The rectification is caused by the asymmetrical structure of the myoendothelial junction. Because the head of the myoendothelial junction is separated from the cell it is attached to by a narrow neck region, a signal generated in the neighboring cell can easily drive a concentration change in the head of the myoendothelial protrusion. Subsequently the signal can be amplified in the head, and activate the entire cell. In contrast, a signal in the cell from which the myoendothelial junction originates will be attenuated and delayed in the neck region as it travels into the head of the myoendothelial junction and the neighboring cell.Micro-anatomical structures in tissues have potential physiological effects. In arteries and arterioles smooth muscle cells and endothelial cells are separated by the internal elastic lamina, but the two cell layers often make contact through micro protrusions called myoendothelial junctions. Cross talk between the two cell layers is important in regulating blood pressure and flow. We have used a spatiotemporal mathematical model to investigate how the myoendothelial junctions affect the information flow between the two cell layers. The geometry of the model mimics the structure of the two cell types and the myoendothelial junction. The model is implemented as a 2D axi-symmetrical model and solved using the finite element method. We have simulated diffusion of Ca Such unidirectional flow is found in e.g. synapses of the nervous system 2+ signal initiated in the SMC layer (e.g. by stimulation with phenylephrine) is apparently transmitted to the ECs by diffusion of IP3, whereas propagation of the signal in the opposite direction does not take place 3-receptor channels and other proteins The functional properties of the MEJ have been studied in mouse and rat mesenteric arteries where they extend from the EC layer to contact the SMC layer To address the apparent asymmetry in myoendothelial signaling we constructed a spatiotemporal model of the EC-SMC contact . The strThe model consisted of three main regions, the SMC, the EC and the MEJ that protrudes from the EC in the present model. The protrusion is in continuity with the EC and the SMC is connected with the MEJ through gap junctions, which in the model initially were assumed to have a lumped pore radius of 100 nm  2+ ions in a non-buffered cytosol in order to quantify the basic properties of the structure. Initially the concentration of Ca2+ was 0.1 µM in all compartments. We simulated an increase in bulk cytoplasmic Ca2+ concentration in either the EC or SMC by increasing the boundary concentrations (2+ was assumed to be isotropic with a diffusion coefficient of 233 µm2/s In Model 1 we simulated diffusion of CaAll parameters are listed in 2+-induced Ca2+-release (CICR) from endoplasmic reticulum (ER) in the EC and MEJ. This model contains the following diffusible species: Ca2+ in the cytosol and in ER, Ca2+ buffers in the cytosol and ER and IP3 in the cytosol , which corresponds numerically to the Ca2+ concentration at which the buffer is 50% saturated. In the cytosol of the SMC, EC and MEJ the Kd was 0.5 µM 2+ buffer binding was also modeled in ER:3 receptor is activated by IP3 and by low concentrations of Ca2+, whereas Ca2+ at higher concentrations inhibits the channel. Using a rapid equilibrium approximation De Young and Keizer ijk) describes if a ligand is bound (1) or not bound (0) to the three independent binding sites. The first place (i) refers to binding of IP3, the second (j) refers to binding of Ca2+ to the activating site and the third (k) to binding of Ca2+ to the inhibitory site.110 for the channel to be open, the fraction of open channels is given by 3 is described with the following equations 3 bound forms are found as:x and dx are constants  was modeled using f and r indicates the forward and reverse modes. All constants are given in Re-uptake of Ca3 concentration was 0.1 µM prior to stimulation and the maximum Ca2+ permeability through the IP3R channel (IP3RP) was fitted to counterbalance the activity of the SERCA pump 3 in the EC or the SMC we increased the concentration of IP3 to 0.4 µM around all external boundaries in either the EC or the SMC . An increase in bulk Ca2+ concentration was simulated by raising the concentration of cytosolic Ca2+ ([Ca2+]cyt) by 0.5 µM above the resting level at all boundaries in either of the cells, except for the boundaries of the MEJ    and diffoupling) . We founoupling) .endoplasmic reticulum (ER) (Model 2). ER is present in the MEJ and it may extend into the cytosol of the EC 3 receptor channels (IP3R) 2+ induced Ca2+ release (CICR) 3 was increased in the SMC there was a pronounced Ca2+ release in the MEJ after less than 100 ms due to Ca2+ release from the ER (2+ concentration increased rapidly (<50 ms) after SMC IP3 was an increased . To model variations in the length of the neck the diffusion coefficient was re-scaled in the direction of the z-axis in the narrow part of the neck  that can pass through gap junctions, the structure of the MEJ leads to significant signaling rectification in the sense that the concentration of the diffusible species in the head responds more rapidly to changes in the SMC than it does to changes in the EC from which it originates.We perturbed the model by simulating an increase in the bulk cytosolic concentration of a diffusible species in either the EC or the SMC. The increase in concentration was implemented as a concentration increase at the boundaries in the respective cells . The lag2+ concentration in the head 0.1 s after stimulation of the EC. It is clear that the longer the neck, the smaller the concentration obtained. The reverse was true for the response of the head to an increase in SMC Ca2+ concentration, where a longer neck hindered the loss of Ca2+ from the head, and therefore led to a higher concentration following the stimulation (The degree of asymmetry depends on two factors, the degree of gap junctional coupling between the head and the SMC and the length of the neck region that connects the head to the bulk of the EC. As can be seen in mulation . The res3, and as in the former case, the head region quickly equilibrated with respect to changes in IP3 levels in the SMC. Because of the presence of the ER, the rise in the IP3 level in the head will lead to Ca2+ release from the ER through the IP3 receptors. Such a localized Ca2+ release may then spread into the EC as a Ca2+ wave mediated by Ca2+ induced Ca2+ release (3 from the SMC can diffuse through the gap junctions in the MEJs and induce a local Ca2+ increase in the EC We next addressed the question of how a change in head concentration of a given substance may affect the EC. One possibility could be the presence of an amplification mechanism within the head region. It is well known that the ER may extend into the MEJ, and we therefore considered the possible role of the ER in signal amplification. In this scenario we modeled the diffusion of IP release . We hypo2+ microdomain, i.e. a restricted volume of the cytoplasm where the local Ca2+ concentration may reach high levels (2+ microdomain is the restricted space between endoplasmic/sarcoplasmic reticulum and the plasma membrane 2+ microdomains where the local concentration of Ca2+ can reach values of more than 10 µM The restricted space in the MEJ is an example of a local Cah levels  and 3. A2+ microdomains have important roles in regulation of processes near membranes including regulation of ion channels 2+ microdomains in the SMCs regulate the activity of BK channels and thereby play a central role in regulation of the tone + channels is tightly regulated + channel type KCa3.1 is highly expressed in the MEJ 2+ in the range 50–900 nM 2+ concentration could activate K+ currents in the MEJ, which in turn would hyperpolarize the entire cell. Activation of the channel would in addition lead to an increase in the K+ concentration in the internal elastic lamina, which could, in principle, activate Kir channels in