Ca2+ loading of the sarcoplasmic reticulum (SR), and administration of blockers caffeine and thapsigargin have been utilized to probe the phenomena surrounding SR Ca2+ release. Here, we develop a deterministic mathematical model of a rat ventricular myocyte under VC conditions, to better understand mechanisms underlying the response of an isolated cell to calcium perturbation. Motivation for the study was to pinpoint key control variables influencing CICR and examine the role of CICR in the context of a physiological control system regulating cytosolic Ca2+ concentration ([Ca2+]myo).The past thirty-five years have seen an intense search for the molecular mechanisms underlying calcium-induced calcium-release (CICR) in cardiac myocytes, with voltage clamp (VC) studies being the leading tool employed. Several VC protocols including lowering of extracellular calcium to affect Ca2+ channels (trigger-channel and release-channel). It releases Ca2+ flux into the cyto-plasm and is in turn enclosed within a negative feedback loop involving the SERCA pump, regulating[Ca2+]myo.The cell model consists of an electrical-equivalent model for the cell membrane and a fluid-compartment model describing the flux of ionic species between the extracellular and several intracellular compartments , in which resides the mechanistic basis of CICR). The DCU is described as a controller-actuator mechanism, internally stabilized by negative feedback control of the unit's two diametrically-opposed Ca2+ release at high Ca2+ gain in a homeostatically-controlled environment where [Ca2+]myo is precisely regulated. We elucidate the importance of the DCU elements in this process, particularly the role of the ryanodine receptor in controlling SR Ca2+ release, its activation by trigger Ca2+, and its refractory characteristics mediated by the luminal SR Ca2+ sensor. Proper functioning of the DCU, sodium-calcium exchangers and SERCA pump are important in achieving negative feedback control and hence Ca2+ homeostasis.Our model reproduces measured VC data published by several laboratories, and generates graded Ca2+ regulating mechanisms in handling various types of induced disturbances in Ca2+ levels by quantifying cellular Ca2+ balance. Our model provides biophysically-based explanations of phenomena associated with CICR generating useful and testable hypotheses.We examine the role of the above  Ca2+ concentration [Ca2+]myo. Sarcolemmal (SL) membrane depolarization triggers Ca2+ influx from the extracellular medium by opening dihydropyridine (DHP)-sensitive L-type Ca2+ channels. Following diffusion across a small sub-membrane dyadic space, this influx activates ryanodine receptors (RyRs) controlling ryanodine-sensitive Ca2+ release channels in the junctional portion of the sarcoplasmic reticulum (jSR). Fabiato and Fabiato myo is returned to resting levels by combination of: (a) Ca2+ buffering in the dyadic space and cytosol; (b) sequestration of Ca2+ by sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)-type calcium pumps lining the longitudinal portion of the sarcoplasmic reticulum (LSR); and (c) Ca2+ extrusion from the cytosol by Na+/Ca2+ exchangers and Ca2+-ATPase pumps on the sarcolemmal membrane.Contraction of cardiac muscle is triggered by a transient rise in intracellular  Fabiato  named thCa2+ release is proportional to the influx of trigger Ca2+ [Ca2+ release flux. Graded Ca2+ release with high gain is somewhat paradoxical according to Stern [Ca2+ current. Stern [Ca2+ release by RyRs were actually the local nanodomains of [Ca2+] generated by nearby L-type channels, rather than the global cytosolic [Ca2+]myo. According to this hypothesis, graded control of macroscopic SR Ca2+ release can be achieved by graded statistical recruitment of individual, autonomous, all-or-none stochastic release events  in the dyadic space and myoplasm. In fact, Ca2+- release from the jSR was blocked by administering a relatively high dose of ryanodine (20 μM) in all experiments. Therefore, to study the additional influence of SR Ca2+ release on Ca,LI, we modified the original Ca,L Imodel to better characterize the process of Ca2+-dependent inactivation. In the modified Ca,L Imodel, majority of the structure for the 6-state dynamic scheme remains the same, however changes have been made to the voltage-dependent inactivation rate function  as well as produce tail currents during repolarization from large clamp voltages (≥ 40 mv).Our previous study  was focuCa2+ binding sites on the inner sarcolemmal leaflet. Following these authors, we included low-affinity (d k= 1.1 mM) and high-affinity (d k= 1.3 μM) Ca2+ binding sites on SL wall boundary of cylindrical dyadic space. The presence of these membrane bound sarcolemmal Ca2+ binding sites in our model has significant physiological implications, in that it prevents local dyadic Ca2+ concentration near the "mouth" of DHP-sensitive Ca,L Ichannel from becoming excessively high. Allowing such a condition to occur can cause a reversal of the Ca,L Icurrent, which does not occur physically during normal jSR release. Addition of these SL Ca2+ binding sites does not significantly slow the build-up of Ca2+ within the dyadic space, nor is the Ca2+-induced Ca2+ release mechanism affected myo. Since many of the kinetic gating schemes derived from lipid bi-layer data fail to support stable E-C coupling in simulations, he concluded that the RyR gating process in situ may differ considerably from that in bi-layers.The gating characteristics of the Ry-sensitive release channel are not only modulated by the dyadic d Levine  with 4 Cand Fill ; (2) theand Fill , which aand Fill , which cn et al.  demonstrn et al.  further Ca2+ ions, whereas inactivation depends on the binding of a single Ca2+ ion. Four additional features have been built into our model: (a) the crucial role of the luminal SR Ca2+ sensor , is adopted to reflect recent observations that the inactivation of the RyR depends on the high local Ca2+ concentration consequential to their own Ca2+ release jSR). The specific equations for the Ca2+ release model are given in Appendix A1 (Equations 73-92).Our gating scheme is patterned after the release channel used in the model of Stern, Pizarro and Rios  Figure , where tr Figure  in assisr Figure ; (b) CaM release -50 is mo release ; and (d)r et al.  and Zuccr et al. . Our inin et al.  was quitn et al. , and furCa2+ release is modulated by a multi-molecular Ca2+ signalling complex which is localized to the junctional SR [Ca2+ conducting pore [Ca2+ binding protein [Ca2+ concentration dependent luminal control of the RyR channel. Our detailed biophysical model of the luminal sensor is strongly based on the recent findings of Terentyev et al. [Ca2+]jSR) dependent, CS mediated mechanistic interaction of the protein triadin with the RyR channel.The RyR ional SR -56. Thising pore ,58, cals protein ,60, and  protein  and junc protein . It was  protein . More re protein . Triadin protein , but the protein  takes a v et al.  which unCa2+ release by sensitizing the RyR to activation by the trigger current Ca,L,TT I. This is incorporated in our model by allowing the rate constants in the 4-state Markovian model for the RyR  available to bind with triadin. This results in a decrease in the extent of interaction between triadin and RyR (A1ls in the model), thus inhibiting the RyR channel and leading to robust termination of SR Ca2+ release in cardiac myocytes  (both on the dyadic side ([Ca2+]ryr) as well as the luminal side ([Ca2+]jSR)) experiences a strong interaction of Ca2+ flow through itself and its gating mechanism described as [Ca2+]ryr induced self-inhibition and the luminal sensor induced inactivation. Hence, the rate constants of the luminal sensor model  and CS  being equal to one is explicitly imposed Appendix A1, Equations 87-88) on the model of the luminal sensor in order to avoid run-off.Due to the lack of in-vivo measurements on individual state transitions, the rate constants in our novel luminal sensor model Table  are chosold true ,69. The l Figure -i are noCa2+ buffers play an important role in sequestering a fraction of the total Ca2+ released during E-C coupling and contraction. These buffers include: (a) calmodulin (CaM), which is assumed to be uniformly distributed in the myoplasm and dyadic space; (b) troponin in the bulk myoplasm; and (c) calsequestrin (CS) in the jSR. The dyadic space has been shown to be accessible to calmodulin (CaM), but mostly inaccessible to fluorescent dyes . The differential equation describing the change in [CaF3] with time is given in Appendix A2 (Eq. 138) which follows from Shannon et al. myo, its transport rate is far too slow for it to be an important factor in Ca2+ fluxes during the cardiac cycle. It might, however be more important in long-term extrusion of Ca2+ by the cell. Our model of the plasma membrane Ca2+ ATPase pump current is adopted from Sun et al. o) to 0 mM in mammalian cardiac muscle can be prevented by including appropriate concentrations of other activator cations of the Na+/K+ pump such as Cs+ in the 0 mM [K+]o bathing solution jSR in CICR. In particular, the relationship of the peak cytosolic calcium transient and the SR Ca2+ content is examined. The effect of modulatory agents like caffeine and thapsigargin on SR release/uptake is studied to understand the significance of these mechanisms in facilitating a normal CICR.The DCU is a fundamental element in the mechanism of CICR. The sequence of events resulting in CICR is triggered by the Ca2+ channel has a key role in initiating CICR. Hence a thorough analysis of its activation and inactivation mechanisms is considered here. Though the activation of the Ca,L Ichannel is solely voltage dependent once activated, the inactivation of the channel is influenced not only by the trans-membrane voltage but also the Ca2+ concentration in the vicinity of the channel jSR , which is of the same order as reported by Wang et. al. jSR - dependent closure of the RyR channel. This mechanism for inactivation of the Ry-sensitive Ca2+ release channel is accounted for in our model.The slow rising foot that precedes the rapid upstroke  the modulation of release due to pulse duration dependent change in the amount of trigger Ca2+ entering the dyadic coupling unit (DCU) and (ii) the pulse duration dependent change in the relative role of the Na+/Ca2+ exchanger co-located in the DCU.Cytosolic rization -102. TheCa2+ entering the DCU via the Ca,L,TT Ichannel causes a large release of Ca2+ from the sarcoplasmic stores via the Ry-sensitive Ca2+ release channel on the jSR lumen that interacts with the DCU. This high gain is essential in producing physiological cytosolic Ca2+ transients when 10000 of these DCU's operate in tandem. Two different definitions of the gain or amplification factor due to CICR have been adopted in our model simulations, namely the ratio of:Besides the graded release, an extremely valuable characteristic of the CICR process is the high gain associ-ated with it. A small amount of 1. average integrated RyR flux to average integrated DHPR flux ; andCa2+ transient in the presence of CICR to the peak calcium transient in its absence, con-tributed by the trigger calcium alone jSR dependent inhibition induced by the protein triadin from the luminal side of the RyR channel. This restraining lock on the RyR channel assists in reloading the jSR.Given the fact that the CICR