Also reported is the existence of “Ca2+ indicator dye (fluo-4) and a protein Ca2+ sensor (yellow cameleon). Using fluo-4 AM dye, we found spontaneous Ca2+ spikes in 18% of rat SCN cells in acute brain slices, but the Ca2+ spiking frequencies showed no day/night variation. We repeated the same experiments with rat (and mouse) SCN slice cultures that expressed yellow cameleon genes for a number of different circadian phases and, surprisingly, spontaneous Ca2+ spike was barely observed (<3%). When fluo-4 AM or BAPTA-AM was loaded in addition to the cameleon-expressing SCN cultures, however, the number of cells exhibiting Ca2+ spikes was increased to 13∼14%.We addressed this issue based on a Ca2+ spiking activity of SCN. Furthermore, our study strongly suggests that the spontaneous Ca2+ spiking activity is caused by the Ca2+ chelating effect of the BAPTA-based fluo-4 dye. Therefore, this induced activity seems irrelevant to the intrinsic circadian rhythm of [Ca2+]c in SCN neurons. The problems with BAPTA based dyes are widely known and our study provides a clear case for concern, in particular, for SCN Ca2+ spikes. On the other hand, our study neither invalidates the use of these dyes as a whole, nor undermines the potential role of SCN Ca2+ spikes in the function of SCN.Despite our extensive set of experiments, no evidence of a circadian rhythm was found in the spontaneous Ca The circadian clock system governs various daily rhythms in physiological activities, from cellular autonomic activities to animal behaviors. The key component of this system is the suprachiasmatic nucleus (SCN) 2+ ions ([Ca2+]c) is a general intracellular messenger regulating a variety of different cellular processes, including membrane potentials and gene expression. Thus, it is possible that [Ca2+]c is a mediator coupling clock gene oscillations and AP firing rhythms in SCN neurons 2+]c was higher during day time than night time by using fura-2 2+]c of cultured mouse SCN neurons that expressed yellow cameleon, a Ca2+-sensing protein 2+]c was persistent. On the other hand, the amplitudes of circadian oscillations, both in AP firing rate and [Ca2+]c, were significantly reduced by ryanodine and 8-bromo-cADP ribose. Thus, the release of Ca2+ from internal Ca2+ stores might have driven AP circadian oscillations in SCN neurons.The concentration of cytosolic free Ca2+]c, there have been reports on Ca2+ spikes that have a duration in the order of several seconds to a minute in SCN neurons. For example, van den Pol et al. discussed SCN Ca2+ spikes in connection with neuro-glia interactions 2+ spikes 2+ spikes in fura-2 stained acute SCN slices In addition to the slow circadian modulation of [Ca2+]c dynamics, we investigated their relationship: In particular, we looked for the existence of circadian modulation in the Ca2+ spiking frequency. We also investigated the nature of spontaneous Ca2+ spikes in SCN based on two different Ca2+ indicators, fluo-4 dye and yellow cameleon.Knowing that SCN cells support two rather temporally distinct c responses from multiple cells in a single slice. We used the second method to make simultaneous measurements of [Ca2+]c concentrations and membrane potentials in single SCN neurons, also by loading fluo-4 through patch micro-electrodes. The patch micro-electrodes were back-filled with an internal solution containing (in mM) 132 K-gluconate, 8 KCl, 8 NaCl, 10 HEPES, 0.5 MgCl2, 4 Mg-ATP, and 0.4 Na-GTP  and filled with an internal solution containing fluo-4 pentapotassium salt  according to the methods described by Eilers and Konnerth 0, where F0 is the initial fluorescent intensity at the cell body of the SCN neuron) was calculated in order to monitor [Ca2+]c variations.We used two different methods for fluo-4 dye loading into SCN neurons. First, we transferred acute SCN slices into a small home-built staining chamber and incubated them for 1 hr in oxygenated regular ACSF containing 20-µM fluo-4 acetoxymethyl ester , following the methods of Ikegaya et al. in vitro.Yellow cameleon genes YC3.6  linked t2+ imaging analysis at a high sampling rate. In this setup, images were acquired by an iXon EMCCD camera  mounted on an inverted microscope  that was equipped with a 10× objective lens , a mercury arc lamp, and a Dual-view optical module . For excitation we used a filter set  that included an excitation bandpass filter (436±20 nm) and a dichroic mirror . In order to minimize the photo-bleaching, two neutral-density filters  were placed in the excitation light path as well. The fluorescent emission was separated by a dichroic mirror  and two emission bandpass filters (480±30 nm and 535±40 nm) installed in the Dual-view module. An electromagnetic shutter  was placed in front of the lamp house. The shutter control, image acquisition, and online image processing were coordinated by software based on the Andor SDK subroutine packages. A SCN slice that had successful YC expression was transferred to the recording chamber and superfused with warmed oxygenated regular ACSF. The fluorescent emission images were collected every 1.5 seconds for more than 5 minutes. The acquired images were further analyzed by ImageJ  with custom-built software.We used two different imaging setups for the cameleon imaging. The primary setup was designed for Ca2+]c levels in SCN neurons 2 incubator, a mercury arc lamp, an excitation filter , an excitation neutral density filter , a dichroic mirror , and a 20× objective lens . The two emission bandpass filters  are switched by a filter changer wheel . We acquired the resulting image pairs through a cooled CCD camera  at a sampling rate of 1 frame per 3 seconds for detecting Ca2+ spikes or of 1 frame per 10 minutes for detecting circadian [Ca2+]c oscillations. Argus-HiSCA imaging software (Hamamatsu) was used to control an electromagnetic shutter , a filter changer wheel, and image acquisition.The second imaging setup was based on the original cameleon imaging system created for the long-term monitoring of [Ca2+ spiking responses to different levels of fluo-4 AM concentration.Means were calculated along with standard errors. Single pairwise comparisons were analyzed with the two-tailed Student's t-test. A one-way analysis of variance (ANOVA) was used to compare the population percentage of differently prepared SCN slices and the different Ca2+]c activities over a duration of 800 seconds. Time traces exhibiting Ca2+ spikes are shown in 2+ spikes. Collectively, our extensive set of data showed no indication of circadian variation. In other words, unlike the AP firing frequency of SCN neurons, the Ca2+ spiking frequency showed no circadian variation  transfected with pCMV/YC and in 0.6±0.6% of the cells (number of slices = 5) transfected with pNSE/YC. In the mouse slice cultures, Ca2+ spikes were observed only in 1.2±0.7% of the cells (number of slices = 5) transfected with pCMV/YC and were not observed at all in cells transfected with pNSE/YC (number of slices = 5). These percentages were much smaller than those for the acute rat SCN slices that were stained with fluo-4 AM  for 5 minutes during different circadian phases  (2+ spikes (9 slices).To understand the lack of CaCN cells . At a co+ spikes  in 14% o slices) . Since b2+]c and the AP firing pattern in individual SCN neurons. Vm). In many cases, this regular pattern changed significantly with an addition of fluo-4 (pipette concentration in the range of 80–120 µM). As the dye diffused from the patch pipette into the target neuron, during the first 100 seconds or so, the AP firing rate gradually decreased from 7 Hz to 3 Hz, and at around 150 seconds the AP activity disappeared rather quickly . When the dye concentration was in the intermediate range of 80–120 µM, about half (9 out of 18 cells in 8 different slices) of the tested SCN neurons exhibited Ca2+ spikes, and they showed a decrease in Vm of 6.7% (P<.05 by Student's t-test).Regarding the Ca2+ spikes was not a simple function of fluo-4 AM concentration. We observed quite heterogeneous responses following a stepwise increase of fluo-4 AM concentration . For the next majority of the cells , there was no systematic trend , while the amplitude could be decreasing, unchanging, or increasing, depending on the individual cells. In other words, the systematic trend in the frequency did not guarantee the same trend in the amplitude.The frequency of induced Caic trend  both in 2+]c of SCN neurons are known to have circadian rhythms. Also reported was the existence of [Ca2+]c transients in SCN having a typical duration of tens of seconds. Here, we termed these Ca2+ transients as “Ca2+ spikes.” One immediate question following these observations would be if the Ca2+ spiking activity of SCN neurons can exhibit any circadian variation similar to the circadian AP firing activity, and here we studied that issue. The level of [Ca2+]c was visualized by Ca2+ sensitive fluorescent protein cameleon as well as fluo-4 AM dye. Many fluo-4-loaded SCN cells exhibited Ca2+ spikes spontaneously, but our extensive set of fluo-4 imaging experiments based on 82 acute SCN slices found no day/night variation in the Ca2+ spiking rates (2+ spiking activities of SCN cells during the night time were as strong (or weak) as those of day time, or vice versa. In our earlier studies that investigated the progressive changes in [Ca2+]c for the duration longer than a circadian cycle, we used organotypic cultures of SCN slices that were transfected by pNSE/YC genes 2+]c was estimated with YC2.1 at every 10 minute in order to minimize photo-bleaching: with this slow image acquisition rate, the presence or the absence of Ca2+ spikes could not be determined 2+]c images were acquired at every 3 seconds to monitor Ca2+ spiking events. The cameleon YC3.6 is a newer version of YC2.1 giving a better spatiotemporal resolution 2+]c, just as in the YC2.1-transfected cultures. However, in the same preparations almost no Ca2+ spike was observed even at the peak of [Ca2+]c circadian oscillation  and concentration are two important factors to be considered. High-affinity chelators trap calcium efficiently, and the presence of them at high concentration can sequester intracellular free Ca2+ significantly. We believe that the affinity (Kd = 345 nM) and the concentration (5∼20 µM) of fluo-4 were high enough to interfere with the intrinsic calcium kinetics of SCN neurons and resulted in non-intrinsic Ca2+ spikes. The affinity of cameleon YC3.6 (Kd = 250 nM) is 1.73 times higher than that of fluo-4 (Kd = 345 nM). Therefore, we suspect that the cytosolic concentrations of cameleon protein expressed within the SCN neurons were quite lower than that of fluo-4 dye that we used. As for the concentration, another important factor to be considered is the stability of the dye. It is well known that cameleon is far more stable than any BAPTA-based calcium indicators with respect to dye leakage, exhaustion, or cumulative photodestruction When the chelation by indicator dye becomes an issue, its affinity , may play a critical role for the function of SCN neurons 2+ buffering proteins and their localization within the SCN may differ depending on the particular animal species 2+ kinetics at a given time.Previous research has shown that Ca2+ dyes often compete with the Ca2+ buffering system of involved cells. In fact, the Ca2+ chelating effects of fura-2 could be used to estimate the capacity of the Ca2+ buffering systems in various types of cells 2+ spikes . On the other hand, the possible existence of such neurons in SCN has never been addressed nor been documented in the past.Our experimental studies demonstrated that CaFigure S12+ spiking activities in cultured rat hypothalamic neurons that express yellow cameleon (pNSE/YC). Time series (sampling rate at 1 frame per 1.5 seconds) of the level of [Ca2+]c in two different hypothalamic neurons exhibit robust synchronized Ca2+ spiking activities.Spontaneous Ca(0.20 MB DOC)Click here for additional data file.