both EC and SMCs. It is known that an increase in interstitial K+ concentration dilates vessels in part due to Kir activation + channels in the MEJ could lead to a hyperpolarization of the SMC, in turn inhibiting voltage sensitive Ca2+ channels leading to vasodilation.Ca3Rs and IP3 phosphatases also can give rise to an asymmetrical IP3/Ca2+ signaling between ECs and SMCs 3Rs in and near MEJ in arterioles from various tissues is limited. However, our conclusions are not dependent on any functional polarization, but only depend on the actual structure of the MEJ. If degradation of IP3 by phosphatases is taken into account it will enhance the reported effect of a unidirectional flow from the SMC to the EC, because IP3 that enters the MEJ from the SMC has a relative short distance to travel before it encounters the ER in the MEJ. IP3 produced in the EC will on the other hand have to travel through the entire MEJ before entering the SMC and this would decrease the amount of IP3 entering the SMC. In the SMC IP3 could activate opening of IP3Rs in the sarcoplasmic reticulum, but as we have shown in 3 produced in the EC could activate IP3R channels in the SMC. If phosphatases in both ECs and SMCs were inhibited and the EC was stimulated for a prolonged period it could be speculated that IP3 from the EC could stimulate a Ca2+ release in the SMC.It has previously been suggested that asymmetrical distribution of IP3 and Ca2+ in the SMC are rapidly and efficiently transmitted to the EC, whereas the reverse is not the case.In conclusion, the micro-anatomical structure of the MEJ, with a long neck region restricting diffusion between the head and the bulk of the cytosol, by itself leads to a rectification of information flow between the SMC and the EC. Changes in IP


We have applied Molecular Dynamics tostudy this process with atomistic details, using Calbindin D9k (CaB) as a modelprotein. The simulations show that in most of the time the Ca2+ion spends within the Debye radius of CaB, it is being detained at the 1st and2nd solvation shells. While being detained near the protein, the diffusioncoefficient of the ion is significantly reduced. However, due to the relativelylong period of detainment, the ion can scan an appreciable surface of theprotein. The enhanced propagation of the ion on the surface has a functionalrole: significantly increasing the ability of the ion to scan the protein'ssurface before being dispersed to the bulk. The contribution of this mechanismto Ca2+ binding becomes significant at low ion concentrations,where the intervals between successive encounters with the protein are gettinglonger. The efficiency of the surface diffusion is affected by the distributionof charges on the protein's surface. Comparison of the Ca2+binding dynamics in CaB and its E60D mutant reveals that in the wild type (WT)protein the carboxylate of E60 function as a preferred landing-site for theCa2+ arriving from the bulk, followed by delivering it tothe final binding site. Replacement of the glutamate by aspartate significantlyreduced the ability to transfer Ca2+ ions from D60 to the finalbinding site, explaining the observed decrement in the affinity of the mutatedprotein to Ca2+.The encounter of a Ca The elevated Ca2+concentration modulates Ca2+ regulatory proteins at key controlpoints in essential physiological pathways, until the Ca2+ is pumpedout of the cytoplasm by a Ca2+ ATPase.Intracellular calcium plays an essential role in the transduction of most hormonal,neuronal and muscular stimuli. Cells have a multi-components calcium signalingtoolkits that can be assembled to create a wide range of spatial and temporalsignals. This versatility is exploited to control processes as diverse asfertilization, proliferation, development, learning and memory, contraction andsecretion 2+ bindingproteins have been the subject of many studies. One of the most prevalentCa2+ binding structures is a conserved motif termed the EF-hand2+ signaling pathways andacts both in modulation, buffering and protein stabilization. The heart of theEF-hand is its Ca2+-binding ‘loop’, consisting of twelveconsecutive residues, six of which provide direct or indirect Ca2+coordination. Bordering the loop are two α-helices which serve to anchor theloop and to communicate the information of Ca2+ binding to distantregions of the molecule. Usually, two EF-hand sites are associated in the sameprotein domain to yield a highly cooperative Ca2+ bindingsystem.Being of crucial importance to all living organisms, Ca2+ vs. Mg2+ andthe rate constants of the process on) values ofthe binding process vary over a wide range for different EF-hand proteins andchelators and are considerably slower than the diffusion-controlled limit. Thisobservation is in apparent contrast to the rapid rate of inner sphere watersubstitution of Ca2+, ranging between 108 and109 sec−12+ ion to the binding site. These events can not beelucidated by measurements of the association rate constants (kon), asthis value is calculated indirectly from the equilibrium constants (Keq)and the dissociation constants (koff) of the process(kon = koff * Keq)on value incorporates the overall binding process and does notgive insights on the detailed mechanism, which for Ca2+ ions, may bequite complex, as evident by the wide range of association rates found in theorganic and inorganic world.Various studies on EF-hand containing proteins have yielded different parameters suchas specificity of the EF-hand to Ca2+ to EF-hand proteinscan be derived from studies on the properties of the Ca2+ ion andits binding with chelators. The dehydration free energy of Ca2+ isas high as +362 kcal/mol 2+ is such that 7or 8 coordinating oxygen atoms can be comfortably accommodated around the ion. Thus,these coordination numbers are most frequently observed in inorganic complexes. Thekinetics of spherical metal ion binding reveals the key molecular details of theprocess by which a solvated ion is engulfed by chelators Several aspects of the binding mechanism of Ca2+ from the binding sites of Calmodulin (CaM) significantlyaltered the stability of the protein. Such conformational changes may expedite thebinding process.The modeling of the complex formation should account for the flexibility of theresidues in the binding site. High flexibility of the residues may slow the rate ofbond formation between the ion and the protein, since the residue may have a lot ofconformational space to search for the ion. On the other hand, a rigid binding sitemay also hinder the binding process as the ion has to perfectly align itself. Whatis more, it is logical to assume that the interaction of the ion with protein causesstructural rearrangements in the protein. This has been witnessed in severalexperimental and computational studies 2+ ion with the bindingsite, which is the first step in the binding process, raises interesting questionsregarding the mechanism by which the ion approaches the binding site. This event mayoccur by a simple Brownian diffusion from the bulk. Yet it is very likely that theion first encounters a negatively charged group on the surface of the protein and isdetained in the group's Coulomb cage. In the detained state the ion may shuttleto nearby negative groups, progressing from one carboxyl to another, until itreaches the correct binding site, a concept known as the antenna effect 2+ ions, having a greater charge density and a more intricatecoordination system than a proton, Na+ or Cl− ions,poses new challenges to the suggested mechanism since the solvent-ion interactionsare more complex.Beside the stepwise bond formation that seems to be the rate limiting step of thebinding process, the encounter of the Ca2+. In theseexperiments, charged surface residues of CaB, which do not serve as direct ligandsof the Ca2+, were neutralized and the overall rate constant forCa2+ binding(kon = koff * Keq) wasshown to reduce significantly in a low ionic strength environment on values ascompared to the same protein where these surface