mechanism has a high gain associated with it, it could be prone to unstable behavior if not for the refractory characteristics of the Ry-sensitive release channel . It is nT2 in Figure Ca,L,TT I) of trace 1  in Figure T1 in Figure T2) was gradually reduced from 250 ms to 50 ms in steps of 25 ms to observe the effects of partial recovery of the RyR channel.A dual stimulus protocol (S1-S2) was employed to study RyR refractoriness. The RyR channel was stimulated initially by stimulus trigger current S1, followed after an interval  and hence robustly avoids re-excitation. However, when the RyR channel is in the relative refractory period, a partial release , duration (50 ms) and frequency of 4.0 Hz which is applied for a period of 2.5 sec. The value of the luminal control of the RyR channel in allowing adequate SR filling can be seen in Figure Ca2+ transient  in diastolic SR level  the amount of trigger Ca2+ entering by means of the Ca,L,TT Ichannel (b) the concentration gradient of Ca2+ between the jSR and the mouth of the RyR channel in the dyadic space and (c) the open probability of the RyR channel modulated by the interaction between the luminal sensor and the RyR protein. The SR Ca2+ released also inactivates the trigger current Ca,L,TT I versus the Ca2+ concentration at the mouth of the RyR channel in the dyadic space [Ca2+]ryr constructed from model-generated data for a pulse of amplitude -40 mv to 10 mv and a duration of 50 ms. The pre-release diastolic [Ca2+]jSR is 918 μM. Following excitation by the trigger current during phase A, the RyR channel begins to open, allowing Ca2+ flux into the dyad and elevating [Ca2+]ryr at the mouth of the RyR channel. The rate of opening of the channel is Ca2+ dependent; hence as [Ca2+]ryr increases, RyR open probability increases first slowly and then ever more rapidly. With this onrush of Ca2+, [Ca2+]ryr rapidly equilibrates with the [Ca2+]jSR, reducing and eventually abolishing the concentration gradient across the channel. [Ca2+]ryr soon reaches its maximum value  Ca2+ fluxing out of the dyad into the cytosol, (b) lack of a drive from SR release due to drastically diminished Ca2+ gradient at the mouth of the RyR channel and (c) continued Ca2+ induced self-inhibition. Decreasing levels of free Ca2+ in the jSR force a strong RyR inactivation via the luminal sensor  RyR release is reduced, as is the basal intracellular Ca2+ concentration, and (b) Ca2+ dependent inactivation of the Ca,L,TT Ichannel is reduced due to the lowered dyadic Ca2+ concentration, which allows for greater Ca2+ entry into the dyadic space via the Ca,L Ichannel. This increase in trigger current results only in a very small increase in Ca2+ release, because of the inhibition of the Ry-sensitive channel caused by the luminal sensor in response to low SR Ca2+ content. Thus, these factors cumulatively result in only a small linear increase in the peak [Ca2+]myo with increasing SR Ca2+ content in Region I. The behavior in Region I corresponds to traces 7-1 in Figure O2ryr vs [Ca2+]ryr for different steady state [Ca2+]jSR concentrations), where the peak RyR open probability shows very small gradual increase with increasing levels of diastolic pre-release [Ca2+]jSR for a constant trigger Ca2+ input via Ca,LI. The steady state diastolic [Ca2+]jSR levels being low, the maximum RyR open probability values attained are low. Although the peak [Ca2+]ryr does not increase substantially as the diastolic [Ca2+]jSR levels increase from 317μM to 673μM, the area enclosed by the [Ca2+]ryr - O2ryr loop (which indicates the amount of SR Ca2+ released into the dyad) increases exponentially due to increasing peak RyR open probability combined with the increasing difference between rate of activation and inactivation with rate of activation increasing faster than the rate of channel inactivation. In Region II of Figure Ca2+ content begins to translate into a substantial increase in peak [Ca2+]myo. This is because increasing diastolic [Ca2+]jSR causes a significant increase in the peak RyR open probability, as shown in Figure Ca2+ transient. The inhibiting role of the luminal sensor is relieved with building [Ca2+]jSR levels. The nonlinearity observed between the diastolic [Ca2+]jSR levels 673μM  increased release owing to large SR Ca2+ content; (b) large values for saturated RyR open probability supported by an increase in the area contained by each loop as seen in traces 6-1 of Figure Ca2+ concentration levels in the cytosol after RyR release [Ca2+]jSR levels increase beyond 948μM, the area enclosed by the [Ca2+]ryr - O2ryr loop begins to decrease despite increasing peak [Ca2+]ryr due to saturation of the peak RyR open probability and increasing rate of RyR channel inactivation due to large values of local Ca2+ concentration ([Ca2+]ryr) assisting in faster recovery [Ca2+]myo and the pre-release [Ca2+]jSR agrees with Figure Figure M Figure  and 734μM Figure  at the t release . As the recovery . This moCa2+]ryr the RyR open probability takes a lower value despite a higher steady state diastolic [Ca2+]jSR level. This decreasing slope with increasing diastolic, pre-release [Ca2+]jSR reflects a faster rate of rise in [Ca2+]ryr for a larger pre-release SR content with a very small accompanying increase in the RyR open probability.As seen in traces 1-10 of Figure Ca,LI-induced Ca2+ release in rat ventricular myocytes in the presence and absence of caffeine, which altered [Ca2+]myo. Using their protocols and data as a guide, we modeled the effect of caffeine on jSR Ca2+ release and its subsequent effect on Ca,L I. Specifically, a Boltzman relationship was adopted to simulate the binding of caffeine to the ryanodine receptor increasing channel conductance, which in turn enhances jSR Ca2+ release. The process is assumed to be dose-dependent, with an open probability of 0.5 at a caffeine concentration of 5 mM .Adachi-Akahane et al.  investigCa2+]myo responses in myocytes dialyzed with 2 mM Fura-2, where a simple voltage clamp pulse (-40 mV to 0 mV) is applied in the presence and absence of caffeine. Ca,L Iwaveforms associated with these two protocols are shown in Figure Ca2+]myo (10 nM), the depolarizing test pulse to 0 mV from the holding potential of -40 mV for 0.1 sec fully activates Ca,L I, which in turn triggers a rapid but small amplitude Ca2+ transient .; and (b) the strong competing role of the luminal RyR sensor in inactivating the RyR channel in response to the post-caffeine, low SR content. The Ca,LI-inactivation characteristics undergo a change as shown in Figure Ca,L I(trace 1). After exposure to caffeine, the overall amplitude of [Ca2+]dhp during release does not increase as much as in the control case, since jSR Ca2+ release is reduced. Consequently, Ca2+ dependent inactivation (during release) of Ca,L Iafter caffeine application is not as strong as that in the control case. However, the baseline level of Ca2+ concentration at the mouth of the DHP channel ([Ca2+]dhp) is elevated following caffeine application, causing sustained inactivation resulting in the crossover of the current trace in Figure Ca2+ induced inactivation is a result of calmodulin mediated sensing of the local Ca2+ concentration.Figures l Figure , due to:Ca2+ uptake is blocked, SR Ca2+ content declines, as does the jSR Ca2+ release with each voltage pulse  is activated, leading to a lower degree of Ca2+ dependent inactivation. Thus, Ca,L Ipeak is enhanced, as shown in Figure Ca2+ entering the cell. This inverse relationship between jSR Ca2+ content and the amount of trigger Ca2+ entering the dyadic space for a given clamp voltage is the essence of the feedback mechanism maintaining the efficacy of CICR. This mechanism is operational even in the presence of high concentrations of cytoplasmic Ca2+-buffers, since the small dimensions of the dyadic space allow jSR released Ca2+ to reach its destination  before being captured by the buffers o causes an increase in entry of extracellular Ca2+ into the cell via the Ca,L Ichannel. This results in an increase in SR Ca2+ content, as shown in Figure Ca2+ as well as a larger SR Ca2+ content, the RyR release is enhanced. This increase in SR release assisted by impaired Na+/Ca2+ exchange due to elevated [Ca2+]o manifests in an increase in the peak Ca2+ concentration levels in the cytosol, as shown in Figure Ca2+ levels causes a decrease in trigger Ca2+ available to cause CICR, which results in a decrease in peak [Ca2+] levels in the cytosol, as shown in Figure Ca2+ entry into the cell via Ca,LI, translating into decreased availability of post-release Ca2+ in the cytosol, causes reduction in SR filling via the bi-directional SERCA pump. The corresponding drop in the SR Ca2+ content is evident in Figure Ca2+ content with a decrease in [Ca2+]o as reported in Figure Ca2+ content with decrease in [Ca2+]o in cells not showing spontaneous release. In this study, we have attempted to isolate the effects of changes in [Ca2+]o on intracellular Ca2+ levels by ensuring that there are no voltage dependent effects on Ca2+ transport via the Ca,L Ichannel, Na+/Ca2+ exchanger or the plasma membrane Ca2+ ATPase pump. The simulation protocol used in our study  results in a decrease in steady state SR Ca2+ content as a result of the dominating effect of reduced Ca2+ entry via the Ca,L Ichannel despite a decrease in net Ca2+ extrusion via the Na+/Ca2+ exchanger per cycle.Increase in Ca+o causesz et al. ),is a puCa2+ stability. Analogous to the experimental protocols of Negretti el al. myo returns to control levels by the end of the long-pulse stimulation protocol. The long-pulse stimulation protocol thus serves as a means of studying the steady-state balance of Ca2+ influx and efflux to and from the cytoplasm, under control conditions. In contrast, this Ca2+ balance is not present in the short-pulse protocol, and [Ca2+]myo is elevated at pulse termination. Our long-pulse simulations show that: (1) the magnitude of individual Ca2+ transients do not change during the 40 pulse sequence; (2) the peak calcium release current (ryrI) has a constant magnitude; and (3) the occupancy of the calsequestrin Ca2+ buffer in the jSR compartment is reduced by 15% as a result of Ca2+ release during each cycle. Although Ca2+ fluxes cross the sarcolemma and SR membranes in either direction, the integral of each of these currents (indicating charge transfer) over the pulse repetition interval is a constant. Figure Ca2+ nor consecutive-pulse Ca2+ depletion (or augmentation) in the jSR compartment under the long-pulse protocol. In Figure Ca2+ influx and efflux through the bounding cell membrane (sarcolemma (SL) and T-tubule) as "external fluxes" , whereas SR membrane Ca2+ fluxes  are called "internal fluxes." By convention, inward fluxes are negative  and outward positive . Figure Ca2+ charge entering the cell via Ca,L Iis 603.28 pC (pico-coulomb), and the sum of averaged Ca2+ charges extruded from the cell via NaCa Iand PMCA Iis 603.28 pC  negative and an outward flux positive . The integral of ryr Iis 751.8 pC, compared with integral of the Ca,L Itrigger current (603.28 pC). The calcium gain of CICR calculated in this way is low owing to the increased entry of Ca2+ via the trigger current Ca,L Ifor a prolonged duration of 800 ms compared to 50 ms previously. The integral of SR Ca2+ uptake is 751.8 pC. These two internal component currents yield flat integral values during long-pulse stimulations