D) of mammalian rod photoresponse recovery is the deactivation of the active phosphodiesterase (PDE6). Physiologically relevant Ca2+-dependent mechanisms that would affect the PDE inactivation have not been identified. However, recently it has been shown that τD is modulated by background light in mouse rods.The rate-limiting step that determines the dominant time constant . We show a novel static effect of calcium on mouse rod phototransduction: Ca2+ shortens the dominant time constant (τD) of saturated photoresponse recovery, i.e., when extracellular free Ca2+ is decreased from 1 mM to ∼25 nM, the τD is reversibly increased ∼1.5–2-fold.We used D during low Ca2+ treatment is not due to increased [cGMP], increased [Na+] or decreased [ATP] in rod outer segment (ROS). Also it cannot be due to protein translocation mechanisms. We suggest that a Ca2+-dependent mechanism controls the life time of active PDE.We conclude that the increase in τ The intracellular calcium concentration ([Ca2+]i) is generally in the order of 100 nM while the extracellular Ca2+ level ([Ca2+]o) is above 1 mM. The steady-state concentration difference is set by delicate control of calcium influx through various types of calcium channels and by the extrusion of calcium ions through Na+/Ca2+ exchange  and Ca2+-ATPases. This kind of dynamic control principle ensures that all the changes in the intracellular [Ca2+] are transient in nature, no matter whether the release of calcium is from intracellular stores or from the extracellular space, and this allows the wide use of calcium ion as internal transmitter in various types of signalling mechanisms.In all eukaryotic cells a very steep gradient of Ca2+]i during photoresponses are used as negative feedback signals in phototransduction. In mouse rods [Ca2+]i is ∼250 nM in darkness and declines to ∼20–50 nM during bright illumination 2+ accelerates both the synthesis of cGMP 2+]i is known to lower the affinity of the cGMP-gated channels to cGMP In vertebrate photoreceptors in darkness, calcium enters the outer segment through cGMP-gated channels and is extruded by the NCKX. Light closes cGMP-gated channels, which reduces the influx of calcium while the calcium extrusion by the NCKX is continued, leading to a decrease in the intracellular calcium concentration. These changes in [Ca2+]i) do not affect the output signal through the mechanisms of the static effect. In this study we demonstrate a novel effect of calcium on mouse rod phototransduction: calcium shortens the dominant time constant of saturated photoresponse recovery (τD). Our data suggests that in mouse rods the intracellular calcium concentration needed to generate this effect may be below the values of [Ca2+]i attained in bright light. Hence we call this effect of calcium on τD static. We show that τD is 1.5–2 times larger in very low free [Ca2+]o (25 nM) compared to that in physiological [Ca2+]o (1 mM). The effect of low external calcium was fully reversible, since the physiological value of τD was completely restored upon return to normal calcium even after long (>1 hour) exposures to low Ca2+. We show that the increase of τD in low Ca2+ was not due to high [cGMP]i that results from the extensive activation of guanylate cyclase (GC) in low Ca2+. Also we show that neither highly increased ATP consumption nor increased intracellular sodium concentration can explain the increase of τD in low Ca2+. We suggest that in mouse rods there is a Ca2+ -dependent mechanism that controls the life-time of active PDE, and that a certain minimum level of intracellular calcium is needed in setting the physiological value of τD.In addition to the dynamic effects described above, calcium ions also may have static effects on signalling. By “static effects” of calcium we refer to calcium-dependent mechanisms that participate in setting the operating point of a system (here phototransduction machinery) while moderate changes in the feedback signal (here [Casat) to the moment when the responses had recovered 20% from saturation (i.e. from the plateau level) as a function of stimulus strength in logarithmic scale. A linear regression line fitted to the data points (black squares) gives a dominant time constant τD of 161 ms  which is comparable to τD-values obtained for mouse rods by the double-flash ERG method in vivoD = ∼0.2 s 2+ solution with ∼25 nM free [Ca2+] (see Methods). When the retina was exposed to low Ca2+ solution, the saturated response amplitude showed a large initial increase (up to ∼4-fold). However, at 37°C these large responses could not be maintained long, and the saturated response amplitudes dropped quite fast (within ∼10 min) to the values ∼50–100% larger compared to normal Ca2+. After this the responses continued to decrease more slowly, being only somewhat larger than in normal Ca2+ after about 30 minutes exposure to low Ca2+. The responses in panel B were recorded between 22–32 minutes after the solution change to low Ca2+. sat data from these responses (red circles), showing that τD increased from 161 in normal Ringer to 231 ms in low Ca2+. A similar increase was observed in all four similar experiments at 37°C . This novel Ca2+-dependent effect on τD is investigated in the following sections.2+ directly increases the single channel conductance of cGMP-gated channels 2+-dependent increase of τD, in HEPES-buffered Ringer at 25°C.The isolated mouse retina is a robust preparation that allows long-lasting treatments and experiments not easily attained with e.g. suction pipette recordings. However, the low external calcium concentration we used exerts a heavy metabolic load on photoreceptors: in these conditions intracellular calcium should go low enough to fully activate the calcium-controlled feedback mechanisms  in the rod outer segment even in darkness. The maximal guanylate cyclase activity leads to a substantial increase in [cGMP], and thus in the number of open cGMP-gated channels. Further, low external Ca2+ solution, the saturated response amplitude showed a very large initial increase that was followed by a decrease to a steady-state value ca. 5 times larger than the saturated photoresponse in 1 mM Ca2+ (measured at the plateau level). In other experiments the steady-state increase varied between 2 and 5-fold being 2.6-fold on average (n = 12). Despite of this large increase in the response amplitudes, the recordings in low Ca2+ were extremely stable. After ca. 10 minutes needed to reach a steady state in low Ca2+, the response amplitudes and kinetics remained virtually unchanged for at least 1 hour in most of the experiments.2+ exposure and C; after low Ca2+ exposure) and in 25 nM [Ca2+]free (B). The collected Tsat data of these responses is presented in D increased from 380 ms in 1 mM calcium to 710 ms in 25 nM Ca2+. The effects of low Ca2+ treatment on photoresponse kinetics, sensitivity and τD were always almost fully reversible, indicating that low Ca2+ treatment as such was not harmful to rods. As evident also in 2+ treatment. In this experiment the return to normal Ringer restored τD to its original value of 380 ms. In 12 experiments τD was (mean ± SEM) 342±28 ms in 1 mM calcium and 662±50 ms in 25 nM Ca2+, corresponding to 94% increase in τD when switched to low Ca2+. This value is somewhat larger than the observed 51% increase in τD at 37°C. To test whether the reason for this difference is that the molecular identity of the rate-limiting process of saturated photoresponse recovery changes (e.g. from PDE deactivation to rhodopsin deactivation) between 25°C and 37°C, we determined τD at three different temperatures (24–38°C) from two retinas in HEPES buffered Ringer. If the identity of the rate-limiting mechanism were different at 25°C and 37°C, and if these mechanisms had different activation energies, the slope of the Arrhenius plot would change between 25°C and 37°C. The semi-saturated and saturated photoresponses of x channels in the rod inner segment 2+  at 100 nM free Ca2+, while the τD values at 10 nM [Ca2+]free were 68 and 56% above those in normal Ringer. Although we do not know how much the declines in [Ca2+]o reduce the intracellular calcium concentration at each [Ca2+]o, the large circulating current even at the lowest external calcium concentrations used in this study suggests, that there remains a substantial inward sodium gradient across the outer segment plasma membrane, that can drive the Na+/Ca2+-K+ exchanger to keep the intracellular calcium level below the [Ca2+]o. Therefore it is probable that the ∼two-fold increase in τD takes place only at intracellular calcium levels below those . Finally, response families were recorded under backgrounds of increasing intensity (range from 20 to 6400 Rh*/s per rod), when a steady-state had been achieved in each background. 2+ under a background producing 640 Rh*/s per rod. Panel D collects the results of panels A–C, and for this retina τD was 360 ms in 1 mM Ca2+, increased to 660 ms in 25 nM Ca2+, and was slightly shorter, 600 ms, in 25 nM Ca2+ under the background. In this experiment response families were recorded under backgrounds producing 20, 64, 200, 640, 2000 and 6400 Rh* s−1 per rod. The corresponding τD values were 690, 680, 780, 600, 580 and 540 ms, respectively. Under the strongest background (6400 Rh* s−1 per rod) the saturated photoresponse amplitude was already smaller than in normal Ringer in darkness. Since lowered calcium is known to strongly increase the conductance of cGMP-gated channels in rods 2+ compared to that in normal Ringer without background. With backgrounds exceeding 640 Rh* s−1 τD shortened somewhat with increasing background intensity, but remained significantly larger compared to that in normal Ca2+. These results suggest that the increase in τD during low Ca2+ exposures is not due to a direct or an indirect effect of elevated [cGMP].One of the major consequences of lowered external calcium is the increase in [cGMP] in the ROS. To investigate whether elevated [cGMP] could explain the increase in τD was to increase [cGMP] concentration in the presence of normal Ringer by inhibiting PDE with IBMX. IBMX is known to boost photoresponse amplitudes and decelerate photoresponse kinetics as low Ca2+ treatment does, but to our knowledge, the effect of IBMX on τD has not been investigated before. We did two experiments in which we used two concentrations of IBMX, 30 and 100 µM. Instead of increasing, IBMX decreased τD slightly, being 270 ms in normal Ringer, and 260 and 250 ms at 30 and 100 µM IBMX, respectively. The same experimental protocol was repeated with another retina with similar results. Our results indicate that the level of steady-state [cGMP] is not important in determining τD.Our second approach to investigate the possible effect of elevated cGMP concentration on τ+ ions flowing into the ROS through the cGMP-gated channels 2+ this load is heavily raised: First, the guanylate cyclase is activated, leading to an increase in [cGMP] and thereby to an excess opening of cGMP-gated channels, and secondly, the conductance of single cGMP-gated channels is increased two- to threefold in low Ca2++/K+ ATPase activity increases about 16-fold as the rat retina was switched from 2 mM Ca2+ to Ca2+-free media 2+ the high ATP consumption might drop the ATP concentration in rods to a level low enough to slow down e.g. the phosphorylation processes involved in the phototransduction machinery, and thereby lead to an increase in τD. Further, the increased cGMP-gated conductance in lowered external Ca2+ most likely leads to an increase in [Na+]i that is assumed somehow to impair phototransduction In dark-adapted mouse rods the major metabolic load in regard to ATP consumption comes from the extrusion of Na+]i on τD, we decreased the external sodium concentration from 140 mM to 8 mM in our low Ca2+ solution by replacing sodium with membrane impermeable choline. 