charges are neutralized. It istherefore clear that a more detailed evaluation of the reaction mechanism isneeded.Site specific mutations of Calbindin D9k (CaB) demonstrate that its surface chargesplay a significant role in the binding of Ca2+ binding toEF-hand proteins. While it is problematic to devise an experimental system suitableto monitor the dynamics of Ca2+ on a protein, one can use moleculardynamics to simulate the process and gain insights on the mechanism of the process.By using the simulations, the underlying processes which make up the overall bindingprocess can be elucidated, breaking up the experimentally measured kon toits constituent components. The ion detainment is a general feature reflecting theeffect of local fields on the diffusion of ions. In the case of S6, where theprotein does not utilize any ion for its activity, the ion detainment appears as asimple consequence of the ions interacting with the protein's electrostaticfield The present research investigates the mechanism of Ca2+ binding domain 2+ binding studies in EF-hand proteins 2+2+ ion spends withinthe Debye radius of CaB, it is detained at the 2nd and 1stsolvation shells. While being detained near the protein, the diffusion coefficientof the ion is significantly reduced. However, due to the relative long period ofdetainment, the ion can scan an appreciable surface of the protein. This surfacescanning may play a functional role, significantly increasing the surface area ofthe protein available for the Ca2+ ion, especially at lowCa2+ concentrations. This mechanism was demonstrated bycomparison of the Ca2+ binding dynamics in CaB and its E60D mutant.In the WT CaB, the side chain carboxylate of E60 was witnessed to be an intermediatestation for the Ca2+ ion arriving from the bulk, on their way to thebinding residues. As witnessed from the simulations, the E60D mutant has asignificantly reduced ability to transfer Ca2+ ions from D60 toimportant binding residues, thereby decreasing the overall binding affinity,explaining the experimental observations.The structure of the CaB protein The mechanism leading to the binding of an ion  toits specific binding site consists of three processes: (1) The encounter of theion with the perimeter of the protein  (2) Wandering of the ion in the immediate proximity of the proteinand (3) The final encounter with the specific binding site and the formation ofthe stable complex. The velocity of the first process is of a diffusioncontrolled reaction and varies with the concentrations of the reactants. Thesecond process is concentration independent and the last one may reflect somerate limiting steps like preparatory conformational changes or those that followthe complex formation and stabilize its structure.nd and 3rdaspects of the binding process, using molecular dynamics simulations. The systemconsisted of a single protein molecule, stripped from the Ca2+ion and fully relaxed, embedded in a box stretching a little more than 1.2 nmfrom the protein in the presence of sufficient Na+ andCl− ions to attain electroneutrality and maintain ionicstrength of ∼0.1 M. The system contained also two Ca2+ ions,sufficient to saturate both binding site. The observed frequency of encounterbetween the Ca2+ ions with the protein was 1–2 per ns, inaccord with the rate estimated by the Debye-Smoluchowski equation. It should bestressed that the nominal concentration of the Ca2+ was ∼25µM, which is ∼3 orders of magnitude above the physiologicconcentration of the ion. However, any attempt to adhere to the physiologicCa2+ concentration will waste most of the simulation timewaiting for primary encounters of the ion with the protein.In this study we focused our attention on the 2The encounters of the ions with the protein are sensitive not only to the totalcharge of the protein but also to its precise distribution over theprotein's surface kBT levelsat panels A, B and C, respectively. The 1 kBT potential, as a function of itsdistance from the protein (defined as the distance between theCa2+ ion and its nearest protein atom). The results,presented in 2+) as the distance fromthe protein's surface changes. Ions located within the first shell . Ions in the second solvation shellare almost as immobile as those in the first shell. The intensity of theelectrostatic potential in the second shell is in the order of 4kBT, explaining the restriction on the translationalmotion of the ions at that distance from the protein. Ions located out of the 1kBT boundary, about 10 Å from the surface ofthe protein, exhibit diffusion coefficient of 1.303×10−5cm2s−1±0.140×10−5, that is close to the one computed fora free ion, indicating that there are no restrictions on their ability topropagate in the diffusion space.To evaluate the effect of the increasing electrostatic potential on thetranslational motion of the Ca−5 cm2s−1±0.022×10−5), with that measured for Lysozyme(0.111×10−5 cm2s−1) 2+ in water is only slightlysmaller than the experimental of 1.584×10−5cm2s−1The accuracy of the calculation is supported by the comparison of the diffusioncoefficient of CaB, derived from the simulations peak thatcorresponds with the second shell is also clearly visible. The accumulation ofthe calculated curve (red) indicates that at ∼65% of the time, theion was located within the first shell of the protein, and at another 20%of the time, it was in the second shell. Close inspection of the histogramreveals hints for more remote shells, however these are hardly ordered and wehave thus defined the remaining shells consecutively with a fixed width of 2.5Å  and3rd (bottom panel) shells. The residence decay of theCa2+ near CaB, as computed from the simulations, is denotedin the black curves. As the residence time is an exponentially decaying process,an exponential fit was applied and is given in the green curve. As can be seen,in both shells the single exponential fit does not accurately describe the decaycurve. This can be explained by recalling that the protein exhibits positive andnegative electrostatic lobes, in which the residence time of theCa2+ ion is expected to be completely different. Thus, wehave performed bi-exponential fits (blue curves) and summarize the results forall shells in rd shell and up, the bi-exponential functiondescribes well the residence dynamics  that a twoexponential function does not accurately describe the residence curve.The preferential dwelling of the ions within the first two discrete shells isreflected by the Ca2+ moleculeslocated in the positive lobe of the electrostatic potential. The slower phase,which relates to long residence times, is attributed to ions located within theattractive negative lobe. The ions located in the first shell exhibit anextremely long residence, in the order of 2.5 ns, while ions in the second shelldwelled in the layer with time constant of ∼200 ps. Further on along theradius vector the dwell time is reduced to ∼15 ps. On the 1st and2nd shells, the residence process is more complex, due to thegreat inhomogeneity of the proteins surface and 2nd (dashed) solvation shells. For this calculation wecompiled the data of all CaB simulations  screening forthe events where the Ca2+ ions resided in a specific shell.The curves in the figure represent the percentage of events (within aspecific shell) in which the Ca2+ ion propagated (measuredas MSD) more than the value in the abscissa, relative to the surface of theprotein.The approach of Ca2+ ions that propagated more than 1 nm (MSD ≥1nm2) while in the 1st solvation shell(∼13%) is larger than that of the ions in the 2ndshell (∼4%). Considering that the length of a glutamate sidechain is ∼0.4 nm, a ∼1 nm displacement cannot be achieved through a“piggy back” riding over the rotameric space of the residues.There must be a net motion on the surface of the protein. In contrast, whenthe ion is detained in the 2nd solvation shell, it moves morethan 1 nm only 4% of the time (dashed line). Thus, despite therestricted diffusion coefficient of the ion in the first