and sum to zero.During repetitive long-pulse (800 ms) stimulation, transient Ca2+]myo observed with stimulation by long pulses, short pulse stimulation produces a negative staircase , which in turn causes a decrease in [Ca2+]jSR resulting in decreased release (ryrI). With successive pulses, cyt,serca Iand ryr Idecline further, mirroring the exponential decline in NaCa Itoward balance. At steady state, the external and internal fluxes reach the new equilibrium, where the sum of the integrals of external currents and the sum of the integrals of internal currents are zero.Decreasing the length of the depolarizing pulse to 100 ms (short pulse) has a pronounced effect on the recovery of contraction Figure  and 20D.e Figure  in agreeCa2+ transients in response to a repetitive 4 Hz  voltage clamp stimulation lasting for 2.5 seconds. The sustained peak and basal Ca2+ levels of [Ca2+]myo over this prolonged time frame are indicative of long tern Ca2+ stability in the model. The corresponding [Ca2+]jSR profile over 2.5 seconds is also shown in Figure Most VC experiments on cardiac cells are usually conducted at low stimulation rates. Our simulations of typical VC experiments at these low rates all exhibit long term calcium stability Figures  and 20. Ca2+ influx called a "tail current", which has been shown to trigger RyR release and hence cause contraction during repolarization myo (panel D). We note from Figure Ca2+ release although an Ca,L Itail current is produced. This tail current produced with the 50 ms pulse slightly augments the [Ca2+]myo transient just after its peak , but there is no corresponding decrease in the [Ca2+]jSR transient  indicating no RyR Ca2+ release. At the end of this short pulse (50 ms), the RyR channel is in fact in its absolute refractory period. The small secondary increase in [Ca2+]myo transient as seen in data corresponding to trace 4 of Figure Ca2+ influx via the Ca,L I"tail current". During this phase, the Na+/Ca2+ exchanger is biased to extrude Ca2+ out of the dyad, and hence cannot be responsible for this secondary rise in the cytosolic Ca2+ transient. As pulse duration is increased beyond 100 ms the "tail current" causes a gradual increase in RyR open probability  to the tail current during repolarization but also allows for the voltage- and time-dependent recruitment of 'silent' Ca2+ channels modeled using a high voltage state C6dhpr. This study proves to be an adjunct to the study presented in Figure Our voltage clamp simulations have dealt with clamp voltages in the range -30 mV ≤ V ≤ 40 mV. Some experimental studies ,2,112 horization ,2. The sn et al. troponin binding site; (c) the Ca2+ - Mg2+ competitive troponin binding site; and (d) the fluorescent indicator dye Fluo3 used to detect changes in [Ca2+]myo. The effects of the component buffers in helping to maintain a low [Ca2+]myo are shown in Figure C O, tc O, tmgc O, tmgmg O in the cytosol, and calse O in the jSR Ca2+ release compartment. Figure Ca2+ release channel is triggered, the jSR releases Ca2+ and the occupancy calse Oin the jSR, declines from 68% to 37%. Ca2+ release from the jSR induces fast Ca2+ binding by calmodulin and troponin in the cytosol, as represented by the increases of c O(0.29) and tc O(0.16) in Figure Ca2+ and the troponin-Mg sites result in an increase in occupancy of these sites by Ca2+ (tmgcO), and a decrease in occupancy by Mg2+ (tmgmgO).In our model, cytosolic buffering is attributed to several factors including: (a) calmodulin (CaM); (b) the Ca2+ uptake compartment), junctional sarcoplasmic reticulum (jSR or release compartment), and the cytosolic fluid compartment). An external bathing medium completes our fluid compartment description of the cell Figure Ca2+ in the cell. In particular, we have focused on the dyadic mechanisms effecting CICR.It is well-established that mammalian cardiac excitation-contraction coupling is mediated by calcium-induced calcium release (CICR). We have developed a comprehensive mechanistic model of CICR under voltage clamp conditions in the rat ventricular myocyte, which includes electrical equivalent circuit models for both the free sarcolemma and that portion involving junctional transmission, as well as, fluid compartment models for several fluid media within the cell  to the dyadic space. After diffusion within the dyadic space, trigger Ca2+ affects the Ca2+ dependent open probability of the jSR Ca2+ release channel over a small range of Ca2+ concentrations. Clamp voltage magnitude strongly affects the Ca,L Icurrent and hence the amount of Ca2+ delivered to the dyadic space, but the range over which the Ca2+ concentration at the mouth of the Ca2+ release channel changes (in the absence of RyR release) is quite small (23.42μM to 8.82μM for a change in VC from 10 mv to 40 mv). The RyR-mediated Ca2+ release is activated by trigger current, but the release itself is affected by the concentration gradient between the jSR and the dyadic space, and the temporal and refractory properties of the ryanodine receptor . It integrates these various stimuli and delivers a trigger current to the small dyadic space. In contrast, the jSR membrane lining the opposite boundary of the dyadic space is concerned with the adequacy of Ca2+ release in CICR. It contains Ry-sensitive Ca2+ channels that require a Ca2+ concentration gradient directed across the channel and into the dyadic space for their operation. Thus, jSR Ca2+ concentration must be maintained within an acceptable range so that calcium is always available for ready release. The relationship between jSR Ca2+ content and peak [Ca2+]myo is shown in Figure Ca2+ from the myoplasm. Pumping rate is controlled by the cytosolic Ca2+ concentration [Ca2+]myo as well as the LSR Ca2+ concentration [Ca2+]LSR as part of a mechanism for replenishing and maintaining jSR Ca2+ stores. Our model also incorporates the Ca2+ induced CaM mediated effects of CaMKII and CaN on targets such as the DHP-sensitive Ca,L Ichannel, Ry-sensitive Ca2+ release channel, as well as the SR Ca2+ ATPase pump. Provision for the effects of phospholamban on SERCA has also been included.The sarcolemmal portion of the dyadic membrane defining the dyadic space contains voltage-sensitive Ca2+ signaling. Although physically separated, the voltage-sensitive Ca2+ channel  that was given the role of initiating RyR recovery. This has been tested in our model by artificially clamping all the variables (including concentration at the mouth of the RyR channel) to the levels reached at T2 in Figure Ca2+ concentration. Our model predicts (data not shown) that this Ca2+ signaling continues even in the presence of high concentrations of Ca2+ buffering agents in the cytosol . Tight Ca2+ concentration [Ca2+]myo is evident at the cellular level. It is affected strongly by the voltage and [Ca2+]myo-dependent properties of the sarcolemmal currents NaCa Iand Ca,LI, as well as the [Ca2+]myo-dependent properties of the PMCA Iand SERCA pumps. We demonstrate a model-generated whole-cell Ca2+ balance, which shows the importance of the Na+/Ca+ exchanger in extruding the Ca2+ that has entered the cell under normal activity, and also any excess that might occur when cytosolic Ca2+ levels rise. The variety of experiments emulated in this study demonstrates quantification of Ca2+ balances for all external and internal Ca2+ fluxes and shows that the model has long-term stability in regulating cytosolic Ca2+, as shown in the 120-sec duration experiments of Negretti et al.  that decrease [Ca2+]myo and hence Ca2+-uptake via the SERCA pump, jSR Ca2+ content decreases. For decreases in jSR Ca2+ content, the opposite occurs, so that regardless of the sign of the perturbation in jSR Ca2+ content, it tends to stay constant in the steady-state. This is an important feature of the dyadic mechanism that preserves the integrity of CICR. The gain of this feedback system about the operating point is visualized as the slope of the peak Ca2+ transient vs SR Ca2+ content characteristic  for a given voltage pulse level. Observing this figure, we note that there is a linear operating range beyond which the system gain increases dramatically. Our model results also show that a decrease of jSR Ca2+ content (simulated either by a decrease of Ca2+ uptake into the LSR by thapsigargin or an increase of Ca2+ leak out of the jSR by caffeine) decreases systolic [Ca2+]myo, and hence the model might serve as a useful adjunct in a study of heart failure, where decreased contractility as a result of diminished Ca2+ transients are commonly observed NaCa, [Na+]NaCa are Concentrations at the mouth of the NaCa-exchanger.where ryr is the Ca2+ concentration at the mouth of the RyR channel on the dyadic side. In the presence of caffeine :where [Gating scheme for the 6-state Markovian model for the Luminal Calcium sensor.A1ls   Fractional occupancy of RyR by Triadin/JunctinI2ls   Fractional occupancy of RyR by Triadin/Junctin and CalsequestrinI3ls   Fractional occupancy of Triadin/Junctin by CalsequestrinI4ls   Free Triadin/Junctin, B5ls - Free CalsequestrinB6ls   Fractional Occupancy of Calsequestrin by CalciumCa2+ binding to CaM and CaM bufferingCaMKII activation:CaN activation:Fluorescent indicator dyeCa2+ buffering:Intracellular calmodulin (bulkmyoplasm):TroponinCa2+):::(Fractional occupancy of troponin-Mg complex by 2+ concentration:Intracellular Ca2+ concentration in the cytosolCa1. dyad Iis the net integrated Ca2+ flux diffusing out of all the dyadic units into the cytosolwhere 2+ concentration in the jSRCa2. 2+ concentration in the LSR3. Ca4. Diffusion equation for Calcium in the dyadic space+ concentration:Intracellular Na+ concentration in the cytosolNa1. + concentration in the dyadic spaceNa2. + concentration:Intracellular Cs+ concentration in the cytosolCs1. + concentration in the dyadic spaceCs2.


A single double-strand break (DSB) triggers the ATR/ATM (Mec1/Tel1)-dependent DNA damage response in budding yeast. These kinases phosophorylate not only histone H2A (γ-H2AX) but also the C-terminal threonine of histone H2B (γ-H2B), both of which contribute to checkpoint responses and repair of the DSB. Despite both being phosphorylated by the same kinases, γ-H2AX kinetics are much more rapid than that of γ-H2B, but when H2A is mutated to prevent its phosphorylation, γ-H2B kinetics adopt the rapid kinetics seen for γ-H2AX. Both modifications are removed the PP4 phosphatase. γ-H2AX and γ-H2B spread over ≥ 50 kb on either side of the DSB but they are nearly absent in strongly transcribed regions. When transcription is turned off, γ-H2AX rises to a high level within 10 min, catalyzed by either Mec1 or Tel1, even 5 h after the break was induced and when Tel1 is reported no longer to be associated with the DSB. We show that if a DSB is created within 15 kb of one yeast centromere that γ-H2AX and γ-H2B spread to the pericentromeric regions of all other chromosomes, demonstrating that the kinases can act in trans to regions of DNA that are located in close proximity.When a DSB is created in a locus that can be repaired by gene conversion, 5’ to 3’ resection leads to the loss of well-positioned nucleosomes adjacent to the DSB. When repair is complete, nucleosomes are re-established, but the pattern is distinct from that seen prior to the break or when the repaired cells are allowed to replicate. Chromatin re-establishment is dependent on the histone chaperones CAF-1 and Asf1.