2+ and 8 mM Na+ is shown in panel B. In low Ca2+-low Na+ the response amplitudes were not much reduced compared to those recorded in our normal Ringer, in spite of the fact that a large fraction of the current carriers through the cGMP-gated channels were removed. The responses remained very stable in these conditions. In this experiment τD was 400 ms in normal Ringer, increased to 760 ms in low Ca2+-low Na+ solution, and returned to 420 ms when Ringer was restored. Similar results were obtained in two other experiments conducted with the same protocol. The average increase in τD was (mean ± SEM) 174±44% when the perfusate was changed from our normal Ringer to low Ca2+-low Na+ Ringer. These results show that even in conditions where sodium accumulation inside the rods and the excessive ATP consumption are prevented, the exposure of rods to 25 nM Ca2+ results in a similar (or larger) increase in τD as observed in the presence of normal Na+.To test the possible effects of increased ATP consumption and increased , 2) accumulation of Na+ inside rod outer segments D in low Ca2+.Reducing intracellular CaD. In this study, however, we showed that a significant increase of τD was evident also when [cGMP]i was strongly lowered  by background light during low Ca2+ exposure. This leads us to conclude that the occupation of PDE non-catalytic binding sites do not explain the increase of τD in low Ca2+.The occupancy of the non-catalytic cGMP-binding sites of rod phosphodiesterase (PDE6) has been shown to control the GTPase activity of PDE, and the termination of PDE activity is slower when the non-catalytic binding sites are occupied and maximal when the sites are empty 2+ supports very high inward Na+ current in ROS through cGMP channels. In toad the cGMP current increased transiently over 20-fold when switching from 1 mM Ca2+ to 0 Ca2+ plus 2 mM EGTA, but this dark current could not be maintained +/K+ ATPase cannot maintain sodium gradient across plasma membrane in the presence of very high inward Na+ current, and that Na+ most probably is accumulated into the cell. Our experiments, however, suggest that elevated intracellular [Na+] itself does not increase τD in our low Ca2+ conditions. Firstly, the increase in τD is evident also in low Ca2+ Ringer containing only 8 mM Na+. Secondly, we did one experiment (not shown) in which we switched background light on just before introduction of low Ca2+ in order to suppress excessive cGMP-gated current. The intensity of the background light was chosen to keep the saturated response amplitude in low Ca2+ at a level approximately the same as in normal Ca2+ in darkness. Still τD increased in low Ca2+ (with background light) 67% from that in normal Ringer without background.Low Ca2+2+ must exert a very high metabolic load on mouse rods. In accordance with this, Winkler and Riley +/K+ ATPase activity in rat rods when exposed to low Ca2+. The high expenditure of ATP in low calcium may lead to the deficiency of ATP, which might retard phosphorylation processes, especially phosphorylation of active rhodopsin, and thus lengthen the lifetime of R*. Our experiments where we prevented the increase in the circulating current in low Ca2+ with 1) by lowering Na+ by more than one log unit or by 2) application of background light suggest that the larger τD in low Ca2+ is not due to the deficiency of ATP in mouse ROS.It has been calculated that the maintenance of ion gradients dominates the ATP consumption in darkness for mammalian rods even in normal CaD in low calcium could be the retardation of diffusion of the membrane proteins involved in phototransduction. This explanation, however, seems quite improbable: As a first approximation, instead of retardation one might expect acceleration of membrane protein diffusion, since the binding of the divalent calcium to the negative surface charges of disc membranes should rather increase than decrease the order parameter of the membranes, and thereby enhance the diffusion constants of the membrane proteins. Further evidence against this explanation comes from experiments on amphibian rods, where lowering the extracellular calcium level to nM range does not affect the diffusion constant of rhodopsin .One possible explanation for the increase in τD in low Ca2+ is that the removal of calcium might induce translocation of a phototransduction protein or proteins between the outer and inner segments. To test this hypothesis we made fast solution change experiments (not shown) where we rapidly switched from 1 mM to 25 nM external calcium, recorded one response at a moment of about 30 s after the solution had changed at the retina, and immediately switched back to 1 mM calcium. These experiments showed that that τD had increased to its final value within 60 s after the solution change to low calcium. This approach ruled out the role of translocation of phototransduction proteins in τD increase.One further possibility for explaining the elevated τD observed in our low calcium experiments could, in principle, be explained by the deceleration of either PDE or rhodopsin deactivation. If the latter case were true, it would mean that in low calcium conditions PDE deactivation would be faster than rhodopsin deactivation, and now rhodopsin deactivation should be the rate-limiting step. This seems to be very unlikely since calcium-bound recoverin inhibits phosphorylation of rhodopsin 2+]i releases this inhibition and accelerates photoresponse recovery D is the same in wild-type and recoverin knockout mice D seems to be PDE. The physiological significance of this novel mechanism may be that a certain amount of calcium is necessary in setting the operation point of the phototransduction machinery in regard to photoresponse recovery.The seminal study by Krispel et al. (2006) The use and handling of all the animals in this study were in accordance with the Finnish Act on Animal Experimentation (62/2006) with guidelines of the Animal Experimentation Committee of Finland.+ 133.9, K+ 3.3, Mg2+ 2.0, Ca2+ 1.0; Cl− 143.2, glucose, 10.0; EDTA, 0.01; Hepes, 12.0, buffered to pH 7.5 (at room temperature) with 5.8 mM NaOH. Sodium-L-aspartate (2 mM) was added to block synaptic transmission to second-order neurons. In the experiments at 37°C the Ringer contained (mM): Na+, 115.3; K+, 3.3; Mg2+, 2.0; Ca2+, 1.0; Cl−, 124.6; glucose, 10.0; EDTA, 0.01; HEPES 10.0; NaOH, 4.8 mM; NaHCO3, 20 mM. DL-2-Amino-4-phosphonobutyric acid  was used to block synaptic signaling from photoreceptors to ON-bipolar cells. The Ringer was pre-heated to ca. 37°C and bubbled with a mixture of 95% O2 and 5% CO2 Leibovitz culture medium L-15 (Sigma), 0.72 mg/ml, was added to improve the viability of the retina. In addition in the experiments at 25°C, BaCl2 (10 mM) was added in the lower electrode space, from where it would diffuse to the retina to suppress glial currents by blocking potassium channels located mainly at the endfeet of Müller cells 2 was added to the perfusate, instead, because the 10 mM BaCl2 in the lower electrode space seemed to be less effective probably due to higher perfusion rate at 37°C. Pharmacological manipulations of the standard solution are explained below. The temperature was controlled by a heat exchanger below the specimen holder and monitored with a thermistor in the bath close to the retina.Pigmented mice (C57Bl/6N) were dark-adapted overnight. The animals were sacrificed, the eyes were enucleated and bisected along the equator, and the retinas were detached in cooled Ringer under dim red light. The isolated retina was placed in a specimen holder The transretinal voltage changes (ERG) were recorded with two Ag/AgCl pellet electrodes, one in the subretinal space and the other in chloride solution connected to the perfusion Ringer through a porous plug. The DC signal was sampled at 200–10000 Hz with a voltage resolution of 0.25 µV.The flashes used to produce short flash stimuli with homogeneous full-field illumination to the distal side of the retina were provided by a dual-beam optical system adapted from the setup used in −2 s−1) incident on the retina (I0) was measured in each experiment with a calibrated photodiode . The amount of isomerisations (Rh*) produced by the stimulating flash light in individual rods was calculated as described earlier The absolute intensity of the unattenuated laser beam (photons µm2+]free (7.5–100 nM) solutions calcium was buffered with EGTA, and the free [Ca2+] was calculated using an “EGTA calculator” 2+] present in our Ringer's solution. The low Na+ solution was prepared by substituting 132 mM [choline chloride] for NaCl, yielding 8 mM [Na+].All the chemicals were purchased from Sigma-Aldrich Inc. In the lowest [CaD was extracted by determining the time for semisaturated or saturated photoresponses to recover to a criterion level (Tsat) which was chosen as 20–30% of the saturated photoresponse amplitude measured at plateau level after the nose sat values were plotted against the natural logarithm of normalized flash intensity (Ln(I/I0)) and τD was then the slope of the fitted straight line.The dominant time constant τD) data as a function of T−1 in the temperature range 24–38°C. There, k is the rate constant of the reaction, Ea is the activation energy of the reaction (J/mol), R is molar gas constant and T is the absolute temperature of the retina and A is a temperature-independent pre-exponential factor. Ea was calculated from the slope of the fitted straight line (Ea  =  R*slope).The Arrhenius equation