solvation shell,the long dwell time allows the ion to translate over the protein'ssurface.The results of this analysis clearly imply that the fraction ofCa2+ ion may endup in quite a different site than the one it first encountered.Based on these observations we conclude that ions located in the firstsolvation shell are mobile enough to skim over the surface of the proteinand search for alternative sites. Thus the Ca2+ ions, as taken from the crystallographic structureby Szebenyi and Moffat 2+ ions on both sites (either directly or via a watermolecule). During the molecular dynamics simulations, the protein was‘stripped’ from the Ca2+ ions and allowed torelax into a solution structure of the Apo state, both for the WT and theE60D mutation. This observation is in accord with our previous report thatthe Amber 94 force field indeed retains the structure of CaB intact, both inthe Apo and Holo states 2+ ions without the need to evaluate the changesexperienced by the protein.2+ ion propagates on the surfaceof the protein was investigated by examining the encounters of theCa2+ ions with the individual residues on the surface ofthe CaB protein. The formal concentration of Ca2+ in thesimulations was 25 mM, leading to an average frequency of one encounterevery 1.3 ns. These encounters, where the ion came to contact with a residueon the surface after diffusion in the bulk, are defined as primaryencounters. We define secondary encounters as events where theCa2+ ion came to contact with a residue due to transferfrom another surface residue, without leaving the first solvation shell. Thecompilations of all these events are given in The mechanism by which a Ca2+ ions were free in solution for 44% of thetime and, when bound to the protein, interacted simultaneously with(average) ∼1.6 nearby residues. Inspection of 2+ ions by the same extent. Fewresidues experienced many encounters and accumulated extended contact time while others, of equal charge, hardlyencountered with the ion . Inspection of the table reveals thatthe high frequency of encounters, and a long binding time is not limited tothe binding sites (marked by bold letters). It should be mentioned that A14,which in the crystalline state of the protein is a ligand to theCa2+ in site I, did not make a single contact with theCa2+ during the whole simulation.2+ ion within few tens of ps.Comparison between the abundance of primary encounters to the secondary onesreveals that the ratio between the total secondary encounters to the totalprimary encounters per residue is ∼0.3, meaning that of all contactsmade by the ion with the protein about one quarter are mediated through aprevious encounter of the ion a with some other site on the protein ratherthan coming directly from the bulk. The average dwell time of the ions onthe binding residues were widely spread; some residues retained contact for few ns, while other residues (like S62) release theCaExamination of the accumulative time of contact of the residues associatedwith binding sites I and II reveals that from all residues making bindingsite I, only E17, and to a lesser extant Q22, were sufficiently attractiveto retain the ion for 14% and 7% of the total simulation time,respectively. The residues of binding site II appeared to be more reactiveboth in accumulated contact time (up to ∼29% for D58 and∼40% for E60/D60) and in the number of primary and secondaryencounters. These findings are correlated with experimental indications thatsite II is occupied first during the binding process 2+ with the oxygen atoms of backbone carbonyls whilethe latter reports encounters with the more polarized side chain oxygenatoms. The overall ratio between secondary and primary encounters for allthe protein oxygens is 0.38, which is somewhat higher than 0.3 ratioobserved at the per-residue resolution. This implies that intra-residueCa2+ transfer occur quite frequently. The overall ratiobetween secondary and primary hits for carboxylates and carbonyls is 0.33and 0.55, respectively.A further breakdown to the specific binding atoms is given in 2+ ions withcalbindin, derived by the Debye-Smoluchowski equation is∼3×1010 M−1s−1and the apparent rate constant at 25 mM Ca2+ will be∼0.8×109 s−1 or encounters ataverage intervals of 1.3 ns. The value derived from the number of primaryhits that occurred during the 2 µs trajectories is quite comparable,∼1.3 ns. Thus, the encounter of the ion with the surface of the proteinis compatible with that of a diffusion controlled reaction and depends onthe concentration of the Ca2+ ions in the solution. Once aprimary encounter occurs, the following secondary encounters are independentof the free Ca2+ concentration and occur at averageintervals of ∼4 ns. Under physiologic conditions, were the freeCa2+ concentration is few µM, the time intervalbetween primary encounters will be stretched to the µs time frame.However, many of the Ca2+ ions that encounter theprotein's surface will be transferred to a nearby site (secondaryencounter) within the ns time frame. Thus, as at physiologicCa2+ concentration, the primary encounters will serve asefficient donors to the nearby sites, rendering the protein's surfaceas a collecting antenna, and increasing the surface of the protein which isscanned by the ions.The approximated rate of encounter between the Ca2+ ion to thespecific binding sites(kon = 2×107M−1s−1) 2+ to the specific sites. Still these simulations aresufficient to reveal how a single mutation can alter the reaction pathway.The measured rate constant for the binding of the Ca2+ shuttling mechanism can beevaluated in the CaB itself by comparing the WT simulations with the simulationsof the E60D mutant. This conservative mutation has a highly similar structure tothe WT CaB, yet at physiologic ionic strength its affinity toCa2+ is reduced by ∼8 fold 2+ , we wished to investigate the mechanism leading to thelower affinity of site II.The functional role of the Ca2+ ion with its surroundingligands are detailed in 2+ is bound in thecrystal structure to the carbonyl of E60, a single carboxylate oxygen of D54, asingle carboxylate oxygen of D58, the amide oxygen of N56 and the carboxylate ofE65. In the NMR structures, only D54 and E65, with one or two carboxylate oxygencoordination appear in all structures. D58 carboxylate and E60 carbonyl appearin the majority of the structures and N56 is missing altogether, replaced by thecarbonyl of G58 or E59.The interactions between the Ca2 order parameter of amide carbonyl and carboxyl groupsin the side chains of aspartic acid, asparagine, glutamic acid, and glutamine ofCa2+-loaded calbindin D9k P43G 2order parameter is model independent, and measured in solution, some of thebiases described above are eliminated. The results of Paquin et. al. show thatthe Ca2+ binding residues  are associated with high S2 values in the rangeof 0.68<S2<0.94. It can also be witnessed that three of thebinding carboxylates, E27, D54 and E65, show exceptionally high S2values of 0.836±0.041, 0.938±0.047 and 0.913±0.056,respectively, indicating an extremely low motional liberation. These values aresignificantly higher than those of the other binding residues, ASN56 and D58,which exhibit values of 0.685±0.037 and 0.681±0.075, respectively.For E27, there was a consensus between the crystal and NMR structures that itbinds Ca2+ in a bi-dentate manner, using both of its carboxylateoxygens. The high S2 values of D54 and E65 suggests that D54 and E65both bind the site II Ca2+ ion using both their carboxylateoxygens. This will form tight binding with very little motional liberation ofboth residues.In order to resolve these apparent discrepancies, we have to consider that bothcrystallographic and NMR structure determination involve inherent biases. Incrystallography, the crystal structure is created in non-physiologicalconditions. The NMR structures reflect some sort of average, and not necessarilya structure that existed in the solution. Both methods carry a bias caused bythe FF used to refine them. In