Activation of the EP1 receptor in neurons results in increased intracellular calcium levels; therefore, we hypothesize that the blockade of the EP1 receptor reduces hemin neurotoxicity. Using postnatal primary cortical neurons cultured from wild-type (WT) and EP1−/− mice, we investigated the EP1 receptor role in hemin neurotoxicity measured by lactate dehydrogenase (LDH) cell survival assay. Hemin (75 μM) induced greater release of LDH in WT (34.7 ± 4.5%) than in EP1−/− (27.6 ± 3.3%) neurons. In the presence of the EP1 receptor antagonist SC-51089, the hemin-induced release of LDH decreased. To further investigate potential mechanisms of action, we measured changes in the intracellular calcium level [Ca2+]i following treatment with 17-phenyl trinor PGE2 (17-pt-PGE2) a selective EP1 agonist. In the WT neurons, 17-pt-PGE2 dose-dependently increased [Ca2+]i. However, in EP1−/− neurons, [Ca2+]i was significantly attenuated. We also revealed that hemin dose-dependently increased [Ca2+]i in WT neurons, with a significant decrease in EP1−/− neurons. Both 17-pt-PGE2 and hemin-induced [Ca2+]i were abolished by N-methyl-D-aspartic (NMDA) acid receptor and ryanodine receptor blockers. These results suggest that blockade of the EP1 receptor may be protective against hemin neurotoxicity in vitro. We speculate that the mechanism of hemin neuronal death involves [Ca2+]i mediated by NMDA acid receptor-mediated extracellular Ca2+ influx and EP1 receptor-mediated intracellular release from ryanodine receptor-operated Ca2+ stores. Therefore, blockade of the EP1 receptor could be used to minimize neuronal damage following exposure to supraphysiological levels of hemin.Although hemin-mediated neurotoxicity has been linked to the production of free radicals and glutamate excitotoxicity, the role of the prostaglandin E Heme is a protein prosthetic group consisting of a porphyrin ring and an iron atom in the center  is oxidized to ferric iron (Fe3+). However, under pathophysiological conditions, disruptions in iron homeostasis can cause an increase in the oxidation of iron from Fe2+ to Fe3+ via the Fenton reaction. The increase in Fe3+ concentration can produce harmful superoxide free radicals in the brain that lead to oxidative stress and neuronal death  is produced by various cell types and is involved in ischemic neuronal injury -induced neuronal injury  , and NeuroCult SM1  and plated onto poly-D-lysine-coated 24-well plates at a density of 5 × 105 cells/well. Cells were maintained in growth medium at 37°C in 95% air/5% CO2-humidified incubator for 10–12 days in vitro before treatment. Fifty percent of the media was exchanged with fresh medium containing B27  every 4 days. Neurons from WT and EP1−/− pups were treated with either vehicle control or hemin  in Neurobasal/B27 minus antioxidant supplemented medium. For direct comparison between WT and EP1−/− neuronal cultures, “sister cultures” were used to increase the reliability of our data.Postnatal mouse neuronal cultures were isolated from 0- to 1-day-old WT and EP1Nco1  using manufacturer protocols. Completion of plasmid digestion was visualized through gel electrophoresis with ethidium bromide staining. Linearized plasmid DNA was gel extracted using the Qiagen gel extraction kit (Qiagen) following the manufactures protocols and quantified via Nanodrop-1000 spectrophotometer . To analyze the copy number of each receptor, eight point standard curves ranging from 4.0 × 107 to 4000 copies per μL was constructed using linearized plasmid DNA obtained from each receptor. Finally, to ascertain the best concentration of cDNA synthesized from the RNA isolated from our neuronal cultures and determine copy numbers, a six point standard curve was developed for all four receptors ranging from 200 to 3.125 ng of total cDNA. The copy numbers for each receptor type was estimated using the following formula: number of copies/μL = 6.022 × 1023 (moles/mole) × DNA concentration (g/μL)/number of base pairs × 660 Dalton's  containing inserts for each receptor (EP1-4) were cultured overnight in Luria-Bertani broth/Amp (100 μg/mL) at 37°C. Plasmid DNA was purified with a QIAprep Spin Miniprep Kit . Purified bacterial plasmid DNA was linearized through restriction enzyme digestion using To assess hemin neurotoxicity, neurons were treated with control  and hemin at concentrations 0, 12.5, 25, 50, 75, and 100 μM for 18 h. In separate sets of experiments, neurons were pre-treated with EP1 receptor selective antagonist SC-51089 (10 μM)  and selective COX-2 inhibitor NS-398 (10 μM) (Sigma-Aldrich) and NMDAR antagonist MK801 (10 μM) , for 15 min in Neurobasal/B27 minus antioxidant supplement media and then with these agents still present, neurons were co-treated with vehicle or hemin (75 μM) for 18 h. Cell viability was assessed using the CytoScan™-Fluoro assay . This is a fluorometric assay (excitation/emission 530/590 nm) for estimating cell cytotoxicity based on the release of LDH from cells with damaged membranes. The low LDH activity (background) in wells subjected to medium exchange only was subtracted from all values to yield the signal specific for the neurotoxic insult-mediated by hemin as described by Koh and Choi  for 1 h and washed twice with HBSS consisting of 2 mM CaCl2 and 1 mM MgCl2. The membrane-permeable fluorescent calcium indicator Fluo-8 AM was used for imaging dynamic changes in [Ca2+]i, an increase in fluorescence intensity of Fluo-8 AM corresponds to an increase in [Ca2+]i. To determine if the change in fluorescence following treatment with hemin reached plateau, calibration of Fluo-8 AM was performed with NMDA (100 μM) and ionomycin (5 μM) after the addition of hemin in the same cultures. Changes in Fluo-8 AM-fluorescence intensity were measured following addition of 17-pt-PGE2 (100 nM and 1 μM)  or hemin (1–25 μM) for 60 s with and without 15 min pre-treatment with SC-51089 (10 μM), NS-398 (10 μM), MK-801 (10 μM), nifedepine (10 μM), ryanodine (50 μM) and vehicle (0.05% DMSO). The intensity of fluorescence was determined with excitation 482 nm and emission 505–530 nm of selected neurons using a Leica DMI6000 B inverted microscope equipped with Leica HC PL FLUOTAR 20×/0.50 objective and Lambda DG-5 Plus. All images were captured using a Hamamatsu electron multiplying charged-coupled device camera and select regions acquired at a scanning time of ~1 s per frame. A time course of changes in fluorescence intensity generated was with MetaFluor Fluorescence imaging software . Fluo-8 AM fluorescence values are presented as Δ F/F0 where Δ F = F − F0, F is the fluorescence recorded following 17-pt-PGE2 or hemin application and F0 is the mean basal fluorescence. To determine statistical analysis between treatments, area under the curve (AUC) was calculated for each neuron recorded using the trapezoid rule as computed by GraphPad Prism. Data for each treatment was from 3 to 6 independent neuronal cultures. All imaging experiments were done at room temperature.Eight to ten-day-old postnatal mouse cortical neuronal cultures were loaded with 3 μM Quest Fluo-8™ (Fluo-8 AM)  in Hanks' balanced salt solution (HBSS) without CaClt-test. Differences were considered significant if p < 0.05. All data are expressed as mean ± SEM. All data were analyzed by GraphPad Prism 6.0 software.One-Way ANOVA was used to test for differences in mean values from multiple samples  with Bonferroni's multiple comparison tests. Statistical difference between two means was analyzed by two-tailed unpaired or paired Student's 2 receptor subtype. EP3  is the most highly expressed receptor. The mRNA expression of EP1, EP2, and EP4 receptors all had the same levels of expression (estimated 4000 copies) , 75 μM (34.7 ± 4.5%), and 100 μM (50.1 ± 4.2%) of hemin were significantly greater compared to control (16.5 ± 1.5%). Using the LDH assay, we also found hemin to be neurotoxic in neurons cultured from EP1−/− mice. Hemin at 75 μM (27.6 ± 3.3%) and 100 μM (44.3 ± 0.8%) significantly increased LDH release as compared to control (12.8 ± 1.7)  or EP1 receptor antagonist SC-51089 (10 μM) for 15 min and then co-treated with hemin (75 μM). Compared to hemin alone (61.8 ± 1.9%), MK-801 and SC-51089 significantly decreased  hemin-induced LDH release  Figure . Howevere Figure . Similar−/− neurons as compared to WT neuronal cultures when treated with 50 and 75 μM of hemin  significantly increased [Ca2+]i   was significantly different between WT and EP1−/− neurons (18.8 vs. 12.4 respectively).   Figure . Next, 1) Figure .2+]i in EP1−/− neurons, separate experiments were conducted in WT neurons using SC-51089. Pre-treatment with SC-51089 (10 μM) reduced 17-pt-PGE2 (100 nM)-mediated [Ca2+]i  . In these experiments, NS-398 attenuated 17-pt-PGE2-mediated increase in [Ca2+]i  , L-type calcium channel blocker, nifedepine (10 μM) and ryanodine receptor (RyR) antagonist, ryanodine (50 μM). In all cases, application of the antagonists lead to a significant decrease of the 17-pt-PGE2-mediated [Ca2+]i . Following nifedepine treatment alone the measured [Ca2+]i slightly but significantly decreased, the AUC has a negative value (−3.4) compared to that when the same antagonist was used with 17-pt-PGE2 (AUC: 0.38). This decrease in [Ca2+]i by nifedepine treatment alone may have been due to the steady-state blockade of calcium influx via the L-type calcium channel.In addition to studying [Ca) Figure . Further) Figure . To dete Figures . Applica2+]i. Thus, following application of hemin (1–25 μM) for 60 s, we measured a dose-dependent increase in [Ca2+]i and AUC in WT neuronal cultures . Using antagonists of the EP1 receptor, NMDAR and RyR, we found that hemin-induced [Ca2+]i decreased. Treatment with SC-51089 (10 μM) (AUC: hemin 8.09 vs. SC-51089 0.90), MK-801 (10 μM) (AUC: 1.96), or ryanodine (50 μM) (AUC: 1.1) significantly decreased or abolished hemin-induced increase in [Ca2+]i. However, treatment with nifedepine (10 μM) (AUC: 3.8) only decreased hemin-induced [Ca2+]i to 64.1 ± 16.6% of the average value following hemin only . However, 17-pt-PGE2 also shows considerable EP3 receptor  agonism at higher concentrations and therefore may explain why there was still Ca2+ signaling in EP1−/− neurons following treatment with 17-pt-PGE2  against the EP1 receptor would allow us to determine the functional implications of higher levels of the EP3 receptor compared to that of the EP1 receptor in vitro. Together with cell viability and Ca2+ imaging experiments, we showed that SC-51089 and MK-801 significantly reduced hemin-induced neurotoxicity suggesting that activation of NMDAR and EP1 receptors are involved in hemin-induced neurotoxicity. Calcium imaging studies presented here also indicate that hemin stimulates RyR-operated Ca2+ release as the blockade of RyR resulted in the decrease of hemin-induced [Ca2+]i in WT neurons.Prostaglandin-mediated inflammation and the accumulation of red blood cell metabolites such as iron and hemin have been recognized in the pathogenesis of stroke, Alzheimer and Parkinson diseases. However, our knowledge of how prostaglandins and hemin accumulation results in neuronal death is unclear. The results presented here show that hemin is neurotoxic and also affects Caαq-proteins, resulting in increased phosphatidyl inositol hydrolysis and the elevation of [Ca2+]i. The increase in [Ca2+]i has an important role in the control of neuronal function and plasticity (Catterall et al., 2+]i that exacerbate neuronal cell death (Kawano et al., 2+]i, pharmacologic blockade of EP1 receptor could be a promising therapeutic intervention against neuronal injury (Gendron et al., in vitro (Liu et al., 2+]i in neuronal activity and excitotoxicity following hemorrhage is unknown; however, data suggest that increased [Ca2+]i contributes to increased cerebral vasospasm commonly seen in intracerebral hemorrhage (Kikkawa et al., −/− mice showed reduced neuroprotection when treated with NMDA in neuron-glia co-cultures (Carlson et al., in vitro data demonstrates that EP1 deletion provides neuroprotection against hemin-induced cell death, recently our group has found that EP1 deletion may exacerbate neurobehavioral impairments in a collagenase-induced mouse model of hemorrhagic stroke by impairing the function of microglial cells surrounding the area of injury (Singh et al., in vitro and in vivo models are so numerous that different cell types that express the EP1 receptor may reflect the overall outcomes. Although the hemin-mediated increase in [Ca2+]i was