In the slice preparation, this form of LTP requires a rise in postsynaptic Ca2+-concentration and activation of Ca2+-dependent signalling pathways. Here, we investigated the signalling mechanisms underlying this novel form of LTP in vivo. We found that the signal transduction pathways causing LFS-induced LTP in vivo include activation of neurokinin 1 and N-methyl-D-aspartate receptors, rise of [Ca2+]i from intracellular stores and via T-type voltage-dependent Ca2+ channels, activation of phospholipase C, protein kinase C and Ca2+-calmodulin dependent kinase II. These pathways match those leading to hyperalgesia in behaving animals and humans. We thus propose that LTP induced by low-level activity in C-fibres may underlie some forms of hyperalgesia.Inflammation, trauma or nerve injury trigger low-level activity in C-fibres and may cause long-lasting hyperalgesia. Long-term potentiation (LTP) at synapses of primary afferent C-fibres is considered to underlie some forms of hyperalgesia. In previous studies, high- but not low-frequency conditioning stimulation of C-fibres has, however, been used to induce LTP in pain pathways. Recently we could show that also conditioning low-frequency stimulation (LFS) at C-fibre intensity induces LTP  In fact, low-level presynaptic activity was either ineffective or induced synaptic long-term depression (LTD) rather than LTP in previous studies.Low-level activity between 1–10 imp·s2+]i and Ca2+-dependent signal transduction pathways -N-(4methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine , the phospholipase C (PLC) blocker 1- -trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione , the neurokinin receptor 1 (NK1 receptor) blocker cis-2-(Diphenylmethyl)-N- [(2-iodophenyl)methyl]-1azabicyclo [2.2.2]octan-3-amine oxalate salt  and the ryanodine receptor blocker dantrolene  were dissolved in DMSO. Dantrolene was further dissolved in standard solution titrated to a pH of 9.5. All other drugs were further dissolved in artificial cerebrospinal fluid to obtain desired concentrations as indicated and applied directly onto the spinal cord i might shift the threshold from LTP towards LTD i, shifting synaptic plasticity from LTP towards LTD. This illustrates that blocking different routes of [Ca2+]i rise may have differential effects on synaptic plasticity.The thresholds for the induction of LTP and LTD have been suggested to be tuned narrowly. Changes in the level of elevation of i may be increased by the release of Ca2+ from intracellular stores i rise is further enhanced by Ca2+-induced Ca2+ release from ryanodine sensitive stores (8.). CaMKII is activated by an increased [Ca2+]i and phosphorylates AMPA receptors, thereby increasing their channel conductance. Its activity is also necessary for translocation of AMPA receptors to the subsynaptic membrane [Based on the results of the present study, the following sequence of events leading to the induction of LTP at synapses of primary afferent C-fibres can be suggested Fig. : LFS of membrane . This ulThe authors declare that they have no competing interests.RD and JS participated equally in the conception, design and interpretation of the study, RD performed all experiments and data analysis and drafted the manuscript. Both authors read and approved the final manuscript.


Voltage ramps applied over 300 ms from -100 to 100 mV resulted in membrane currents with marked inwardly- and outwardly-rectifying components. Paxilline (2 μM) abolished the outwardly-rectifying current. Outwardly-rectifying on-cell patch currents were increased markedly by menthol (100 μM) added to the bath. The estimated on-cell conductance of these channels was 253 pS. Kinetic analysis showed that added menthol increased channel open probability and mean open frequency after 5 min. In a similar time course menthol increased [Ca2+]i, and this increase was abolished either by added paxilline, tetraethylammonium ion or by Ca2+-free external solution. Finally, menthol stimulated the rate of DBTRG cell migration into scratch wounds made in confluent cells, and this also was inhibited by paxilline or by tetraethylammonium ion. We conclude that menthol, a TRPM8 agonist, increases DBTRG cell [Ca2+]i that in turn activates membrane BK ion channels. Inhibition of BK channels by paxilline reverses menthol-stimulated increase of [Ca2+]i and of cell migration. Thus, BK channels function to maintain elevations in [Ca2+]i needed to sustain increases in DBTRG cell migration.This study examined the effect of menthol, an agonist for transient receptor potential melastatin 8 (TRPM8) ion channels, to increase intracellular Ca Glioblastoma multiforme (GBM) has a particularly grim prognosis with a median survival time of 15 months i, that is necessary for cell migration and, presumably, tumor invasion i [Others have shown that the large-conductance Cama cells . These B [Ca2+]i ,8. They  [Ca2+]i .2+]i necessary for stimulating glioblastoma cell migration, and that blocking BK channels abolishes the menthol-stimulated increase in [Ca2+]i as well as cell migration.In this study we report the integrative function of BK ion channels in relation to the increase in cell migration by menthol, the putative agonist of TRPM8 ion channels. We show that menthol increases , 10 glucose, and pH 7.4 (1N HCl). In some instances KCl was increased from 6 mM to 60 mM by isosmotic substitution for NaCl. Whole-cell voltage clamp pipettes were fabricated by means of a Brown/Flaming micropipette puller  and were filled with (in mM): 140 KCl, 6 CaCl2 , 2 MgCl2, 11 EGTA, 50 HEPES, and pH 7.2 (1N KOH). The pipettes were 4-5 MΩ in the bath solution, and whole-cell voltage-clamp measurements were performed by standard technique i was measured by inverted microscopy (AccuScope 3030) [2+] was determined by interpolation from a standard curve generated from a Ca2+ calibration buffer kit #2 (Molecular Probes) and Fura2/K5-salt.Cells on glass coverslips were loaded with Fura2 and i and increases the rate of migration of DBTRG cells [Since added menthol increases Ca+i and in2+]i and, therefore, to BK channels, we measured effects of menthol on DBTRG [Ca2+]i. Menthol increased DBTRG [Ca2+]i within 5 to 10 min, Figure 2+]i increased markedly and appeared in two waves. The increase in [Ca2+]i reversed rapidly and completely either by eliminating nominal Ca2+ from the external solution, Figure 2+]i since it inhibits BK channels in human glioma cells [2+]i, but the inhibition was slower and smaller than that of paxilline, Figure To test further whether this effect of added menthol on ion channel activity might be attributable to increased [Cama cells . TEA als2+ influx into DBTRG cells. As an initial step to test this possibility we determined that the membrane depolarization from a ten-fold increase in external [K+] resulted in a reversible decrease in menthol-stimulated [Ca2+]i, Figure It is likely that BK channel activity increases or sustains the transmembrane potential, which in turn maintains a favorable electrochemical potential for Ca2+]i in DBTRG cells, we performed experiments to determine whether paxilline or TEA affect menthol-stimulated migration of DBTRG cells i and, thus, increases the rate of cell migration [2+]i activate membrane BK ion channels. The latter are widely expressed in human glioma cells i. The latter increases BK channel open probability and open frequency. Surprisingly, the rise in [Ca2+]i did not increase channel mean open time, which suggests an alternatively spliced isoform of the α subunit gene i is relatively high, and the onset of this increase varies with time. The latter may result from autocrine/paracrine secretion of hepatocyte growth factor, whose concentration in the medium and ancillary effects on [Ca2+]i may depend on cell density [2+]i by its release from the endoplasmic reticulum and by its influx. So, it is noteworthy that menthol-stimulated rise in [Ca2+]i occurred in successive waves. Whether this involves store-operated Ca2+ entry requires further investigation; however, it also is noteworthy that Orai1 and STIM1 are critical for breast tumor cell migration and metastasis i by both added paxilline/TEA and Ca2+-free external solution indicates that BK channels play a role in maintaining elevated [Ca2+]i. Here the activation of BK channels may sustain or increase the magnitude of the transmembrane potential and, thus, maintain a favorable electrochemical potential for cation influx. Indeed, increasing external K+ from 6 to 60 mM by isomolar substitution for Na+ resulted in rapid diminution of [Ca2+]i that reversed on restoration of control solution.The reversal of menthol-stimulated increases in [Ca2+]i is necessary for DBTRG cell migration, but the present study does not differentiate between global versus local increases in [Ca2+]i. It is becoming increasingly evident that Ca2+-activated K+ channels are controlled by nano/micro domains of [Ca2+]i [2+-activated K+ channels may function to maintain local increases in [Ca2+]i necessary for molecular events of cell migration either at the membrane microdomains of the advancing lamellipodium [2+]i and cell migration.The rise in [Ca [Ca2+]i ,26. This [Ca2+]i . Scratch [Ca2+]i , which ilipodium . We cannlipodium ,16. More2+]i which, thereby, increases activity of BK ion channels. Inhibition of BK channels by paxilline or TEA abolishes the menthol-stimulated increases in [Ca2+]i and cell migration. We conclude that BK ion channels play an important role in cellular mechanisms that increase [Ca2+]i needed to increase the rate of cell motility and tumor invasion.We have shown that menthol, a known agonist of TRPM8 ion channels found in DBTRG human glioblastoma cells, stimulates the migration of DBTRG human glioblastoma cells into scratch wounds and increases i: intracellular Ca2+ concentration; FBS: fetal bovine serum; HEPES: N-(2-Hydroxethyl)piperazine-N'-(2-ethanesulfonic acid); TEA Cl: Tetraethylmmonium chloride.GBM: Glioblastoma multiforme; DBTRG cells: human glioblastoma cells; TRPM8: transient receptor potential melastatin 8 ion channel; BK: large-conductance CaThe authors declare that they have no competing interests.RW conducted the electrophysiology, cell migration studies, and statistical analyses and drafted the manuscript. JWB carried out calcium measurements and helped draft the manuscript. Both authors read and approved the final manuscript.


