basal intracellular Ca2+ levels and refills the ER Ca2+ stores. In PDEC, the Ca2+ signalling is not characterized in such detail; however, the overall polarity of the ductal cells including the ion channels and transporters, IP3 receptors and mitochondria [2+ signalling, like in acinar cells. Further studies are required for the clarification of these questions.Agonist binding  to G-protein-coupled receptors activate phospholipase C β (PLCβ) in pancreatic acinar and ductal cells. The activated PLCβ releases inositol trisphosphate (IPe (PIP2) . Under plization ,39, mostchondria , suggest(b)2+ influx to orchestrate non-excitable cell functions has been established several decades ago [et al. found that agonist-mediated depletion of the intracellular Ca2+ stores induced a Ca2+ selective sustained inwardly rectifying current, which was termed ICRAC  [2+ sensor stromal interaction molecule 1 (Stim1) [2+ channel Orai1 [2+ entry (SOCE) consist of the following elements. In resting conditions the ER Ca2+ stores are refilled and Stim1 distributes in the ER membrane. However during physiological stimulation the ER Ca2+ stores are quickly depleted, which induces the dissociation of the bound Ca2+ from the EF hand of Stim1. This is followed by a conformational change and translocation of Stim1 to defined ER-PM junctions, termed as puncta formation [2+ influx channel Orai1, where the Stim Orai1-activating region (SOAR) and polybasic domains of Stim1 interact with different binding sites of Orai1 that results in clustering and activation of the channel [2+ entry channels that seem to play a role in Stim1-mediated SOCE are the TRPC channels [2+-permeable non-selective cation channels mediating receptor evoked Ca2+ influx in many cells [et al. found that intracellular Ca2+ elevation, caused by the activation of SOCE might play a role in exocytosis in pancreatic ductal cells isolated from dog main pancreatic duct [The complex role of extracellular Caades ago ; howevercurrent) . The rea (Stim1)  and the el Orai1 ,44. Brieormation . This tr channel . In addichannels ,48. Thesny cells . SOCE hany cells –52. Intetic duct ,54; howe4.2+ signalling, mitochondria takes up Ca2+, which has been shown to directly increase energy output by enhancing the activity of tricarboxylic acid cycle dehydrogenases and the ATP synthase [et al. highlighted that the cytosolic Ca2+ signals propagate to the mitochondria [et al. found that ER membrane and the outer mitochondrial membrane form a quasi-synaptic connection [2+ hotspots [2+ signalling the molecular background of the process was not known. In 2011, two groups independently identified the mitochondrial Ca2+ uniporter (MCU), an inner mitochondrial membrane protein that is responsible for the mitochondrial Ca2+ uptake [2+ efflux from the mitochondria is mediated by the mitochondrial Na+/Ca2+ exchanger (NCLX) [2+ level is tightly regulated under physiological conditions. However, pathophysiological signals can lead mitochondrial injury, which can activate both apoptosis and necrosis. The classical mitochondrial apoptotic pathway involves the outer membrane permeabilization by Bax and Bak (two members of the pro-death Bcl-2 family) that will allow apoptotic factors like cytochrome c, Smac/DIABLO and apoptosis inducing factor to be released from the intermembrane space into the cytosol, leading to cell death by apoptosis [2+ overload or increased reactive oxygen species (ROS) production can cause the opening of mitochondrial permeability transition pore (MPTP) that results in the loss of mitochondrial inner membrane potential, uncoupling of the respiratory chain with a consequent drop of mitochondrial ATP synthesis, and increased permeability of the inner mitochondrial membrane that eventually leads to mitochondrial swelling, rupture and necrotic cell death [2+ efflux [1F0 ATP synthase or the proapoptotic Bax/Bak proteins [et al. demonstrated that both genetic and pharmacologic inhibition of MPTP opening  significantly ameliorated pancreatic damage in different experimental AP models in mice [During physiological Casynthase . The piochondria  and a connection  that is hotspots . Despite+ uptake ,60. The r (NCLX) , thus thpoptosis . On the ll death ,64. Notall death , mitocho+ efflux  and ROS + efflux  as well.+ efflux . The his+ efflux . However+ efflux ,70). A g+ efflux . CypD is+ efflux  and phar+ efflux . On the proteins  are requproteins , whereasproteins . At the  in mice . Importa in mice .5.3-mediated Ca2+ release from the ER is an essential component of the physiological response to agonist stimulation, but it could also contribute to the pathological Ca2+ overload of the cells evoked by toxic factors that induce AP  [3Rs due to multiple actions that include the inhibition of phospholipase C-mediated production of IP3 [3Rs [3Rs that reduce the channels open-state probability [3-mediated Ca2+ signalling may be protective in AP [et al. recently studied the effects of caffeine and its xanthine metabolites on pancreatic acinar IP3R-mediated Ca2+ signalling and experimental AP [3-mediated Ca2+ oscillations in response to uncaged IP3 or toxins, prevented mitochondrial depolarization and necrotic cell death in vitro and significantly impaired the severity of experimental AP in three different models. These observations suggest that caffeine, or its metabolites might be suitable starting points to develop therapy for AP  . Caffeinn of IP3 , antagonIP3 [3Rs  and direbability . Interesve in AP . Based oental AP . They foy for AP .Figure 2+ entry could be a key component in the development of cellular Ca2+ overload. Earlier Kim et al. showed that genetic [2+ elevation in pancreatic acinar cells evoked by cell stressors (bile acid or fatty acid ethyl ester). In addition, it prevented the pathological inhibition of digestive enzyme secretion and markedly reduced intracellular trypsin activation and excessive actin depolymerization in vitro and the severity of pancreatitis in vivo. Recently, Gerasimenko et al. demonstrated the pharmacological inhibition of another Ca2+ entry channel Orai1 by a specific inhibitor called GSK-7975A which prevents acinar cell necrosis in vitro [et al., who tested the effects of two specific Orai1 inhibitors (GSK-7975A and CM_128) in isolated human and rodent pancreatic acinar cells and in different experimental AP models [2+ elevation in vitro and significantly impaired signs of pancreatic injury including pancreatic oedema, inflammation and necrosis in all tested experimental models.As discussed above, store operated Ca genetic  or pharm genetic  of TRPC3in vitro . This imP models . They shet al. tested the effect of MPTP inhibition on the severity of AP in rodent experimental AP models [Ppif gene  significantly decreases the severity of AP in different independent models. These observations suggest that the MPTP inhibition might be potentially beneficial in the AP therapy. Other indirect evidence for this hypothesis has been provided by Judak et al., who showed that the supplementation of cellular ATP in vitro diminished the inhibitory effect of ethanol metabolites on the ion transport activities in isolated guinea pig pancreatic ductal cells [Mitochondrial permeability transition is a key feature of cellular damage in many cell types and diseases (see above); therefore, MPTP blockers are under detailed clinical investigation in different studies. In a recent clinical study, the efficacy and safety of TRO40303 (an MPTP inhibitor) have been evaluated for the reduction of reperfusion injury in patients undergoing revascularization for ST-elevation myocardial infarction (MITOCARE study) . This stP models . They haal cells . These r6.Although there are several promising results and potential drug targets that play a role in the pathogenesis of AP, it remains a great challenge for researchers and clinicians. A number of unanswered questions are waiting for answers. Moreover, it will take several years to test the experimental results on clinical patients as well. To be able take up these challenges, clinicians and researchers should work closely together in the future.