significantly different between WT and EP1−/− neuronal cultures, the EP1 receptor pathway may not be the single excitotoxic pathways activated by hemin.Previous reports have suggested that prostaglandins and their cognate receptors are involved in mechanisms of neurodegeneration in cerebral ischemia and excitotoxic brain injury Doré, . When ac+ and Ca2+ results in the rapid depletion of neuronal energy, with a significant reduction in adenosine triphosphate (Tsuji et al., 2+-permeable NMDARs (Lafon-Cazal et al., 2+ homeostasis with NMDA treatment by impairing the function of Na+/Ca2+ exchanger mechanisms (Kawano et al., 2+]i. The measured decrease in hemin neurotoxicity and [Ca2+]i after blockade of NMDARs suggests that NMDAR activation is also involved and perhaps more so during the initial phases of hemin-induced [Ca2+]i in neurons. Interestingly, our data also show that the hemin-induced increase in [Ca2+]i was partially decreased with nifedepine, a selective blocker of L-type Ca2+ channels. The involvement of Ca2+ channels in hemin neurotoxicity is possibly due to presynaptic facilitation of glutamate release and consequent overactivation of NMDARs. Considering the predominate effects of hemin on NMDAR-mediated neuronal death, we postulate that Ca2+ entry via postsynaptic and/or extrasynaptic NMDARs may alter the neuronal excitability and cellular signaling resulting in a deficit in overall neuronal networks.The overactivation of postsynaptic glutamate receptors and the neuronal loading of Na2+]i through its capacity to generate ROS (Munoz et al., in vivo (Bostanci and Bagirici, 2+ release through its capacity to generate ROS. In this study, following treatment with RyR antagonist, hemin-induced increase in [Ca2+]i was abolished suggesting that RyR is involved in the hemin-induced increase in [Ca2+]i following stimulation of NMDARs. These data show that hemin-induced [Ca2+]i signaling is rapidly initiated and is a lasting process that may have a role in neuronal death.Iron, as a neurotoxic breakdown product of heme and Hb, has previously been shown to stimulate RyR and mediate an increase in [Ca2+]i via activation of multiple mutually dependent processes involving NMDARs, L-type Ca2+ channels, and calcium-induced calcium release via activation of intracellular RyR and COX-2 pathway. Also, the data that 17-pt-PGE2 and hemin-mediated increase in [Ca2+]i were inhibited by blockade of the EP1 receptor suggest involvement of this receptor as an important counterpart in hemin-induced toxicity.In summary, we have demonstrated that pharmacologic blockade and genetic deletion of the EP1 receptor can affect the outcome of hemin-induced cell death in cultured postnatal cortical neurons. Our data suggest that hemin-induced neurotoxicity is mediated by an abnormal increase in [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.


Saccharomyces cerevisiae has earlier been reported to respond to blue light via activation of the stress-regulated transcription factor Msn2p. Environmental changes also induce activation of calcineurin, a Ca2+/calmodulin dependent phosphatase, which in turn controls gene transcription by dephosphorylating the transcription factor Crz1p. We investigated the connection between cellular stress caused by blue light and Ca2+ signalling in yeast by monitoring the nuclear localization dynamics of Crz1p, Msn2p and Msn4p. The three proteins exhibit distinctly different stress responses in relation to light exposure. Msn2p, and to a lesser degree Msn4p, oscillate rapidly between the nucleus and the cytoplasm in an apparently stochastic fashion. Crz1p, in contrast, displays a rapid and permanent nuclear localization induced by illumination, which triggers Crz1p-dependent transcription of its target gene CMK2. Moreover, increased extracellular Ca2+ levels stimulates the light-induced responses of all three transcription factors, e.g. Crz1p localizes much quicker to the nucleus and a larger fraction of cells exhibits permanent Msn2p nuclear localization at higher Ca2+ concentration. Studies in mutants lacking Ca2+ transporters indicate that influx of extracellular Ca2+ is crucial for the initial stages of light-induced Crz1p nuclear localization, while mobilization of intracellular Ca2+ stores appears necessary for a sustained response. Importantly, we found that Crz1p nuclear localization is dependent on calcineurin and the carrier protein Nmd5p, while not being affected by increased protein kinase A activity (PKA), which strongly inhibits light-induced nuclear localization of Msn2/4p. We conclude that the two central signalling pathways, cAMP-PKA-Msn2/4 and Ca2+-calcineurin-Crz1, are both activated by blue light illumination.Light in the visible range can be stressful to non-photosynthetic organisms. The yeast  Saccharomyces cerevisiae cannot utilize specialized tissue for protection against a wide range of environmental challenges, such as variation in temperature, pH, toxic agents, osmolarity, and UV or visible light illumination. Instead, the whole organism must be able to quickly adapt to environmental changes to counter damage. Naturally, one set of mechanisms optimized to counteract a certain external stress might not be appropriate for a different type of disturbance, i.e. many of the stress responses are specific and often highly complex. For example, signalling pathways react differently to different types of stresses and their responses may also depend on previous cellular “states” 2+-signalling and visible light illumination and report on a comparative study of the temporal localization dynamics of these transcription factors in individual cells subject to the same level of light-induced stress.Yeast cells are continuously exposed to a variety of external conditions that they need to cope with to maximize growth and survival in their natural environment. To maintain optimal internal conditions (i.e. homeostasis) a diverse set of mechanisms, control functions and strategies have evolved. In contrast to higher eukaryotes, unicellular organisms like the budding yeast 2+, which fulfils critical functions in both uni- and multi-cellular organisms 2+-dependent signalling pathways can either be induced by increased cytosolic levels of Ca2+ recruited from secretory compartments, which in eukaryotic cells contain high concentrations of Ca2+, or from increased influx from extracellular sources S. cerevisiae Ca2+ signalling is critical for cell survival during exposure to a variety of environmental stresses, including high salt concentrations, alkaline pH, and cell wall damage. These conditions cause an increase in cytosolic Ca2+2+ concentration is held within 50–200 nM in actively budding cells, even when the extracellular conditions vary from <1 µM to >100 mM 2+ pumps, exchangers and channels 2+ levels increase, the Ca2+ sensor protein calmodulin binds to and activates calcineurin, a well-conserved protein phosphatase. Calcineurin, in turn, dephosphorylates Crz1p 2O2 and heat shock Stress responses in yeast involve multiple integrated signalling cascades 2+The transcription factors Msn2p and Msn4p 2+ levels 2+. Apart from delineating a novel role for Crz1p in the cellular response to illumination, our results highlight the light-induced activation of two central signalling pathways, cAMP-PKA-Msn2/4 and Ca2+-calcineurin-Crz1.By following the temporal and spatial dynamics of the transcription factor Msn2p tagged with green fluorescent protein (GFP) using fluorescence microscopy, we previously demonstrated that continuous exposure to blue light leads to cumulative cellular stress, most likely due to accumulation of toxic photoproducts S. cerevisiae (BY4742) yeast cells with the genotype MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 were used throughout this study. The single deletion strains, except for nmd5Δ (see below for details), were acquired from the EUROSCARF yeast deletion collection ADH1 promoter and containing a LEU2 marker. Crz1-GFP was expressed from the pKK249 plasmid MET25 promoter and containing a URA3 marker. Msn4-GFP was expressed in the same way as Msn2-GFP using the plasmid pGR247 MSN2 ORF had been replaced by the MSN4 ORF from the plasmid pAL32-45. Strain YAJ92 (nmd5Δ) was created by deleting NMD5 in strain BY4742 by the one-step PCR targeted gene disruption method nmd5Δ fw TTTCAGCATAATCAATCATTTTTCGTCTGGATATTTGACACAATTTTGATTTTGACGAAGACATTTTATTCTCGATAATACGATTTAGGTGACACTATAG, nmd5Δ rev TTGAGCATAATATCCTCTCTCTTCTATCTAAATTATGTAATCCAGTGTTTGTTTCAATATACAATCGCCATTTAATTCAAATACGACTCACTATAGGGAG) and plasmid pFA6-kanMX4 as a template. Correct integration was verified by diagnostic PCR. Strains YAJ85 (wild-type Cmk2-GFP) and YAJ89 (crz1Δ Cmk2-GFP) were generated by crossing BY4741 Cmk2-GFP to BY4742 crz1Δ::kanMX4 (both from EUROSCARF) and selecting for methionine prototrophic, histidine prototrophic, lysine auxotrophic and G418 sensitive (YAJ85) or G418 resistant (YAJ89) progeny. YAJ85 is MATa and YAJ89 is MATα.Haploid 2+], but ammonium sulphate was replaced with 3.5 g/l ammonium chloride (Sigma) and CaCl2 (Sigma) was used to set the [Ca2+]. Medium without CaCl2 still contained trace amounts of Ca2+ originating from calcium pantothenate (0.4 mg/l ∼ 0.8×10−3 mM). Overnight cultures were inoculated in the morning and grown to an OD600 of 0.4–0.5 at the start of the microscopy experiment. Where indicated addition of EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid) to a final concentration of 5 mM was made from a stock solution of 200 mM (pH 8.0).Strains were grown at 30°C in synthetic defined medium containing Yeast Nitrogen Base (Formedium™) with ammonium sulphate (0.5%), glucose (2%) and complete supplement mixture without the appropriate amino acids and nucleotide bases , buffered to pH 5.8 with 1% succinic acid and 0.6% NaOH. The same medium was used in experiments with different : 0.8×10−3 mM, 0.9 mM (standard medium), 5 mM and 10 mM. Cells were exposed to continuous blue light illumination  during 60 minutes and the nuclear localization of the transcription factors were analysed. No cells displayed nuclear localization at the start of the experiment as a result of the altered Ca2+ concentration compared to standard medium. Note that Ca2+ can potentiate the response in particular for Crz1p and Msn2p.(TIF)Click here for additional data file.Figure S42+ chelation by EGTA inhibits the nuclear localization response of Crz1p during illumination.Ca Addition of EGTA to the culture medium to a final concentration of 5 mM just prior to illumination increases the response time and lowers the number of cells with nuclear localization of Crz1p (blue curve) compared to wild-type cells (red curve) or cells grown in trace amounts of Ca2+ (black curve). In a pmc1Δ strain, Crz1p did not localize to the nucleus (green curve), indicating that both extracellular Ca2+-influx and vacuolar stores are important for the wild-type response.(EPS)Click here for additional data file.Figure S5Crz1p nuclear localization in pmr1Δ cells. (A) The nucleocytoplasmic localization profile of Crz1-GFP extracted from fluorescence microscopy images. Cells were continuously illuminated with blue light . (B) Time to first Crz1p nuclear localization. Deletion of PMR1 decreases the response time of Crz1p. The differences are statistically significant .(TIF)Click here for additional data file.Figure S62+-calcineurin-Crz1 signalling cascade or in PKA activity.Altered vacuolar morphology seems independent of perturbations in the Ca (A) Altered vacuolar morphology in response to illumination  was quantified by manual inspection in (B) wild-type and cnb1Δ cells, (C) pde2Δ cells, (D) pmc1Δ cells, (E) cells treated with 5 mM EGTA just prior to light-induced stress and (F) cells grown in higher extracellular Ca2+ levels. The plots show the mean value of two independent experiments with error bars representing 1 standard deviation. These data indicate that altered vacuolar morphology seen upon illumination with blue light does not seem to be linked to the Ca2+-calcineurin-Crz1 signalling cascade or to reduced PKA activity.(EPS)Click here for additional data file.Datasheet S1Experimental data. Nuclear localization trajectories for individual cells.(XLSX)Click here for additional data file.Datasheet S2Experimental data. Nuclear localization trajectories for individual cells.(XLSX)Click here for additional data file.Datasheet S3Experimental data. Nuclear localization trajectories for individual cells.(XLSX)Click here for additional data file.