MicroRNAs (miRNAs) are short non-coding RNAs that have key functions in a wide array of critical cell processes, including haematopoiesis by regulating the expression of multiple genes. Aberrant miRNA expression has been described in acute myeloid leukaemia suggesting a role in leukaemogenesis. In this review we summarise the current knowledge. FLT3) gene, mutations in the nucleophosmin (NPM1) gene, CCAAT/enhancer binding protein-α (CEBPA) gene and in the Wilms’ tumour gene and partial tandem duplication (PTD) of the mixed-lineage leukaemia (MLL) gene (reviewed by ERG (v-ets erythroblastosis virus E26 oncogene homologue) and BAALC  genes has been found in CN-AML (NPM1 mutations predict favourable outcome in young CN-AML in the absence of FLT3-ITD (reviewed by CEBPA mutations identifies a group of patients with a better prognosis within the young high-risk CN-AML group (patients with FLT3-ITD and/or wild-type (wt) NPM1)  without effect on overall survival (reviewed by Acute myeloid leukaemia (AML) arises from myeloid progenitor cells that are arrested at early stages of differentiation. It is a cytogenetically heterogeneous disorder with acquired recurrent chromosomal alterations detected in about 55% of adult patients, such as translocations ; t; t), inversions (i.e. inv(16)), deletions (i.e. del(7q)), trisomies (i.e. +8) and monosomies (i.e. −5/−7)  microarrays have been used to interrogate whole gene expression in AML samples. Indeed, several groups have reported distinctive signatures associated with particular cytogenetic and molecular groups of AML (miR-15a/miR-16-1 in chronic lymphocytic leukaemia (CLL)  has been reported in different human B-cell neoplasms suggesting a role for miR-155 in B-cell lymphomagenesis  followed by a full-blown malignancy.MicroRNAs (miRNAs) are non-coding RNAs of 19–25 nucleotides in length that regulate gene expression by repressing translation or accelerating mRNA decay . MicroRNOver the past 3 years, several groups have described miRNA signatures associated with recurrent cytogenetic abnormalities, molecular aberrations and outcome in AML. In this review we discuss the current miRNA profiling studies in AML and outline future prospects and potential therapeutic applications.Four recent large-scale studies have reported miRNA expression in primary AML blasts and CD34+ selected cells  method  may influence the results. Further studies will be needed using highly pure populations, including CD34+ selected leukaemic cells, to assess miRNA changes with respect to CD34+ normal cells.Only few miRNAs were commonly (in at least two studies) down-regulated in AML patients, such as FLT3-ITD (miR-34b (CDK4 and CCNE2), miR-15a (BCL-2), the let-7 family (RAS), the miR-29 family (MCL-1 and TCL-1) and miR-372 (LATS2). Interestingly, miR-155 was found up-regulated in AML patients with high white count and FLT3-ITD mutations. This miRNA has been recently described to block ‘in vitro’ human myeloid colony formation, halt megakaryopoiesis (let-7b and let-7c (known to target the oncogenes RAS) were found down-regulated in CBF leukaemias , t, inv(16), 11q23 translocations) in 100 AML samples. In particular, acute promyelocytic leukaemias bearing a t had a distinctive signature including the up-regulation of seven miRNAs located on chromosome 14q32 which is not implicated in the translocation , inv(16), t and MLL rearrangements , three  and seven miRNAs (from the polycistronic miR17-92 cluster) are sufficient to predict CBF AML, t and MLL AML respectively, resulting in a diagnosis accuracy better than 94%  AML.Li than 94% . Some ofNPM1 mutation which is the most common genetic alteration in CN-AML. They profiled 85 de novo AML patients (55 with NPM1 mutation and 30 with NPM1 wt) and found a strong miRNA signature that distinguishes CN-AML with NPM1 mutation from NPM1 wt  predicted outcome independent from other variables, including age and cytogenetics. FLT3-ITD and/or an NPM1 wt). This subgroup of AML represents more or less a third of young AML patients. The prognostic signature included miR-181a and miR-181b which were inversely correlated with risk of event and miR-124, miR-128-1, miR-194, miR-219-5p, miR-220a, miR-320 which were positively associated with the risk of event. This prognostic signature was independent from other factors. Furthermore, using mRNA gene-chip microarray analysis, the authors showed an inverse correlation between the expression of miR-181 and predicted target genes. Many of these genes encode for proteins involved in pathways of innate immunity mediated by Toll-like receptors  and interleukin-1β . Activation of Toll-like receptors induces production of inflammatory cytokines through nuclear factor κB and interleukin-1β promotes the survival and the proliferation of AML blasts. No miRNA was found to be associated with outcome in low-risk group  (vs 45 years (range 19–45). This is critical because FLT3-ITD and NPM1 prognostic implications are age dependent. FLT3-ITD has been shown to have a negative prognostic impact only in young AML patients , miR-10a and 196a-1 in HOXB (at chromosome 17q21), miR-196a-2 in HOXC (at 12q13) and miR-10b in HOXD (at 2q31) clusters. A distinctive correlation between the miR-10a, miR-10b and miR-196-1 and the HOXA/HOXB gene expression was first reported in 30 AML patients suggesting a common regulatory mechanism (HOX cluster in a murine leukaemia model (engineered by over-expression of NUP98/HOXD13 fusion gene with the oncogenic collaborator MEIS1).miR-10a, 10b and miR-196b have recently been linked to molecular or chromosomal alterations in subtypes of AML, in particular CN-AML with mutated NPM1. miR-10a, miR-10b in CN-AML with NPM1 mutation. The over-expression was correlated with high expression level of HOX genes (mainly HOXB genes)  is commonly involved in chromosomal translocations (with more than 60 different fusion partners reported) responsible of AML. Leukaemias related with MLL are in most of the cases characterised by an over-expression of a subset of HOX genes including HOXA9  translocation detected in about 15% of AML. The miR-29b in AML cell lines and primary AML blasts down-regulates the expression of DNA methyltransferases; DNMT1, DNMT3A and 3B. MiR-29b targets directly DNMT3A and 3B and indirectly DNMT1 through its activator Sp1. Restoration of miR-29b leads to global DNA hypomethylation and re-expression of tumour suppressor genes such as the cyclin-dependent kinase inhibitor p15INK4b and the oestrogen receptor ESR1 genes , inv(16) and t. Because most of the strategies to identify distinctive miRNA signatures were based on two-way comparisons (i.e. inv(16) vs all other AMLs), the heterogeneity of the other AMLs will impact greatly on the analysis. This bias is less evident when the authors focus on well-characterised cytogenetic and molecular subgroups of AML. Common miRNAs have been found deregulated in CN-AML cases with FLT3-ITD, NPM1 and CEBPA mutated and in patients with t, inv(16) and t. These results are significant because they will provide with a set of validated miRNAs whose functions will be further investigated. These studies may lead to a better molecular classification, understanding of AML biology and discovery of novel targets for treatment. In addition to improving diagnosis, miRNA signatures may enhance current prognostic markers and help to better stratify patients for treatment. But before any clinical applications, miRNA profiling studies require to be extensively validated and more standard, reliable and reproducible platforms will need to be developed. Future miRNA expression studies should use very well cytogenetically and molecularly characterised AML subtypes to minimise biology heterogeneity. Generation of small RNA libraries and next-generation deep sequencing will enable not only to quantify the whole miRNA transcripts but also to obtain sequences information and discover new miRNAs.In this review, we described four recent large-scale global miRNA profiling studies in AML. The differences among the miRNA signatures reported by these studies can be explained mainly by the use of various cytogenetic and molecular AML subgroups and by the type of platform used to interrogate miRNA expressions. The frequencies of cytogenetic/molecular abnormalities were clearly different among the studies. For example, Garzon We also believe that miRNAs studies have potential relevant therapeutic implications. Synthetic miRNAs or anti-miRNAs alone or in association with chemotherapy could be promising in future AML therapies. Synthetic miRNAs can be used to restore lost tumour suppressor miRNAs expression. The other strategy (so far more developed) is to silence oncogenic miRNAs (with ‘antagomirs’ or anti-miRNA oligonucleotides or locked nucleic-acid-modified oligonucleotides) which are over-expressed in AML (Altogether, the data suggest that miRNAs may complement and enhance our current knowledge about AML. MicroRNA expression profiling has been associated with cytogenetic and molecular subtypes of AML. Functional studies suggested miRNAs may play a role in AML pathogenesis being involved in essential pathways for myeloid differentiation. It is now well demonstrated that miRNAs represent a class of genes with a great potential for diagnosis, prognosis and therapy in AML. MicroRNA-based therapy will certainly play a role in the future of treatment of AML.