a recent study by Paquin et. al., NMR was used tomeasure the S2 values of the ASN56 and D58 residues indicate that they bindthe Ca2+ less tightly. For the ASN56 this is probably due to theweaker electrostatic attraction of the amide carbonyl oxygen. For the D58 thisis probably due to being bound by only one of the carboxylate oxygens . Toconclude, the experimental evidence suggests that the key residues involved in astable Ca2+ binding in site II are D54 and E65.This conclusion also conforms well to the fact that these two residues appear asbinding residues in all crystallographic and NMR structures. The lowerS2+attractor in CaB. This is mostly due to its side chain carboxylate . The E65, on the other hand, shows arelatively high binding time (∼16%). It is interesting that in themutant both these residues show substantial decrease in the overall bindingtime. As demonstrated in In contrast to the increased affinity shown by D58 and D60, the two key residuesfor Ca2+ mediated interactions between specific pairs ofresidues: D54 and D58, D54 and D/E60, E65 and D58, E65 and D/E60. It can be seenthat there is a significant reduction in Ca2+ transfers betweenthese residues, which can quantitatively explain the reduction in binding timeobserved for D54 and E65. However, when looking at the Ca2+transfers between the carboxylates of these residues, it can be seen that theCa2+ transfer between the carboxylates of residues D60 andE65 was only mildly reduced. This discrepancy can be explained by the majorreduction observed between the E65 carboxylate and the D60 carbonyl. It is alsoworth noting that a 2-fold increase in Ca2+ transfer iswitnessed between D60 and D58 carboxylates (data not shown).2+ arrives from the bulk mainly to the carboxylate sidechain of E60. E60 can than transfer the Ca2+ ions to the keybinding residues D54 and E65. Shortening the length of its side chainsignificantly reduces the efficiency of these Ca2+ transferreactions and alternatively increases the Ca2+ transfer of D60with D58. Effectively, in the E60D mutant, both D54 and E65 become“starved” for Ca2+ and the overall binding affinityin site II diminishes significantly.All the above data leads to a mechanism by which in the WT CaB,Ca2+ binding is due to a reduction ofCa2+ transfers between its carboxylate and the carboxylatesof D60, D58 and E65 (the latter is a consequence of its own decreasedaffinity).For D54, the reduction in Ca2+ transfers observedbetween these three species (the carboxylates of D60 and D58 and the carbonyl ofD60), reducing their availability to transfer Ca2+ to E65.For E65, the reduction is due to a reduction in both inter-carboxylateinteractions with the carboxylates of D60 and D58, and also from a reduction inits interaction with the backbone carbonyl of D60. The latter case can beexplained by the increased level of Ca2+ ion with the protein starts when theion encounters the Coulomb cage of the protein. Out of the Coulomb cage the ionis unaware of the protein, having a diffusion coefficient of a free ion. Once itis within the cage, the motion is restricted by the local electrostatic fieldsand its diffusion coefficient decreases as it gets closer to the protein.Although the mobility of the ion is hindered by the decreased diffusioncoefficient, as the ion gets closer to the protein's surface, its residencetime increases, enabling it to skim over longer distances in the 1stshell than in the 2nd one. Thus, the sites first encountered by theCa2+ ion need not necessarily be the final binding site. TheCa2+ can propagate on the surface by hopping from residue toresidue.The interaction of the Ca2+ bindingmostly occurs by hopping from a nearby residue. For carboxylates, the encounteroccurs mainly from the bulk, with 33% from nearby residues. In lowCa2+ concentration, when encounters are rare, the secondaryencounters may significantly increase the protein surface available to theCa2+ ion, thus making the region around the binding site acollecting antenna.For carbonyls, which are weaker binding species, the CaOn average, the ion on the surface is interacting with more than single residue,keeping it bound for a long time, reducing the probability of getting lost tothe bulk.2+ shuttling mechanism was observed in simulations ofCalbindin D9k where the main Ca2+ attractor is the side chaincarboxylate of E60, which is not a part of the binding site, according toexperimental structures. Thus, this site only serves as a midway station forCa2+ arriving from the bulk on their way to the properbinding site. In the calbindin E60D mutant, the ability of D60 to transfer theCa2+ ion to key binding residues in site II (D54 and E65) issignificantly reduced, which we suggest as a possible cause for the reducedaffinity observed in experiments.The CaThe number of simulations that were carried out, their lengths and compositions aregiven in 2+ ion with carboxylate wemodulated the Lennard–Jones parameters as described in −1. The TIP3P 2+ depleted CaB (Apo-CaB), the two Ca2+ ionswere removed from their binding sites and added at random locations. The water boxextended to at least 1.2 nm from the molecule, for CaB simulations, and at least 2.0nm from the Ca2+ ion for the free Ca2+ simulations.D, E and LYS residues were charged to reflect their state at physiological pH.Electroneutrality and physiological ionic strength of ∼0.150 M for the CaBsimulations and 0.1 M for the free Ca2+ simulations were reached byadding appropriate amounts of Na+ and Cl− ions tothe simulation boxes  The force field selected for the simulation was Amber94 2+ at specific shells from the protein, the MSD statisticsused for calculation of the diffusion constant was such that it contained at least 8different simulations which contributed to the MSD average and the MSD graph wasnear linear. The diffusion coefficient errors stated in the manuscript are estimatesbased on multiple calculations using different sets of simulations.The diffusion coefficients were calculated from the mean square displacement (MSD)curves between specific time points selected to give the most reliable results. ForCa2+ ion was found at the desired distance from theprotein up to a given time span marked by the he abscissa. In these calculations anevent where the Ca2+ ion changed its distance from theprotein's surface for a period of 2 ps or less was not considered astermination of its residence period. All Apo-CaB simulations were used to generatethese curves. For the exponential fitting procedure, only sections of the graph witha count higher than 10 were considered.Residence times were calculated by fitting single and double exponential functions tothe residence time decay curves. The residence time decay curves denote the numberof times a CaElectrostatic potential calculation were performed using the APBS software 1.2.1Table S12+ ion to carbonyl oxygen atoms, calculated from ten100 ns WT and ten 100 ns E60D simulations.Total binding times, primary hit count and secondary hit count ofCa(0.06 MB DOC)Click here for additional data file.Table S22+ ion to side chain oxygens of charged and polarresidues (carboxylate oxygens treated as one), calculated from ten 100 ns WTand ten 100 ns E60D simulations.Total binding times, primary hit count and secondary hit count ofCa(0.07 MB DOC)Click here for additional data file.