The Ugi 4- and 3-component reactions in particular , have pr


The equine infectious anemia virus (EIAV) is a lentivirus of the Retrovirus family, which causes persistent infection in horses often characterized by recurrent episodes of high fever. It has a similar morphology and life cycle to the human immunodeficiency virus (HIV). Its transmembrane glycoprotein, gp45 , mediates membrane fusion during the infection. However, the post-fusion conformation of EIAV gp45 has not yet been determined. EIAV is the first member of the lentiviruses for which an effective vaccine has been successfully developed. The attenuated vaccine strain, FDDV, has been produced from a pathogenic strain by a series of passages in donkey dermal cells. We have previously reported that a V/I505T mutation in gp45, in combination with other mutations in gp90, may potentially contribute to the success of the vaccine strain. To this end, we now report on our structural and biochemical studies of the gp45 protein from both wide type and vaccine strain, providing a valuable structural model for the advancement of the EIAV vaccine.d position within the heptad repeat, which protruded into a 3-fold symmetry axis within the six-helix bundle. Our crystal structure analyses revealed a shift of a hydrophobic to hydrophilic interaction due to this specific mutation, and further biochemical and virological studies confirmed that the mutation reduced the overall stability of the six-helix bundle in post-fusion conformation. Moreover, we found that altering the temperatures drastically affected the viral infectivity.We resolved crystal structures of the ecto-domain of gp45 from both the wild-type EIAV and the vaccine strain FDDV. We found that the V/I505T mutation in gp45 was located in a highly conserved Our high-resolution crystal structures of gp45 exhibited high conservation between the gp45/gp41 structures of lentiviruses. In addition, a hydrophobic to hydrophilic interaction change in the EIAV vaccine strain was found to modulate the stability and thermal-sensitivity of the overall gp45 structure. Our observations suggest that lowering the stability of the six-helix bundle (post-fusion), which may stabilizes the pre-fusion conformation, might be one of the reasons of acquired dominance for FDDV in viral attenuation. Lentiviruses infect mammals and induce various diseases characterized by persistent infection and progressively degenerative pathology. For example, the equine infectious anemia virus (EIAV) triggers EIA characterized by anemia, thrombocytopenia, edema, and other degenerative symptoms in horses; and the infection of human immunodeficiency virus (HIV) causes acquired immune deficiency syndrome (AIDS) -4. For HLentiviruses rely on two glycoproteins, gp120/gp90 and gp41/gp45, that mediate attachment to the host cell membrane and viral entry -13. In td position of the heptad repeat, protruding toward the central axis within the six-helix bundle, where high levels of conservation are observed for a range of lentiviruses including HIV and SIV. Along with biochemical and virological data, we discuss the potential association and involvement of this mutation within the vaccine strain.Several achievements have been accomplished in vaccine development against virus-induced diseases ,31,32. E489), pointed outwards and formed an open pocket at the tip of the six-helix bundle  Figure A and conComparative studies of EIAV gp45 and HIV gp41, were carried out by superimposition of the gp45 onto a readily available crystal structure of HIV gp41 (PDB code 2X7R), which contained the fusion peptide proximal region (FPPR) and was the most complete HIV gp41 structure resolved to date. Remarkably, the FPPR at the N-terminus of the NHR of HIV gp41 adopts an open conformation, as in gp45, but the width of the pocket is less pronounced than that of the latter , the NHR helix in gp41 is bent in the middle . However, the early stage of EIAV infection, during which the balance between the virus and host immune system is adjusted, is characterized by febrile episodes . To deted position within the heptad repeat, has prompted investigations into the association of this mutation within the viral attenuation mechanism.EIAV gp45 and HIV gp41 are members of type I membrane fusion proteins, which are conserved in most RNA viruses . In thesThe membrane fusion activity of gp41 is generally proportional to its post-fusion stability . Mutatioin vitro. In the absence of pressure from the host immune system, the V/I505T mutation in gp45 may offer an advantage for the virus in adaptation in specific cells (such as FDD) by increasing the stability of Env pre-fusion conformation, a tendency that cannot be acquired easily in an in vivo environment.The decreased stability of gp45 by V/I505T mutation might also favor the viral replication in specific cell lines, such as donkey dermal cells, a hypothesis which requires further elucidation. Our previous study identified a truncating mutant of gp45 in specific vaccine strains, which resulted in the lack of the C-terminal cytosolic region. This mutant has lower replication in monocyte-derived macrophage (MDM) but higher replication in fetal donkey dermal cell (FDD), indicating an adaptation depending on the host cell context . The vacOur structures of EIAV gp45 also revealed differences between EIAV gp45 and HIV gp41. EIAV gp45 had a more loosely packed structure compared with HIV gp41, reflected by a higher number of coordinated water molecules at the center of the gp45 trimer and observed lower melting temperatures of the six-helix bundle. The EIAV is known to utilize the ELR1 as its sole receptor , in contin vitro at 37°C, encouraging viral adaptation to this temperature but making it considerably more sensitive to temperature fluctuations during febrile episodes. This alteration may allow the host to control viral load easily, while simultaneously maintaining a continuous low level of viral replication asymptomatically and as a result driving the mutual co-evolution of the virus and its host.Additionally, the melting temperature (Tm) of gp45 is also likely to play a role in EIAV vaccine development. We found that the Tm of WT gp45 was 58°C, much lower than that of the HIV gp41 (~80°C) . The gp4in vivo to achieve the effective protection required by the host. Importantly, the level of immunity observed with the vaccine correlates with that of the divergence evolved from the original viral Env [In EIAV vaccine, the attenuated vaccine virus provides a high level of immunity, presumably due to the continuous antigen presentation and consistent optimization of the host immune system. Recent EIAV vaccine studies have indicated that the attenuated virus must achieve a critical level of replication to sufficiently drive the maturation of the host immune . Craigo,iral Env . Hence, in vitro attenuation, and direct a strategy for further development of an effective lentiviral vaccine. Recently, the full-length gp140 structures consisting of all the variable regions have been reported, using both crystallographic and cryo-electron microscopy techniques [d positions can have a direct impact on the stability of viral spikes, offering new implications for the design of envelope protein immunogens.In summary, we have carried out structural and biochemical studies of the EIAV gp45 from both wide-type and vaccine strain. A hydrophobic to hydrophilic interaction change in the EIAV vaccine strain was found to modulate the stability and thermal-sensitivity of the overall gp45 structure. Our studies suggested that the unique features of gp45, such as loose packing and low Tm, contribute to the success of FDDV in EIAV vaccine development. Our studies provide useful information on the underlying mechanism of viral adaptation during chniques ,54. ThesE.coli DH5α competent cells for plasmid propagation. Using this method, a 6× His-tag and a TEV cleavage site derived from the LIC vector were fused upstream of gp45. The QuikChange site-directed mutagenesis kit (Stratagene) was used to generate point mutations. All procedures were performed in accordance with the manufacturer’s instructions.For crystallography structure analysis, the EIAV gp45 was constructed by overlapping the NHR and CHR regions with a GGSGG linker. Both the NHR and CHR were amplified by polymerase chain reaction (PCR) using the gene encoding full-length template of EIAV gp140 (gp90 + gp45) and further cloned by employing the ligation-independent cloning (LIC) technique. The pET30-TEV/LIC was digested with SspI and extracted using a gel extraction kit (Axygen). Subsequently, the cleaved plasmids and purified PCR products were digested with a T4 DNA polymerase (Promega) in the presence of dGTP or dCTP, respectively. The annealed mixture containing pET30-TEV/LIC-gp45 was transformed into E.coli Rosetta™ (DE3) and cultured in Luria-Bertani (LB) medium, supplemented with kanamycin (50 μg/mL). The cells were grown at 37°C until an OD600 of 0.8 was reached. Recombinant protein expression was induced by the addition of 200 μM isopropyl β-D-1-thiogalactopyranoside (IPTG) and allowed to proceed for 12 h at 20°C. The cells were harvested by centrifugation  and re-suspended in a buffer consisting of 25 mM Tris, 500 mM NaCl, 20 mM imidazole, at pH 8.0. Subsequently, cells were lysed by sonication and the crude extract was clarified by centrifugation at 38,000 × g, for 40 min at 4°C.6× His-tagged gp45 was expressed in The supernatant was applied onto a pre-equilibrated Ni–NTA resin, consisting of buffer A  and transferred into a column. The column was washed five times with buffer A containing 40 mM imidazole. Subsequently, proteins bound to the column were eluted with the buffer containing 500 mM imidazole. The eluent was further purified using a Q-Sepharose HiTrap HP column  with a linear concentration gradient of NaCl ranging from 100 to 500 mM. Peak fractions were collected and polished using a Superdex-200 gel filtration column . The purified protein was concentrated to 15 mg/mL and its purity was determined to be >95%, as analyzed by SDS-PAGE and Coomassie blue staining (data not shown).WT (derived from the sequence of strain LN40) were grown using the sitting-drop vapor diffusion method at a constant