The major DNA repair pathways operate on damage in double-strand DNA because they use the intact strand as a template after damage removal. Therefore, lesions in transient single-strand stretches of chromosomal DNA are expected to be especially threatening to genome stability. To test this hypothesis, we designed systems in budding yeast that could generate many kilobases of persistent single-strand DNA next to double-strand breaks or uncapped telomeres. The systems allowed controlled restoration to the double-strand state after applying DNA damage. We found that lesions induced by UV-light and methyl methanesulfonate can be tolerated in long single-strand regions and are hypermutagenic. The hypermutability required PCNA monoubiquitination and was largely attributable to translesion synthesis by the error-prone DNA polymerase ζ. In support of multiple lesions in single-strand DNA being a source of hypermutability, analysis of the UV-induced mutants revealed strong strand-specific bias and unexpectedly high frequency of alleles with widely separated multiple mutations scattered over several kilobases. Hypermutability and multiple mutations associated with lesions in transient stretches of long single-strand DNA may be a source of carcinogenesis and provide selective advantage in adaptive evolution. A variety of error avoidance mechanisms assure low mutation rates across the genome. Genetic defects in DNA replication or repair can lead to genome-wide increase in mutation frequency that may result in cancer predisposition and genetic disease. Transient localized hypermutability drastically differs in its biological consequences from genome-wide mutators. Since genome-wide hypermutability can cause reduced fitness due to accumulation of dysfunctional alleles, mutators are under negative selection pressure. By contrast, there would be less selection against temporary hypermutability within limited genomic regions, suggesting a special role in adaptive evolution and carcinogenesis. Mechanisms of transient hypermutability are poorly understood. Long stretches of single-strand DNA have been implicated but not demonstrated as a source of localized transient hypermutability. Using sophisticated yeast genetic systems that we developed, we found that transient stretches of chromosomal single-strand DNA at double-strand breaks and that telomeres can tolerate multiple lesions and are highly prone to damage-induced mutations, including a very unusual class of widely spaced multiple mutations. The hypermutability relied on error prone translesion DNA synthesis. Our work demonstrates a simple in vivo mechanism for localized transient hypermutability extending over several kilobases that can result in widely spaced multiple mutations without severe mutation load in the rest of the genome. Accurate DNA synthesis and repair of DNA lesions assure low rates of mutation. Genetic defects in either lead to increased, genome-wide mutator phenotypes which can manifest in cancer predisposition Examples of unregulated transient hypermutability include multiple mutations Saccharomyces cerevisiae for the generation of defined, persistent long stretches of transient ssDNA in the vicinity of a unique DSB or an uncapped chromosomal telomere. Lesions could be induced in the ssDNA before restoration to dsDNA. The consequences of restoration could be assessed with genetic reporters located within the regions that give rise to ssDNA. We demonstrate here that damage tolerance in stretches of long ssDNA formed at DSBs and uncapped telomeres results in extremely high frequencies of single and widely-separated multiple mutations caused by two major types of DNA damaging agents.While error-prone restoration of damaged long stretches of ssDNA to dsDNA might explain many examples of transient hypermutability, this hypothesis has yet to be substantiated. Long tracts of ssDNA formed at DSBs and uncapped telomeres  are known to cause checkpoint responses, where the fate of the cell is determined by a balance of arrest, adaptation, recovery and repair processes SceI site-specific endonuclease, there is 5′→3′ resection extending up to 20 kb from the break, after which repair can be mediated by a short oligonucleotide spanning the region of repair around DSB SceI and a DSB target was inserted into either LYS2 on the centromere (DSB-cen) side or URA3 on the telomere (DSB-tel) side of the forward mutation reporter CAN1. Placing cells in galactose medium for 3–6 hr led to induction of the DSB and resection. DNA damage was then induced by ultraviolet light (UV) or methyl methane sulfonate (MMS). The cells were subsequently transformed with a pair of 95 nt self-complementary oligonucleotides that also had sequence homology to both sides of the break to allow DSB repair. As described in + or Ura+ colonies (<10−9) were observed in controls lacking oligonucleotides (data not shown).Recently, we demonstrated that, following the induction of a DSB in a yeast chromosome by the I-−5 for the cells with a break at the DSB-cen position and from 14 to 26×10−5 for a break at the DSB-tel position . DNA damage (UV or MMS) increased transformation frequencies in the no-DSB controls, possibly by introducing recombinagenic DNA lesions. However, cells experiencing a DSB followed by exposure to UV or MMS yielded transformation frequencies that were still considerably greater (40–160 times) than for the “no-DSB+DNA damage” controls. Thus, selection of individual DSB repair events resulting from restoration of a disrupted gene by oligonucleotides (Lys+ or Ura+ colonies) provides the opportunity to specifically examine subpopulations that had undergone DSB repair.In the absence of additional DNA damage the median frequencies of DSB-induced transformation in the wild-type varied from 48 to 205×10position  and S2. some VII  and S2, can1 mutants in the total populations of the DSB strains as well as in “no-DSB” control strains that were carried through all stages of transformation were 0.1–1.0×10−4 (can1 mutation frequencies were 50–300 times greater among the Lys+ (or Ura+) selected colonies that arose after DSB induction and oligonucleotide repair. This is in agreement with observations of increased spontaneous mutation rates in the vicinity of a DSB repaired by recombination with a homologous chromosome or with a repeat in the same chromosome can1 mutations among the cells that repaired a DSB were 180- to 1800-fold greater than in the population of the control, no-DSB strains treated with the same agents  did not affect mutagenesis in CAN1  resulting in G2/M arrest. This arrest is associated with up to 15 kb 5′→3′ end resection that can be detected by ssDNA formation in 3–10% of individual chromosome ends We utilized the special features of the LYS2 was inserted near the de novo telomere (LYS2 reporter cdc13-1 cells were UV-irradiated and plated onto rich (YPDA) medium. Mutagenesis was assessed by replicating colonies to –Lys medium. The frequency of lys2 mutants among Lys− colonies or sectors provided an estimate of mutagenesis next to a telomere. The appearance of Lys− mutants in genes other than LYS2 provided a comparison of telomere-associated vs. genome-wide mutagenesis within the same cell population. In addition to LYS2, there are at least 7 genes scattered across the genome mutations in which can lead to lysine auxotrophy (http://db.yeastgenome.org/cgi-bin/locus.pl?locuslys). Frequencies and allelism of Lys− mutants were also determined in UV-irradiated cells that were not subjected to the 37°C arrest (−4) were observed in the no-UV controls. Exposure to 45 J/m2 had little impact on survival of the cdc13-1 mutant regardless of irradiation at 37° or 23°C. However, the frequency of UV-induced lys2 mutants in the 37°C-arrested cells was at least 60 times that in the non-arrested control cells kept at 23°C. The low frequency of genome-wide UV-mutagenesis, as indicated by Lys− auxotrophic mutations at other loci (“Lys− (LYS2 WT)” bars), was not affected by the cdc13-1 arrest at 37°C. Genome-wide location of mutations in the Lys- (LYS2 WT)-category was confirmed by allelism test which assigned more than a half of them to 6 out of 7 genes with expected Lys- mutant phenotype . The density of mutations in NPR2 derived from this direct measurement is 0.36 mutations per kb, which is close to the density of mutations in the more distant telomere LYS2 calculated on the basis of truncated Poisson distribution (χ2)<0.9) indicating that the mutations were independent and that there was no preferential loss of ssDNA molecules with higher density of damage within the population of damaged molecules. However, the relative survival of damaged vs undamaged ssDNA remains to be established using systems that would provide synchronous generation of ssDNA by 5′→3′ resection in all cells (such as site-specific DSB caused by inducible HO-endonuclease).The high incidence of multiple mutations indicates that the level of UV-induced mutagenesis associated with DSB-repair or uncapped telomere arrest is even greater than estimated based simply on frequencies of loss-of-function utations  and 5. Tutations . To estiutations . In the telomere . We lookG1-cells ; howeverribution . The fitmut = 1−P0). This is 10 to 100 times greater than the observed frequencies of UV-induced mutants associated with DSB repair or uncapped telomere arrest  or uncapped telomere arrest (lys2) could be attributed to pyrimidine bases of the unresected strand (cen and DSB-tel). The large majority of pyrimidines mutated in the unresected strand were associated with di-pyrimidines . This supports the view that multiple mutations resulted mostly from independent UV-photoproducts rather than from extended, error-prone DNA synthesis triggered by a single UV lesion.The primary products of UV-damage as well as sources of mutations are cyclobutane pyrimidine dimers and 6-4 photoproducts resulting from covalent linkages of adjacent pyrimidines d strand . Importa1 arrest . Additiotitution  revealedcen strains the URA3 gene was located telomere proximal to CAN1 , a potentially lower probability of URA3 inactivation by mutations as well as to more distant location from a DSB than CAN1. Importantly, all 22 base substitutions identified in URA3 resulted from mutations in pyrimidines in the strand that would remain after 5′→3′ resection into this gene. In total can1 ura3 double mutants  most of which were strand-specific. Based on reported lengths for 5′→3′ resection at a site-specific DSB  of mutation during restoration of damaged ssDNA to complete chromosomes, we investigated the roles of genes involved in translesion DNA synthesis (TLS) and post-replication repair (PRR) on mutagenesis associated with DSB-repair  and S2.REV3, encoding the catalytic subunit of Pol ζ eliminated most of the UV- and MMS-induced mutagenesis while removal of the translesion polymerase Pol ζ (rad30Δ) had no detectable effect  UBC13 or RAD5 genes, responsible for steps in PCNA poly-ubiquitination and error-free damage avoidance by template switching Similar to the other forms of damage-induced mutagenesis e effect  and 3. TWe considered the possible contribution of greater variability and apparent overall reduction in transformation frequencies, regardless of DNA damage, to these results. For example, if transformants arose through recombination between unbroken chromosomal DNA and oligonucleotides LYS2 associated with uncapped telomere arrest depended on the translesion Pol ζ (REV3) and was not affected by elimination of Pol ζ (RAD30) (rad30 mutation caused a moderate increase in the frequency of Lys− (LYS2+) cells independently of uncapped telomere arrest. This could be due to a stronger role for Pol η in error-free TLS among the unidentified LYS genes as compared to LYS2.)Similar to the observations with DSB-repair associated mutagenesis, the UV-induced hypermutability of  (RAD30) .  