The extracellular calcium-sensing receptor (CaSR) belongs to family C of the G protein coupled receptors. Whether the CaSR is expressed in the pulmonary artery (PA) is unknown.2+]i) was detected by a laser-scanning confocal microscope.The expression and distribution of CaSR were detected by RT-PCR, Western blotting and immunofluorescence. PA tension was detected by the pulmonary arterial ring technique, and the intracellular calcium concentration  or Gd3+ (an agonist of CaSR) induced an increase of [Ca2+]i and PAs constriction in a concentration-dependent manner. In addition, the above-mentioned effects of Ca2+ and Gd3+ were inhibited by U73122 (specific inhibitor of PLC), 2-APB (specific antagonist of IP3 receptor), and thapsigargin .The expressions of CaSR mRNA and protein were found in both rat pulmonary artery smooth muscle cells (PASMCs) and PAs. Increased levels of [Ca2+]i through G-PLC-IP3 pathway.CaSR is expressed in rat PASMCs, and is involved in regulation of PA tension by increasing [Ca Moreover, we also found that 10 mM Ca2+ increased the FI of [Ca2+]i to 1.297 ± 0.150 at 30 s, 1.357 ± 0.176 at 60 s, 1.402 ± 0.183 at 90 s, and 1.419 ± 0.176 at 120 s in the absence of NiCl2, CdCl2 and NPS2390. The FI of [Ca2+]i in both the NiCl2 + CdCl2 + CaCl2 group and the NPS2390 + CaCl2 group was decreased but higher than that in controls (p < 0.01 versus control), and the FI of [Ca2+]i was decreased significantly in the NiCl2 + CdCl2 + NPS2390 + CaCl2 group (p < 0.01 versus CaCl2 group)  Figure , n = 20.2+ is from extracellular Ca2+ entry and release of intracellular Ca2+ store. To verify that the change in [Ca2+]i induced by activation of CaSR is dependent on the intracellular Ca2+ store, the PASMCs were incubated with 10 mM caffeine and 10 μM thapsigargin for 30 min, then 10 mM CaCl2 or 300 μM GdCl3 were added into the media. It was found that Ca2+ FI/FI0 was significantly reduced in the presence of caffeine and thapsigargin (p < 0.01 versus CaCl2 or GdCl3 group)  Figure , n = 20.2+ group, FI/FI0 of [Ca2+]i was decreased in the 2-APB and U73122 pretreated groups. However, U73343 had little effect on [Ca2+]i FI/FI0  was regarded as 100% (vehicle). We observed that an increase in the o causes an elevation of [Ca2+]i mediated by CaSR, Fluo-3/AM was used to measure [Ca2+]i. The EC50 for Ca2+ activation of CaSR is 3-4 mM [2+]o from 1.8 to 2.5 mM had no effect on [Ca2+]i, and a [Ca2+]o from 5 to 12.5 mM induced an elevation of [Ca2+]i in a concentration-dependent manner. This means that in PASMCs, the increase of [Ca2+]o can cause an elevation of [Ca2+]i. Additionally, in the presence of NiCl2 and CdCl2, the FI of [Ca2+]i has decreased, it is still higher than control group. Furthermore, NPS2390 also decreased the FI of [Ca2+]i. However, the elevation of [Ca2+]i induced by 10 mM CaCl2 was nearly abolished in the NiCl2 +CdCl2+NPS2390 group. These results indicated that CaSRs were involved in the elevation of [Ca2+]i induced by an increased [Ca2+]o, or that CaSRs at least played a partial role in this process.To confirm that [Cas 3-4 mM . In the 2+]i induced by elevated [Ca2+]o or [Gd3+]o in PASMCs. It is well known that caffeine is a depletion agent of the ryanodine receptor operated at the Ca2+ store and that thapsigargin is a blocker of sarcoplasmic reticulum calcium ATPase. This suggests that increased [Ca2+]i induced by CaSR activation is from thapsigargin and caffeine sensitive intracellular Ca2+ stores.In the present study, we found that the pretreatment with caffeine and thapsigargin for 30 min prevented a significant increase of [Caet al reported that elevated [Ca2+]o, Gd3+ or spermine can cause Ca2+ release from the sarcoplasmic reticulum of rat myocardium via the G protein-PLC-IP3 signal transduction pathway [2+]i in PASMCs. The results showed that, compared with the 10 mM Ca2+ group, the FI/FI0 of [Ca2+]i was markedly decreased in the 2-APB and U73122 pretreated groups. However, preincubation with U73343 did not alter 10 mM [Ca2+]o -induced elevation of [Ca2+]i. Pretreatment with 300 μM Gd3+ induced responses similar to those observed in Ca2+-treated cultures. These results suggested that activation of CaSR induced the increase in [Ca2+]i in PASMCs through the PLC-IP3 signal transduction pathway.Wang 2+, as an excitation contraction coupling factor, is involved in regulating myocardial contraction and angiotasis. To demonstrate the functional expression of CaSR in PAs, evidence showing that CaSR activation is related to PA tension change needs to be provided. Therefore, we observed the effects of the CaSR agonist, antagonist and other calcium signal-related factors on PAs tension. The results showed that vasoconstriction appeared in a concentration-dependent manner in PAs when [Ca2+]o was increased from 5 mM to 12.5 mM, and Gd3+ also induced a similar response. In addition, the vasoconstriction was not reversed by an inhibitor of the Na+-Ca2+ exchanger and L-type Ca2+ channels, antagonist of CaSR. These findings suggest that an increased [Ca2+]o or [Gd3+]o evoked vasoconstriction at least in part by the CaSR. In subcutaneous artery a biphasic response was observed. That is increasing [Ca2+]o from 0.5 to 2 mM induced a small vasoconstriction followed by progressive vasodilation from 3 to 10 mM [2+]o caused a biphasic vasoconstriction in the spiral modiolar artery o or [Gd3+]o, suggesting that [Ca2+]o or [Gd3+]o induced constriction of PAs related to the Ca2+ release from thapsigargin and caffeine sensitive intracellular stores.The signal transduction mechanism linked to the CaSR is known to involve the release of Cac stores . Therefo2+]o or [Gd3+]o-induced PA vasoconstriction were significantly inhibited by U73122 and 2-APB, but not U73343. Thus, the increases in PAs tension induced by Ca2+ and Gd3+ are linked to the PLC-IP3 signaling pathway.In the experiment with pulmonary artery rings, we also found that the increases in [Ca2+]i increase and vasoconstriction through the G-PLC-IP3 signal transduction pathway. Pulmonary artery constriction contributes to pulmonary hypertension, so it is expected that CaSR activation could be involved in the development of pulmonary hypertension..We have demonstrated that functional expression of CaSRs exists in rat PAs and PAMSCs, and that CaSR activation is involved in [CaThe authors declare that they have no competing interests.All authors participated in the design, interpretation of the studies and analysis of the data and review of the manuscript. B-FY, L-YW, RW and C-QX conducted the experiments. C-QX supplied critical reagents. G-WL, Q-SW wrote the manuscript. G-DY and W-hZ finished necessary language corrections to this manuscript. G-WL, Q-sW, J-HH and W-JX are equally contributed.


Eukaryotic NCX contains Ca2+-binding regulatory domains, CBD1 and CBD2. Ca2+ binding to a primary sensor (Ca3-Ca4 sites) on CBD1 activates mammalian NCXs, whereas CALX, a Drosophila NCX ortholog, displays an inhibitory response to regulatory Ca2+. To further elucidate the underlying regulatory mechanisms, we determined the 2.7 Å crystal structure of mammalian CBD12-E454K, a two-domain construct that retains wild-type properties. In conjunction with stopped-flow kinetics and SAXS  analyses of CBD12 mutants, we show that Ca2+ binding to Ca3-Ca4 sites tethers the domains via a network of interdomain salt-bridges. This Ca2+-driven interdomain switch controls slow dissociation of “occluded” Ca2+ from the primary sensor and thus dictates Ca2+ sensing dynamics. In the Ca2+-bound conformation, the interdomain angle of CBD12 is very similar in NCX and CALX, meaning that the interdomain distances cannot account for regulatory diversity in NCX and CALX. Since the two-domain interface is nearly identical among eukaryotic NCXs, including CALX, we suggest that the Ca2+-driven interdomain switch described here represents a general mechanism for initial conduction of regulatory signals in NCX variants.Na Buried in the molecular center is a four-ion-binding site cluster with one site for Ca2+ and three for Na+. Two apparent passageways allow separate access for Na+ and Ca2+ ions +/Ca2+ exchange 2+ regulatory element encoded by a large cytosolic domain (∼500 amino acids) between transmembrane helices 5 and 6 i.e. the repeated membrane motif, designated as f-loop 2+-binding regulatory domains (CBD1 and CBD2), connected by a short linker 2+ sites (Ca1-Ca4) on CBD1 2+ sites (CaI-CaII) on CBD2 The mammalian Na2+2+ interaction with CBD1 activates the brain (NCX1.4) and cardiac (NCX1.1) variants, whereas Ca2+ has no sustained effect on the kidney variant (NCX1.3) 2+ interaction with CBD1 of CALX1.1 (a Drosophila exchanger) inhibits the exchanger activity, whereas CALX1.2 is