temperature of 20°C; by mixing equal volumes (1:1 μL) of both the protein  and reservoir  solutions. EIAV gp45VACCINE crystals (gp45WT with the V505T mutation) were grown at 20°C in 0.2 M NaCl, 0.1 M sodium acetate trihydrate, 30% v/v (+/-)-2-methyl-2,4-pentanediol, at pH 4.6. Following this, the gp45WT crystals were immersed in 100% paraffin oil for several seconds and the gp45VACCINE crystals were washed in the reservoir solution, prior to freezing in liquid nitrogen for storage.Crystals of EIAV gp45Data collection was performed at the Shanghai Synchrotron Radiation Facility. All datasets were collected at a wavelength of 0.9792 Å (at 100 K), processed using the HKL2000 package , and conWT was solved by molecular replacement using the HIV gp41 structure (PDB code 2X7R) as a search model. Initial models were refined with Phenix [VACCINE was solved following similar procedures by using gp45WT as the initial model. All models had low R and Rfree factors, good deviations from ideal geometry and no Ramachandran outliers. The refinement statistics are summarized in Additional file The structure of EIAV gp45h Phenix  accompanh Phenix . SimulatThe protocol was adapted from a previously published method ,59. Brieg for 10 min, the supernatants were separated through a 0.45 μm filter and stored at -80°C. Viral titers were determined by quantitative PCR (qPCR). Quantified viruses were used for infection of the next passage of ED cells.Wild-type (WT) EIAV was generated from an infectious molecular clone. The clone EIAV UK3  was geneViral RNA was isolated from a 140 μl supernatant using the Qiagen Viral RNA Mini Kit and the whole-cell RNA was isolated using Trizol reagent. In general, the monolayer cultures were rinsed with ice-cold PBS  and lysed upon the addition of 100 μl Trizol in each well, when using a 96-well plate. Phase separation was performed by adding chloroform to the wells and the RNA was precipitated using isopropanol. Finally the RNA pellet was washed in 75% ethanol, air-dried, and re-dissolved in water.The isolated RNA was reverse transcribed and amplified using reverse transcription PCR (RT-PCR) with a PrimeScriptTM RT reagent kit, gDNA Eraser (TaKaRa) and SYBR Premix Ex TaqTM (TaKaRa); SYBR standards RSq > 0.98, 95% < Eff. <105%.6 copies/well). In this case, the supernatant was harvested in 2-day intervals, up to 12 days post-infection. Based on the isolated viral RNA, the virus number was quantified to produce replication curves.To measure the replication kinetics of EIAV, ED cells  were infected by wild-type (UK3) and mutant EIAV viruses  Superimposed structures of EIAV gp45WT  between them. (B) Superimposed structures of EIAV gp45WT  Surface charge potentials of the EIAV gp45, HIV CRF07 gp41, and SIV gp41. Negatively charged residues are colored in red and positively charged residues in blue.Supporting data for the EIAV gp45Click here for fileVACCINE NHR and HIV gp41 NHR derived from crystal structure of Env trimer (PDB code 4NCO). Side-view for (A) and top-view for (B). gp45VACCINE NHR is shown as in Figure Superimposition of gp45Click here for filea, characterized by CD. (B) Thermostability results monitored by CD (at 222 nm) for the a mutants. (C) Sequence alignment of the EIAV gp45 ecto-domain. The LN40 represents the WT pathogenic strain isolated in Liaoning Province, China. The DV/DLV34/DLV61 series is classed as a pathogenic strain; the DLV121/DLV137/FDDV4/FDDV13/FDDV23 as non-pathogenic; with DLV91 depicted as the intermediate. The UK3 pathogenic strain is used in this study to construct the infectious clone. The specific residue at the 505 position is shown in green shadow.Supporting data for the EIAV gp45 mutant proteins. (A) Secondary structure representation of the EIAV gp45 mutants at position Click here for filea (as depicted in Figure Replication analyses of the EIAV mutants at position Click here for fileX-ray crystallographic data and refinement statistics for EIAV gp45.Click here for file


V1.4F1705I) has been linked to cold aggravated myotonia. The functional effects of altered regulation of hNaV1.4F1705I are incompletely understood. The location of the hNaV1.4F1705I in the CT prompted us to examine the role of Ca2+ and calmodulin (CaM) regulation in the manifestations of myotonia. To study Na channel related mechanisms of myotonia we exploited the differences in rat and human NaV1.4 channel regulation by Ca2+ and CaM. hNaV1.4F1705I inactivation gating is Ca2+-sensitive compared to wild type hNaV1.4 which is Ca2+ insensitive and the mutant channel exhibits a depolarizing shift of the V1/2 of inactivation with CaM over expression. In contrast the same mutation in the rNaV1.4 channel background (rNaV1.4F1698I) eliminates Ca2+ sensitivity of gating without affecting the CaM over expression induced hyperpolarizing shift in steady-state inactivation. The differences in the Ca2+ sensitivity of gating between wild type and mutant human and rat NaV1.4 channels are in part mediated by a divergence in the amino acid sequence in the EF hand like (EFL) region of the CT. Thus the composition of the EFL region contributes to the species differences in Ca2+/CaM regulation of the mutant channels that produce myotonia. The myotonia mutation F1705I slows INa decay in a Ca2+-sensitive fashion. The combination of the altered voltage dependence and kinetics of INa decay contribute to the myotonic phenotype and may involve the Ca2+-sensing apparatus in the CT of NaV1.4.Mutations in the cytoplasmic tail (CT) of voltage gated sodium channels cause a spectrum of inherited diseases of cellular excitability, yet to date only one mutation in the CT of the human skeletal muscle voltage gated sodium channel (hNa V1.4 have been reported to cause skeletal muscle disorders V1.4  distinctly modulate inactivation of different isoforms of NaV channels through interaction with structural motifs in the CT although the mechanisms are not fully understood V1.4 channel gating V1.5 and NaV1.1 has been shown to influence Ca2+ regulation of channel gating 2+ sensitivity of channel gating as well 2+2+2+/CaM mediated regulation of gating 2+/CaM sites are also present in the CT of NaV1.4; however, their roles in channel regulation and relevance to disease causing mutations of the CT-NaV1.4 are not known. The goals of this study are to understand the pathogenesis of temperature sensitive myotonia caused by the F1705I mutation, and to use this naturally occurring mutation (and the species specific differences) to better understand the regulation of the NaV channels by Ca2+. We demonstrate that the hNaV1.4F1705I alters the voltage dependence of inactivation and the temperature sensitivity of current kinetics. In addition, key residues in the EFL alter the CaM and Ca2+ dependence of channel gating which may also contribute to the myotonia phenotype.There is substantial evidence that the CT of NaV1.4-EYFP was prepared as described previously V1.4F1698I, was made by site directed mutagenesis using primer pairs:The EYFP fused channel construct NaForward (F): 5′CATGGAGGAGAAGATTATGGCAGCTAACCCTTC3′ andV1.4F1705I, was made by site directed mutagenesis of the human hNaV1.4-EYFP construct using primer pairs:Reverse (R): 5′GAAGGGTTAGCTGCCATAATCTTCTCCTCCATG3′. The human myotonia construct, hNaV1.4F1705I Reverse (R): 5′ GTTGGCTGCCATGATCTTCTCCTCCATG3′. All the clones were sequence verified.Forward (F): 5′ CATGGAGGAGAAGATCATGGCAGCCAAC3′ and hNa6 Human embryonic kidney cells  were cultured in 6-well tissue culture dishes in DMEM supplemented with 10% FBS, L-glutamine (2 mmol/L), penicillin (100 U/mL), and streptomycin (10 mg/mL). The cells were co-transfected with plasmids encoding the β1 subunit and the appropriate fluorescently–tagged NaV1.4 and CaM variants: human or rat wild type NaV1.4-EYFP, hNaV1.4F1705I-EYFP, rNaV1.4F1698I-EYFP, hNaV1.4F1705I+GA/SD-EYFP and ECFP-CaM or ECFP-CaM1234 alone or in combination. Cells were transfected using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer’s instructions and were studied 48 to 72 hours post-transfection. The total amount of DNA for all transfections was kept constant.Approximately 0.75×10V1.4 channels were selected for recording. In some experiments, cells expressing both yellow and cyan fluorophores that were co-transfected with tagged NaV1.4 channel variants and CaM or CaM1234 were selected for patching. Cells were patch clamped with an Axopatch 200B patch-clamp amplifier using pipettes with tip resistances of 1–3 MΩ and typical series resistance compensation of >90% to minimize voltage clamp errors. Current recording was initiated 10 minutes after establishing whole-cell configuration, and currents were filtered at 5 kHz.HEK293 cells expressing wild type or mutant EYFP-tagged Na2, 1 MgCl2, 10 glucose and 10 Na-HEPES (pH 7.4). The Ca2+ free patch pipette solution contained (in mmol/L): 10 NaF, 100 CsF, 20 CsCl2, 20 BAPTA, 0 CaCl2 and 10 HEPES, pH adjusted to 7.35 with CsOH. The 0.5 µmol/L Ca2+ patch pipette solution contained (in mmol/L): 10 NaF, 100 CsF, 20 CsCl2, 5 BAPTA, 4 CaCl2 and 10 HEPES, pH adjusted to 7.35 with CsOH. The osmolarity of the bath and pipette solutions were equalized using glucose. The free [Ca2+] in the solutions was estimated with WEBMAX Standard software (http://www.stanford.edu/~cpatton/webmaxcS.htm) and found to be about 0.5 µM. The free [Ca2+] in the solutions was verified by measurement using a Kwik-Tip Calcium ion-selective electrode (WPI)  and fit to a Boltzmann function of the form (y = [(A1−A2)/(1+e(x−x0)/dx))]+A2) to determine the membrane potential eliciting half-maximal inactivation (V1/2), where A1 and A2 are maximum and minimum availabilities, respectively, x0 is equivalent to V1/2, and dx represents the slope factor. Activation curves were generated from a family of 50 ms test pulses from −100 mV to +80 mV in 5 mV increments from a holding potential of −120 mV. Peak currents at each membrane potential normalized to Imax, were plotted to generate the I–V curves. Conductance (G) was calculated for peak current (IPeak) at each membrane potential (Vm) using the equation ). G was normalized to GMax and fitted to a Boltzmann distribution to determine the membrane potential eliciting half-maximal activation (V1/2). Deactivation was assessed using tail currents elicited