can be efficiently restored to the ds-state in spite of the presence of multiple UV-induced lesions. Lesions in ssDNA along with ssDNA itself were hypermutable and hypermutability relied on TLS Pol ζ. We established the involvement of a ssDNA intermediate in transient hypermutability based on the strand-biased spectrum of UV-induced single and multiple mutations. Importantly, strand biased multiple mutations provide a useful tool for detecting in vivo mutagenesis in single molecules containing long stretches of damaged ssDNA. UV or any other mutagen with a bias towards certain DNA bases would create a permanent signature of strand-biased multiple mutations in chromosomal regions containing long stretches of ssDNA at the time of acute induction of lesions. Importantly, the density of UV-induced mutations was estimated to be ∼1 per 1.5–3 kb, which is comparable to the density of pyrimidines dimers induced by UV as determined in yeast Transient long stretches of ssDNA can form in several ways. Tens of kilobases of ssDNA can be formed in yeast by 5′→3′ resection at site-specific DSBs In addition to end-resection, there are several possible sources of ssDNA. Long ssDNA has been detected in cultured human lymphoma cells, although the mechanism of generation has not been ascertained We observed a high incidence of widely-spaced multiple mutations caused by damage in ssDNA. Multiple mutations are an important genetic phenomenon that may affect species evolution and cancer incidence MATα ade5-1 his7-2 leu2-3,112 trp1-289 ura3Δ All strain constructions were performed using methods of PCR-based gene disruption and direct genome modification by oligonucleotides as described in (kanMX4) module in place of the chosen ORF. Double-deletion strains were obtained by switching the G418 resistance marker to nourseothricin resistance (kanMX4 to natMX4) followed by kanMX4 insertion to replace the second ORF. All construction steps were verified by phenotype and by PCR. The site-specific pol30-K164R mutant was generated as described in TRP5 gene in the chromosome VII used in control experiments has been described All yeast strains were isogenic to CG379 LYS2 gene was inserted after position 34,193 nt in the chromosome V with the help of the tailed PCR primers: oYY_a - TTCTTACTCAGTGTGAACGTGTTCTAAATAAGTTCTTGTTCTAATTAATT-TAAGCTGCTGCGGAGCTTCC and oYY_b - AGATACGATTACTCCAGTTCCTCTTACAAGAAATGCATAAAAATAGTTAC-AATTACATAAAAAATTCCGGCGG . Then, a CORE-I-SceI cassette containing the I-SceI gene under control of the GAL1 promoter, the hygromycin resistance gene (Hyg) and an I-SceI site was placed inside the LYS2 flanked by the following sequences: GCTTGCCGACGGCGGCTAAGCTCATAACATTGATAGTTGAAATAACATTTGGA - telomere proximal and CACAGTTGATATAATTATCCATAATGGTGCGTTAGTTCACTGGGTTTATCCATATGCCAAATTGAGGGA–centromere proximal (insertion strategy and sequences of oligonucleotides are available upon request). At the second step the URA3 gene was inserted after position 29,682 in chromosome V with the help of the tailed PCR primers: oYY_5 - CAGTCTTTATATCAGTCTTTGCATGGCTTTGCATCTGATGCTGGCTCTACCGACTTCTCG-CAGAGCAGATTGTACTGAGAGTGCACC and oYY_6 - ACTCAAATATTTATCCACTTTGGATAGTATATACGTCAAATTCTTTTGGTATTTTATCGC-CGCATCTGTGCGGTATTTCACACCGC . Two independent isolates, YY-22 and YY-24 were used as wild type DSB-cen strains for this study. The control “no DSB” strains (YY_122 and YY_124) differ from YY_22 and YY_24 in that they contain uncuttable, incomplete  I-SceI sites.At the first step of construction the CAN1 (DSB-tel) we first transformed YY_22 and YY_24 strains to Lys+ with “repairing” oligonucleotides oYY_15 and oYY_16 (see below) and selected the removal of the Gal-I-SceI-CORE insert from the LYS2 gene. Then, a CORE-I-SceI cassette containing the I-SceI gene under the GAL1 promoter, hygromycin resistance gene (Hyg) and an I-SceI site was placed inside the URA3 gene flanked by the following sequences: CAAGGAATTACTGGAGTTAGTTGAAGCATTAGGT  and TATCCACATGTGTTTTTAGTAAACAAATTTTGGG  to produce two identical DSB-tel strains YY-266 and YY-267.In order to place the self-generating DSB cassette on the telomere side of de novo telomere we inserted Gal-I-SceI CORE after 34 193 nt at the left end of chromosome V (sequence context: TTCTTACTCAGTGTGAACGTGTTCTAAATAAGTTCTTGTTCTAATTAATT–on the telomere side and GTAACTATTTTTATGCATTTCTTGTAAGAGGAACTGGAGTAATCGTATCT–on the centromere side). After induction of the DSB, cells were transformed with the oligonucleotide TGTGTGTGGGTGTGGTGTGTGTGTGGGTGTGGTG-GTAACTATTTTTATGCATTTCTTGTAAGAGGAACTGGAGTAATCGTATCT consisting of a short stretch of T(G)1–3 telomeric repeats (shown in bold) followed by sequence homologous to the DSB-end on the centromere side. Loss of CORE was selected and isolates with end-truncation and de novo telomere V were identified by both Southern hybridization of the PstI-digested genomic DNA with NPR2-probe and by PCR with the following pair of primers: CA-16–CACCACACCCACACAC, telomere repeat-specific and Tel5-FS-npr2–GAACATTTTGCCCAGCCTAGTA, NPR2-specific. Based on these tests the size of added telomeric repeats was 300–500 nt (not shown). The LYS2 ORF with promoter was amplified with targeting tailed primers oYY_9 (TAAGCTGCTGCGGAGCTTCCTAATATTACAACTTATTTCCGTAAATAAAGATAGTACACACGAATCCAAACGTTTATATAGTTAGCTCTG-) and oYY_10 (AATTACATAAAAAATTCCGGCGGAGACAGAAGAGAAGGGTGTGAAACCACCTCTACCAAACACACCAAGAGATGAACCTAAATCAAATTTTCA-) and inserted between NPR2 and CIN8 close to the de novo telomere . cdc13-1 strains were always grown at permissive temperature 23°C. Deletion mutants in the genes rev3 and rad30 controlling TLS polymerases were obtained in the strain DAG_760. Two independent deletion isolates was studied for each genotype.In order to generate a chromosome end-truncation and also create a telomere . Strain SceI expression for generation of a site-specific DSB. After 3 or 6 hr of growth in galactose cells were washed once with 50 ml dH2O and DNA damage was applied to the yeast suspension. The “No-DSB” control strains went through the same incubation and transformation steps as the strains that experienced an inducible DSB. In the case of UV, yeast were suspended in 25 ml of dH2O (∼106 cells/ml), placed into a 150 mm Petri dish and irradiated with UV-C  with continuous agitation for 20 or 45 seconds. In the case of MMS, cells were suspended in 5 ml of 50 mM sodium phosphate buffer (pH 7.0) (∼107 cells/ml) and then treated with 11.8 mM (0.1%) MMS for 15 or 30 min. MMS was inactivated by adding Na2S2O3 to a final concentration of 5% (w/v) for 2 minutes. Treated and untreated yeast were washed once with 50 ml water followed by transformation to Lys+ or Ura+ in DSB-cen or DSB-tel strains, respectively, with a pair of complementary oligonucleotides (AvaII (DSB-cen) or ApaI (DSB-tel). DSB-cen strains were transformed with a pair of oligonucleotides oYY_15–GGAcCCAAATGTTATTTCAACTATCAATGTTATGAGCTTAGCCGCCGTCGGAATGGTGCGTTAGTTCACTGGGTTTATCCATATGCCAAATTGAG and oYY_16 - GgTCCCTCAATTTGGCATATGGATAAACCCAGTGAACTAACGCACCATTCCGACGGCGGCTAAGCTCATAACATTGATAGTTGAAATAACATTTG; DSB-tel were transformed with a pair of oligonucleotides oYY_47–GGgCCCAAAATTTGTTTACTAAAAACACATGTGGATATCTTGACTGATTTTTTGTTCGTACCACCAAGGAATTACTGGAGTTAGTTGAAGCATTA and oYY_48 - GGGcCCTAATGCTTCAACTAACTCCAGTAATTCCTTGGTGGTACGAACAAAAAATCAGTCAAGATATCCACATGTGTTTTTAGTAAACAAATTTT. can1 mutants were screened among Lys+ or Ura+ transformant colonies after replica plating onto media with 60 mg/ml of L-canavanine.Yeast strains were grown with agitation in liquid rich media (YPDA) for approximately 20 hr and then diluted 37 fold with fresh 2% galactose synthetic complete media to induce Gal-I-leotides  introduccdc13-1 strain DAG_760 or its derivatives with additional mutations in TLS polymerases, carrying LYS2 next to the de novo telomere in the truncated left arm of the chromosome V . All suspensions were plated onto YPDA to yield around 100–500 colonies per plate. Colonies were replica plated onto synthetic complete and lysine deficient media. Single cell isolates from Lys− colonies or sectors were then verified for the phenotype and checked for allelism to the LYS2 gene.The mosome V , were grTable S1can1 mutants associated with DSB-repair.Frequencies of spontaneous and UV-induced (0.03 MB PDF)Click here for additional data file.Table S2can1 mutants associated with DSB-repair.Frequencies of spontaneous and MMS-induced (0.02 MB PDF)Click here for additional data file.Table S3− mutants associated with uncapped telomere arrest.Frequencies of UV-induced Lys(0.02 MB PDF)Click here for additional data file.Table S4can1 - no DSB; UV, 45 J/m2”.Mutation spectrum in the category “(0.02 MB PDF)Click here for additional data file.Table S5can1 - DSB-cen; no UV”.Mutation spectrum in the category “(0.02 MB PDF)Click here for additional data file.Table S6can1 - DSB-cen; UV, 20 J/m2”.Mutation spectrum in the category “(0.02 MB PDF)Click here for additional data file.Table S7can1 - DSB-cen; UV 45 J/m2”.Mutation spectrum in the category “(0.02 MB PDF)Click here for additional data file.Table S8can1 (from can1 ura3 set) - DSB-cen; UV, 45 J/m2”.Mutation spectrum in the category “(0.02 MB PDF)Click here for additional data file.Table S9ura3 (from can1 ura3 set) - DSB-cen; UV, 45 J/m2”.Mutation spectrum in the category “(0.02 MB PDF)Click here for additional data file.Table S10can1 - DSB-tel; UV, 45 J/m2”.Mutation spectrum in the category “(0.02 MB PDF)Click here for additional data file.Table S11lys2, 23°C (no arrest) UV, 45 J/m2”.Mutation spectra in the category “subtelomeric (0.04 MB PDF)Click here for additional data file.Table S12lys2, 37°C (arrest) UV, 45 J/m2”.Mutation spectrum in the category “subtelomeric (0.04 MB PDF)Click here for additional data file.Table S13can1 alleles.Separation between mutations within double and triple mutant (0.03 MB PDF)Click here for additional data file.Table S14lys2 alleles.Separation between mutations within double and triple mutant (0.03 MB PDF)Click here for additional data file.Table S15Calculated densities of UV-induced mutations.(0.02 MB PDF)Click here for additional data file.Table S16Simple base sustitutions.(0.03 MB PDF)Click here for additional data file.Table S17Simple indels.(0.03 MB PDF)Click here for additional data file.Table S18Base substitutions within complex mutations.(0.03 MB PDF)Click here for additional data file.Text S1Supplemental calculations.(0.02 MB PDF)Click here for additional data file.