insensitive to regulatory Ca2+2+-dependent regulation either in NCX 2+ sensor (Ca3-Ca4 sites), either in isolated CBD12 2+ from Ca3-Ca4 sites of CBD12 2 state) of intact NCX observed in electrophysiological experiments Eukaryotic NCX variants differ in their response to regulatory Ca2+ binding to isolated CBD12 2+ binding results in more compact conformation of CBD12 with decreased Dmax, suggesting that Ca2+ interaction with CBD1 changes the electrostatic potential to drive the conformational transition 2+ binding to CBD1 and thus, suggest a more extended conformation of CBD12 2+ binding to CBD1 results in rigidification of CBD12, by restricting the interdomain linker’s flexibility rather than inducing global changes in their relative reorientation Distinct techniques have been used to elucidate the structural effects of Ca2+-bound state, these structures display small differences in interdomain angles, which were suggested as the structural basis for their differential regulatory responses. According to this rationale, the interdomain angles of CBD12 might significantly differ in CALX1.1 and NCX1.4, since they exhibit diverse regulatory response to Ca2+. In light of these results, two critical mechanistic questions emerge. First, how precisely does Ca2+ binding/occlusion at the primary sensor (Ca3-Ca4 sites) couple conformational transitions in CBDs, and second, how can this coupling account for the regulatory diversity of NCX and CALX.Recently, high-resolution X-ray structures of CBD12 from CALX1.1 and CALX1.2 were obtained 2+ binding to the primary sensor. To test this working hypothesis, we crystallized and determined the crystal structure of a two-domain CBD12 tandem from the brain splice variant (NCX1.4). In conjunction with stopped-flow kinetics and SAXS analyses of relevant mutants, we demonstrate that the two-domain interface controls Ca2+-induced tethering of CBDs and slow dissociation of occluded Ca2+. In Ca2+-bound conformations, the interdomain angle of CBD12 is very similar for NCX1.4 and CALX1.1, meaning that the interdomain angle and/or distance between the two domains cannot account for the regulatory diversity in NCX and CALX. Therefore, Ca2+-binding to a high-affinity sensor (Ca3-Ca4 sites) induces a disorder-to-order transition, producing a more rigid conformation of CBD12. This interdomain switch appears to be a common module for regulation even for NCX variants exhibiting opposite responses to regulatory Ca2+.Here, we posit that the two-domain interface initiates conduction of regulatory signals upon Ca(i) The E454K substitution replaces the Ca2+ ion at Ca1 site of CBD1, thus, stabilizing the protein’s structure through a charge compensation mechanism (ii) The full-length NCX1-E454K mutant exhibits WT [Ca2+]-dependent regulation in a cellular system (iii) The Ca3-Ca4 sites retain their affinity for Ca2+ binding in an isolated CBD1-E454K In order to crystallize the tandem, we employed a previously characterized mutant, E454K The overall structure of CBD12-E454K , Table 1Similar to previously published crystal structures of isolated CBD domains, the FG loops of both domains (residues 469–481 and 600–618) cannot be fully visualized due to presumed high flexibility of these areas 2. This small interface surface area is in agreement with our previous observations that isolated CBDs do not interact in solution and that a short linker is obligatory for interdomain interactions 2+ binding loops with the interdomain linker, the CBD2 flexible FG loop and the strictly conserved CBD2 BC loop  and E451 (hydrogen-bonded), replacing Ca1 of CBD1-WT. The overall coordination is similar to that reported for isolated CBD1 2+ from the Ca3-Ca4 sites are observed, with slow dissociation of one Ca2+ ion (ks = 0.5 s−1). This sequential dissociation of Ca2+ is not observed either in isolated CBD1, mixture of isolated CBD1 and CBD2 or in CBD12-7A , and thus, serves as a functional hallmark of domains coupling 2+ is observed, although with ∼ 10 fold increase in kf (fast phase) representing the dissociation of the first Ca2+ ion from Ca3-Ca4 sites of CBD12  of CBD12-WT  and maintains only the Ca2+-bound Dmax of CBD12-WT according to our SAXS analysis . Consistent with this interpretation, Ca2+ binding to Ca3-Ca4 sites seems to be obligatory for robust interdomain interactions, since D499 and D500 are disordered in the apo-form 2+ binding to CBD1 restricts the flexibility of the CBD1-CBD2 linker to rigidify interdomain movements without global changes in CBDs reorientation 2+-induced tethering of CBDs, described here, represents a new mode for ligand-induced rigidification of CBDs. Most probably, both mechanisms contribute in a cooperative way to the decoding of the regulatory message.The most important structural finding is that the two domains are tethered in a Caand CBD2 . The bur2+ binding  and two Ca2+ coordinating residues (D499 and D500) at Ca3-Ca4 sites of CBD1. The R532 mutant of CBD12 lacks the slow Ca2+ dissociation  and no interaction of R532 with CBD1. Therefore, the R532 mutation results in “intrinsic uncoupling” of interdomain interactions, exhibiting a phenotype of non-interacting CBDs. Thus, CBD decoupling may impart decreased affinity for [Ca2+]-dependent activation in a NCX1-R532C mutant due to the low affinity binding of Ca2+ to CBD2 Our findings clearly indicate that the interface participates in slow dissociation of occluded Caociation  and Ca2+ by SAXS . We spec2+ sites. Cadherin studies demonstrated that Ca2+ rigidifies the protein 2+ fluctuations may physiologically regulate cadherin activity 2+-dependent rigidification. In addition, the tandem architecture is reminiscent of the arrestin family, where tandem β sandwiches are apposed by a polar core of buried charged residues. Disruption of this polar core activates arrestin for high-affinity binding to its GPCR target Interface conservation and composition points to a general mechanism for the NCX family. Importantly, the architecture of this interface differs from the tandem C2 domains of synaptotagmin and PKC +/Ca2+ exchanger from Drosophila in which the binding of Ca2+ to CBDs inactivates the exchanger (1.1 variant) or has no effect (1.2 variant) 2+ cannot be attributed solely to the orientation of CBDs in the CBD12 tandem. It is also possible that the conformational dynamic responses to Ca2+ binding differ in CBD12 of NCX and CALX, despite the similar orientation of CBDs present in these crystal structures. We hypothesize that additional structural elements in the regulatory f-loop and/or membrane domain are probably involved in specifying their differing regulatory effects. Identification of these structural elements is a crucial task for further defining the mechanism underlying Ca2+ regulation.Recently, the crystal structures of CBD12 tandems from the two CALX splice variants (1.1 and 1.2) were determined 2+-driven communication between CBDs. Most importantly, occupation of Ca3-Ca4 sites by Ca2+ induces a disorder-to-order transition (40) due to charge neutralization and coordination, thereby constraining CBD conformational freedom, rigidifying the NCX1 f-loop, and triggering regulatory signal transmission to the membrane domain. Undoubtedly, additional structural motifs are involved in transmitting the regulatory signal in NCX variants.In summary, we solved the crystal structure of a two-domain tandem mutant from NCX1’s intracellular regulatory loop and found that the CBDs communicate via a complex network of electrostatic interactions at their interface. We found a correlation between the dynamic properties of CBD12 and structural data revealing CaE. coli Rosetta2 (DE3) competent cells (Novagen), as described The DNA constructs of CBD12 (encoding residues 371–657) of canine NCX1  were cloned into pET23b vector and expressed in 2, 10 mM β-mercaptoethanol and 10 mM Tris-HCl at pH 7.2. Initial crystallization screens were performed at 19°C with Macrosol MD1-22 screen  using the sitting drop vapor diffusion method. Initial