by a test pulse of 0.5 ms to +40 mV followed by repolarization to a family of voltages ranging from −180 to −50 mV. INa decay rates were assessed by fitting to a single exponential decay , where y0 is the offset and A is amplitude. Significance was assessed using unpaired student’s t-test , and p<0.05 was considered significant.Standard two-pulse protocols were used to generate the steady-state inactivation curves. The voltage dependence of steady-state fast inactivation was studied using 500 ms inactivation pre-pulses over a voltage range from −140 to +30 mV in steps of 5 mV, followed by a 50 ms test pulse at −20 mV. Currents were normalized to the maximal current  of activation of the wild type and mutant channels did not differ in the presence of 0.5 µM [Ca2+]i (A mutation (F1705I) in the structured portion of the CT of hNamyotonia . We studmV Inset . The norn muscle . The vol [Ca2+]i .V1.4F1705I. Compared to the wild type channel, the V1/2 of the steady-state inactivation curve of hNaV1.4F1705I was significantly shifted ∼+11 mV in 0.5 µM [Ca2+]i  curves were unchanged in the absence of Ca2+  in the depolarizing direction in the absence of intracellular Ca2+ compared with the 0.5 µM [Ca2+]i  and Ca2+ binding-deficient hNaV1.5 channels that are mutated in the EFL + current inactivation exhibited by hNaV1.4F1705I compared to wild type hNaV1.4 are due to functional alterations in CaM interaction with the IQ motif. We examined the effects of CaM over expression on hNaV1.4F1705I and wild type hNaV1.4 gating. As Ca2+ can bind to CaM and modulate channel gating, we also studied effect of Ca2+ binding deficient CaM or apo-CaM (CaM1234) to delineate direct effects of Ca2+ on the channel compared to Ca2+ effects through CaM. In contrast to the wild type rat NaV1.4 channel 1234 with wild type human NaV1.4 shifts the channel’s I–V relation in the depolarizing direction in 0.5 µM [Ca2+]i  shifted in the depolarizing direction (∼+12 mV) compared to wild type rNaV1.4 in 0.5 µM [Ca2+]i  and alanine (A), and the corresponding residues in rNaV1.4 are serine (S) and aspartic acid (D) respectively . hNaV1.4F1705I+GS/AD and hNaV1.4 have comparable peak current I–V relationships, and in both cases the voltage dependences of activation were not altered by the absence or presence of 0.5 µM Ca2+  reestablished Ca2+ and CaM sensitivity of inactivation gating mimicking that of wild type rat NaV1.4 (V1.4 (rNaV1.4SG/DA) or mutant rNaV1.4F1698I (rNaV1.4F1698I+SG/DA) background restored Ca2+/CaM regulation similar to that displayed by hNaV1.4 or hNaV1.4F1705I respectively  is ∼5 times faster than at RT and is nearly identical to the decay rate of wild type hNaV1.4 current at 37°C , abolishes the Ca2+ sensitivity of hNaV1.4F1705I+GS/AD mimicking that of rNaV1.4F1698I. Similarly, mutating the non-conserved amino acids of the wild type hNaV1.4 EFL region to match that of the rat sequence (G1613S and A1636D) restores the Ca2+ sensitivity of the channel mimicking that of wild type rat NaV1.4  has been reported, implicating the EFL region of the CT in inactivation gating 2+ sensing. Hydrophobic helical interactions in the cardiac NaV1.5 EFL, particularly H1–H4, appear to stabilize the structure of the proximal CT of the channel which is postulated to be a prerequisite for durable inactivation The biophysical effects of hNat NaV1.4 , Table 12+ regulation of gating by residues in the CT is not fully understood and the role of direct Ca2+ binding to this region is debated. In fact the crystal structure of a ternary complex of the CT-hNaV1.5, CaM and FHF fails to show Ca2+ in the EFL region 2+ sensitivity of hNaV1.5 gating 2+ is binding to this region; an alternative is that the mutation(s) produce an allosteric change that alters Ca2+ sensitivity of gating.The mechanism of the CaV1.4 and it remains tethered to the mutant channels in all conformations. An alternative explanation is that the IQ-containing H6 interacts with the EFL V1.4F1698I or hNaV1.4F1705I in H5 could alter the conformation and disrupt Ca2+-sensing but this would have to occur without altering the interaction between CaM and the IQ as myotonic mutant channels of both the species are normally regulated by over expression of CaM  or mutated residues in the EFL (rNaV1.4SG/DA) are insensitive to [Ca2+]i. This study also demonstrates that detailed regulation of inactivation of variants of NaV1. 4 channels by CaM over expression is determined by the composition of the EFL and the ability of intracellular Ca2+ to regulate gating  or insensitive . The Ca2+ sensitivity is in part determined by the amino acid sequence in the EFL. This sequence divergence is associated with differences in the baseline voltage dependence of inactivation as well as the response to CaM over expression Click here for additional data file.


After 24 h of treatment with Ghrelin (10 nM) or GHRP-6 (100 nM) enhanced BS secretory activity; GH secretion stimulated by GHS through the activation of GHS-R because treatment with the antagonist of GHS-R  blocked the GH secretion, and the effect was dose and time dependent . GH secretion stimulated by GHRP-6 was abolished by nifedipine (0.5 μM), a blocker of L-type HVA Ca2+ channels, and KN-62 (10 μM), an inhibitor of Ca2+/CaM-KII. After 72 h in culture, all recorded BS exhibited two main Ca2+ currents: a low voltage-activated  and a high voltage-activated  current. Interestingly, HVA and LVA channels were differentially upregulated by Ghrelin. Chronic treatment with the GHS induced a significant selective increase on the Ba2+ current through HVA Ca2+ channels, and caused only a small increase of currents through LVA channels. The stimulatory effect on HVA current density was accompanied by an augment in maximal conductance with no apparent changes in the kinetics and the voltage dependence of the Ca2+ currents, suggesting an increase in the number of functional channels in the cell membrane. Lastly, in consistency with the functional data, quantitative real-time RT-PCR revealed transcripts encoding for the Cav1.2 and Cav1.3 pore-forming subunits of L-type channels. The treatment with Ghrelin significantly increased the Cav1.3 subunit expression, suggeting that the chronic stimulation of the GHS receptor with Ghrelin or GHRP-6 increases the number of voltage-gated Ca2+ channels at the cell surface of BS.Activation of the growth hormone (GH) secretagogue receptor (GHS-R) by synthetic GH releasing peptides (GHRP) or its endogenous ligand (Ghrelin) stimulates GH release. Though much is known about the signal transduction underlying short-term regulation, there is far less information on the mechanisms that produce long-term effects. In the current report, using an enzyme-linked immunosorbent assay for GH detection and whole-cell patch-clamp recordings, we assessed the long-term actions of such regulatory factors on voltage-activated Ca Growth hormone (GH) synthesis and secretion are regulated by two hypothalamic hormones with opposite actions, the GH releasing hormone (GHRH) and somatotropin release inhibiting factor  . However2+channels (Cav LVA-T and HVA-L-type) and Na+ tetrodotoxin-sensitive (Nav TTX) channels play a role in the rising phase of the AP (depolarization) in the cell membrane whose result is the increase of the [Ca2+]i and the GH release. Diverse intracellular signaling systems are suggested to mediate the acute actions of the GHS. After binding of ligand, GHS-R acts through the Gα11 subunit of G-protein to activate phospholipase C (PLC) resulting in hydrolysis of PIP2 to generate inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) [2+]i increases due to a transient release of Ca2+ from IP3-responsive cytoplasmic storage pools and a sustained Ca2+ influx due to activation of voltage-activated Ca2+ channels and blockade of K+ channels [2+]i due to intermittent firing of nifedipine-sensitive action potentials. Membrane potential (Vm) and Ca2+ oscillations are interdependent during continued gonadotropin-releasing hormone action, and Ca2+ entry is necessary for the maintenance of [Ca2+]i spinking. The initial and sustained Ca2+ transients induced by the agonist and Vm oscillations are abolished by blockade of endoplasmic reticulum Ca2+-ATPase, consistent with the role of Ca2+ reuptake by internal stores in the oscillatory response during both phases. Such a pattern of synchronization of electrical activity and Ca2+ spiking in cells regulated by Ca2+-mobilizing receptors shows that the operation of the cytoplasmic oscillator can be integrated with a plasma membrane oscillator to provide a long-lasting signal during sustained agonist stimulation i i  and GH r2+]i  in somat release , 42. Basin, CREB . In hipprylation . Signalirylation  include 2+ influx [2+/CaM-K II/IV [2+/CaM-K II, KN-62 abolished the GHS-induced GH secretion in bovine somatotropes. These results suggest that Ca2+/CaM-K II plays a role in the signaling pathway from GHS-R activation to an increase in Ca2+ channel functional expression. Work by other groups it has shown that using isoquinolinesulfonamides KN-62 and KN-93  and KN-92  blocked basal prolactin release in a dose- and time-dependent manner, suggesting that calmodulin-dependent protein kinase could mediate the coupling of electrical activity and secretion. However, a similar effect on basal prolactin release was observed on application of KN-92, which does not inhibit this kinase; and therefore caution should be taken when interpreting data from studies using isoquinolinesulfonamides to evaluate the role of calmodulin-dependent protein kinases in excitable endocrine cells, because inactive compounds exhibit comparable effects on action potential secretion coupling to those of active compounds [2+ channels expressed in bovine somatotropes will be needed to define the role of nuclear Ca2+ and Ca2+/CaM-K II in intracellular protein cascades leading to activation of gene transcription after GHS treatment.The actions of GHS treatment on pituitary somatotropes are linked to G-protein-coupled GHS-R , and GHS+ influx , 46, 47.-K II/IV , 49; anoompounds . Additio2+ channels at the cell surface of bovine somatotropes.In conclusion this study demonstrates that the chronic stimulation of the GHS receptor with Ghrelin or GHRP-6 increases voltage-gated Ca