FK506 modulates the RyR by dissociating FKBP12 or FKBP12.6 from the RyR complex. FKBP12 is also associated with inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ release channels (IP3 receptors: IP3Rs) on the endoplasmic reticulum (ER) of non-muscle cells. The IP3R-FKBP12 complex binds to CN, which dephosphorylates the protein kinase C (PKC) phosphorylation site on the receptor. When FKBP12 is dissociated from the IP3R complex by FK506, CN is also dissociated from the IP3R. Thereby, the IP3R is phosphorylated by PKC, and the receptor is modulated. Recently, it was found that FK506 itself induces Ca2+ release through RyRs in some tissues.Tacrolimus (FK506), which was isolated from the fermentation broth of Streptomyces tsukubaensis No. 9993, has an immunosuppressive effect. In T-lymphocytes, FK506 binds to the intracellular receptor, a 12-kDa FK506-binding protein (FKBP12). The FK506-FKBP12 complex binds to the phosphatase calcineurin (CN) and inhibits the activity of CN. By inhibition of the activity of CN, dephosphorylation of a nuclear factor of activated T-cells (NFAT) is inhibited, and translocation of the NFAT to the nucleus is suppressed. Thereby, the production of T-cell-derived mediators such as interleukin 2 (IL-2) is inhibited, and the proliferation of cytotoxic T-cells is suppressed. In muscle cells, FKBP12 and FKBP12.6 are associated with ryanodine-sensitive Ca The 06 was isty of CN  and induty of CN . Therefo2+ channels (ryanodine receptors: RyRs) on the sarcoplasmic reticulum (SR) of skeletal muscle [2+ release through the channel [3)-sensitive Ca2+ channels (IP3 receptors: IP3Rs) on the endoplasmic reticulum (ER) of cerebellum tissues [3R-FKBP12 complex binds to CN [3R [3R complex by FK506, CN is also dissociated from the IP3R [3R is phosphorylated by PKC, and Ca2+ release through the receptor is increased [2+ release from the RyR in pancreatic acinar cells, probably via an FKBP-independent mechanism [It was shown in the 1990’s that FKBP12 is tightly associated with ryanodine-sensitive Cal muscle , 9. FK50l muscle  and to i channel , 11. In  channel . FKBP12  tissues , 15. Theds to CN , which dncreased . Recentlechanism , 18.2+ release channels of the SR or ER membrane are described in detail.In this review, the effects of FK506 as an immunosuppressant are described briefly, and the effects of FK506 on Ca44H69NO12·H2O (Mw: 804.02). The chemical structure was determined by Tanaka et al. [Tacrolimus , a 23-mea et al. . The drucis-trans peptidyl-prolyl isomerase (PPIase) activity. It has been shown that the PPIase activity is inhibited by the binding of CsA and FK506 to immunophilins [2+-activated phosphatase CN and inhibit the enzyme activity of CN [2+ concentrations following T-cell receptor stimulation by antigens and dephosphorylates a cytosolic component of NFAT (NFATc) in the cytoplasm [c dephosphorylated by CN is translocated to the nucleus [n) [c-NFATn complex binds to the regulatory site on the IL-2 promoter to activate transcription of the IL-2 gene [c to the nucleus via inhibition of CN activity [Cyclosporin A (CsA), a fungal metabolite, was a powerful immunosuppressive agent in the 1980’s and has been used as a therapeutic agent to prevent graft rejection following organ transplantation. CsA inhibits the production of T-cell-derived soluble mediators such as IL-2, which is induced by antigen stimulation in T-lymphocytes. FK506 inhibits the production of IL-2 at about a one hundred-times lower concentration than that of CsA . CsA andophilins , 24, 25. nucleus  and combleus [n) , 26. TheL-2 gene . CsA andactivity  and ther2+ stores play an essential role in the regulation of cytosolic Ca2+ concentration, the elevation of which triggers many cellular events, such as muscle contraction, enzyme secretion, cell proliferation and egg fertilization. Two distinct classes of Ca2+ release channels, which induce the release of Ca2+ from the stores into the cytosol, have been identified.In a variety of cell types, intracellular Ca3 that is formed by stimulation of a cell surface receptor with hormones or neurotransmitters [2+ channels (receptors) sensitive to IP3 (IP3Rs) are widely distributed on the ER of many tissues, including T-lymphocytes. The channel protein has been purified [3R isoforms  are expressed [3R1, the major type of IP3R, is widely expressed in the rodent brain, predominantly in cerebellar Purkinje cells. IP3R2 is expressed in glial cells. IP3R3 is expressed in the kidney, pancreatic islets and intestinal epithelium.One is sensitive to the ubiquitous second messenger IPsmitters . Ca2+ chpurified  and clonpurified , 31 in bxpressed , 33. IP32+ channels (receptors) sensitive to ryanodine (RyRs) are activated by caffeine, ryanodine and Ca2+. The channel protein has been purified [+ metabolite cyclic ADP-ribose (cADPR) induces Ca2+ release from RyRs in sea urchin eggs [3 [The other is sensitive to the plant alkaloid ryanodine. Capurified , 35 and purified , 37 in tpurified , liver cpurified  and exocpurified . It has purified –43. RyR1hin eggs , 45, carhin eggs , brain chin eggs  and panchin eggs . This co eggs [3 . It has  eggs [3 , 50, 51.2+ release channel [50 value for dissociation of FKBP12 from the RyR1 complex in skeletal muscle has been reported to be in the concentration range of 0.12 to 0.5 μM FK506 [2+ or caffeine sensitivity of the channel is enhanced [FK506 is known to modulate RyRs. In the skeletal muscle SR, RyR1 is tightly associated with FKBP12 , 9. It h channel . FK506 h channel . The EC5μM FK506 . By the μM FK506 , and theenhanced , 52. Com2+ channels did not increase the open probability for single channel recordings as was the case of FKBP12-deficient skeletal SR Ca2+ channels [2+ channels) in coronary arterial smooth muscle cells, in which FKBP12.6 was detected [2+ release from RyR2 of pancreatic islet microsomes by dissociating FKBP12.6 [45Ca2+ release from the microsomal vesicles of rat pancreatic acinar cells to the left [2+ release through the RyR by FK506. It has been found that cADPR as well as FK506 can bind to FKBP12.6 and dissociate FKBP12.6 from pancreatic islet microsomes to release Ca2+ [2+ channel. It has been found in rat pancreatic acinar cells that cADPR shifts the dose-response curve of ryanodine- or caffeine-induced 45Ca2+ release to the left by the same extent as that in the case of FK506 and that the stimulatory effects on ryanodine- or caffeine-induced 45Ca2+ release by cADPR and by FK506 are not additive [2+ leak through the RyR2 [In cardiac type RyR (RyR2), FKBP12.6 binds to the receptor , 53. FK5channels . Althougdetected . This rethe left . Since athe left , 56, FKBase Ca2+ . These fadditive . The resadditive . The RyRadditive , 58. In additive . The hypadditive , 59 and the RyR2 , 61.3R1, a structurally related tetramer that has up to 40% sequence identity with RyR1 [3R1 of the rat cerebellum at residues 1400–1401, a leucyl-prolyl dipeptide that is an FK506-like domain [50 value for dissociation of FKBP12 from the IP3R1 complex in the rat cerebellum has been shown to be in the concentration range of 10 to 100 nM FK506 [3R1-FKBP12 complex can associate with CN [3R1 [3R1 complex by FK506, binding of CN to the FK506-FKBP12 complex is stimulated [3R1 is phosphorylated by PKC, and Ca2+ flux through IP3R1 is increased [2+ release from the ER to the cytosol through the IP3R by PKC activation activates CN, and the activated CN decreases Ca2+ release by dephosphorylation of the receptor. Thus, the increase and decrease in Ca2+ release through IP3R mediate Ca2+ oscillations [IPith RyR1 , 31, 33,ith RyR1 , 15. It e domain . The EC5 with CN . CN has 2+ release from the ER of non-muscle cells. It has been shown that FK506 induces Ca2+ release through the RyR in pancreatic islets by the removal of FKBP12.6 as mentioned above [45Ca2+ release from the ER of pancreatic acinar cells [45Ca2+ release was seen at concentrations up to 10 μM (Km = 0.5 μM), and the second phase of the release was seen at concentrations over 10 μM (Km = 55 μM). The first phase of the release was stimulated by the presence of cADPR [45Ca2+ release in pancreatic acinar cells is due to the dissociation of FKBP. FK506 is a compound with a macrocyclic lactone ring structure (45Ca2+ release in pancreatic acinar cells may be explained by a direct activation of the RyR by FK506. The second phase of the FK506-induced 45Ca2+ release in pancreatic acinar cells was inhibited by heparin, an inhibitor of the IP3R [3R in pancreatic acinar cells.It is known that FK506 itself induces Caed above . Recentlar cells . The firof cADPR . The FK5tructure . It has the IP3R . Althoug2+ release from the receptor to the cytosol. FK506 dissociates FKBP12 or FKBP12.6 from the RyR complex and increases Ca2+ release through the receptor. In addition, the FK506-FKBP12 complex binds to the phosphatase CN and inhibits the enzyme activity of CN. In T-lymphocytes, dephosphorylation of an NFAT by CN is inhibited by FK506. Therefore, translocation of the NFAT to the nucleus is inhibited, and the immunoreaction of T-cells is suppressed. FKBP12 and CN bind to the IP3R on the ER. When FKBP12 is dissociated from the IP3R complex by FK506, CN is also dissociated from the receptor. Thereby, the IP3R is phosphorylated by PKC, and the Ca2+ release through the IP3R is increased. FK506 has been used as a tool to elucidate the pathway of immunoreaction and the modulation mechanism of Ca2+ release from the SR or ER. With elucidation of the action of FK506 and the cellular functions of FKBPs, FK506 may become more widely used in biomedical research.FKBPs are abundant and ubiquitous proteins within cells. FKBP12 or FKBP12.6 binds to the RyR on the SR or ER and regulates Ca