crystals were obtained with 12% PEG-8K and 200 mM NH4SO4. Crystallization was optimized by adding 10% sucrose and lowering the PEG-8K concentration to 7%. Crystals were slowly dehydrated by raising the PEG-8K concentration in the reservoir solution by 2%/day. When the PEG-8K concentration reached 30%–32%, crystals were flash-frozen in cryo-loops. Data were collected from the flash-frozen crystals cryocooled to 110° K at beamline ID29 of the European Synchrotron Radiation Facility .Purified CBD12-E454K at a concentration of ∼30 mg/mL was prepared in buffer composed of 100 mM KCl, 10 mM CaClhttp://www.rcsb.org/).Data were processed using HKL2000 2+ dissociation from proteins was monitored with Quin-2 as described free = 5–10 µM) was mixed with 150 µl of 200 µM Quin-2 in TK-buffer.The stopped-flow assays were performed with a three-syringe/two mixer SFM-3 instrument  as described 2+. A FoXS web-server was used for computation and fitting of SAXS profiles SAXS data were measured at beamlines 12-ID-B and 12-ID-C of the Advanced Photon Source, Argonne National Laboratory, USA and at ID14-3 of the European Synchrotron Radiation Facility . The following samples were used for the experiments: the wild-type protein CBD12 and its mutants at three concentrations  in buffers containing 20 mM Tris-HCl, pH 7.5, 100 mM KCl, 20 mM β-mercaptoethanol with10 mM EDTA or 10 mM CaThe Ensembl server Figure S1Multiple Sequence Alignment of CBD12 orthologs. Numbering according to canine NCX1 cardiac splice variant.(PDF)Click here for additional data file.Figure S22+ binding sites.Crystal packing involves CBD2 Ca (A) Interaction between symmetry mates in the CBD12-E454K crystal. (B) Salt-bridges are formed between symmetry mates, involving residues that participate in coordination of Ca2+ in isolated CBD2 .(EPS)Click here for additional data file.Figure S3SAXS analysis. Comparison of SAXS scattering curves (left column) and normalized pair distance distribution functions (PDDF) (right column) for the wild type CBD12 , CBD12-7Ala  CBD12-R532A  and CBD12-E454K , respectively, in the absence (black) or presence of Ca2+ (red). The scattering profiles were normalized and superimposed against the first data points.(EPS)Click here for additional data file.Table S1CBD12 Interface Conservation.(DOCX)Click here for additional data file.Table S2Overall SAXS parameters for CBD12 and its mutants.Rg, Dmax, and NSD are, respectively, the radius of gyration derived from Guinier plotting, maximum inter-atomic dimension, normalized shape discrepancy for DAMMIN calculation. Note that NSD scores for all bead models are less than 0.8, indicating high convergence of the bead model calculations.(DOCX)Click here for additional data file.Information S1Experimental procedures.(DOCX)Click here for additional data file.


Isolated adult rat cardiomyocytes were maintained in normal  or high glucose  media for 6-12 hours. Contractile indices were measured including peak shortening (PS), maximal velocity of shortening/relengthening (±dL/dt), time-to-PS (TPS), and time-to-90% relengthening (TR90). (3) Cardiomyocytes maintained with HG displayed abnormal mechanical function including reduced PS, ±dL/dt, and prolonged TPS, TR90 and intracellular Ca2+ clearance. Expression of intracellular Ca2+ regulatory proteins including SERCA2a, phospholamban and Na+-Ca2+ exchanger were unaffected whereas SERCA activity was inhibited by HG. Interestingly, the HG-induced mechanical anomalies were abolished by elevated extracellular Ca2+ (from 1.0 to 2.7 mM). Interestingly, the high extracellular Ca2+-induced beneficial effect against HG was abolished by the CaM kinase inhibitor KN93. (4) These data suggest that elevated extracellular Ca2+ protects against glucose toxicity-induced cardiomyocyte contractile defects through a mechanism associated with CaM kinase.(1) Hyperglycemia leads to cytotoxicity in the heart. Although several theories are postulated for glucose toxicity-induced cardiomyocyte dysfunction, the precise mechanism still remains unclear. (2) This study was designed to evaluate the impact of elevated extracellular Ca The heart was perfused for 20 min with KHB containing 176 U/mL collagenase II  and 0.5 mg/mL hyaluronidase. After perfusion, left ventricles were removed and minced. The cells were further digested with 0.02 mg/mL trypsin before being filtered through a nylon mesh (300 μm). Extracellular Ca2+ was added incrementally back to 1.25 mM. Isolated cardiomyocytes were cultured on glass coverslips precoated with laminin (10 μg/mL) and maintained for 6 or 12 hours in a defined medium consisting of Medium 199 with Earle's salts containing 25 mM HEPES and NaHCO3 supplemented with albumin (2 mg/mL), L-carnitine (2 mM), creatine (5 mM), taurine (5 mM), insulin (0.1 μM), penicillin (100 U/mL), streptomycin (100 μg/mL), and gentamicin (100 μg/mL). This medium also contained either normal glucose (NG: 5.5 mM) or high glucose (HG: 25.5 mM) concentrations. The high glucose concentration is comparable to serum glucose levels in severely diabetic rats -N-(4-methoxybenzenesulfonyl)amino-N-(4-chloro-cinnamyl)-N-methylbenzylamine   associated with unchanged intracellular Ca2+ decay. Similar to the mechanical response, elevated extracellular Ca2+ levels effectively attenuated or nullified glucose toxicity-induced derangement of intracellular Ca2+ handling  (The above-mentioned data were collected under an extracellular Ca in FFI) .2+ and high glucose-induced changes in intracellular Ca2+ handling, expression of intracellular Ca2+ regulatory protein was evaluated. Our results revealed that neither high extracellular Ca2+ nor high glucose, or both, produced any notable changes in the levels of SERCA2a, Na+-Ca2+ exchanger and phospholamban. However, measurement of SERCA activity revealed that high extracellular glucose significantly dampened the thapsigargin-sensitive 45Ca2+ uptake under low extracellular Ca2+ environment, the effect of which was nullified by elevated extracellular Ca2+. Last but not least, increase in extracellular Ca2+ concentration itself did not affect expression or activity of intracellular Ca2+ regulatory proteins . Our data revealed that KN93 effectively abolished high extracellular Ca2+-offered protection against high glucose-induced cardiomyocyte contractile anomalies (decrease in PS and ±dL/dt as well as prolonged TPS and TR90). In addition, KN93 also abolished high extracellular Ca2+-induced cardiomyocyte contractile responses  under low extracellular Ca2+ environment , the effect of which may be abolished by inhibition of CaM kinase using KN93. Several mechanisms may be speculated for elevated extracellular Ca2+-induced protection against glucose toxicity. Elevated extracellular Ca2+ levels are capable of facilitating cytosolic Ca2+ cycling and dynamics thus promoting Ca2+ influx and resequestration, regardless of the extracellular glucose environment. This is supported by the increased rise of intracellular Ca2+ in response to electrical stimuli, facilitated intracellular Ca2+ decay, improved SERCA activity , and shortened TR90 under high extracellular Ca2+ environment. Our observation of depressed cardiomyocyte contractile capacity and maximal velocity of contraction/relaxation along with prolonged duration of contraction and relaxation in high glucose-treated cardiomyocytes is supported by compromised intracellular Ca2+ handling under high glucose environment, consistent with the findings from in vivo diabetic condition [2+ ions promote cell survival through a CaM kinase-dependent activation of Akt [2+ improves cardiac performance is through increased phosphorylation state. It may be speculated that Ca2+ ions promote contractile inotropism first by bolstering cardiac energy state, which occurs rapidly within minutes. The CaM kinase cascade is essential to many physiological processes, the defect of which can lead to a variety of disease states. Calmodulin acts as a primary receptor for Ca2+ in all eukaryotic cells whereas Ca2+/CaM kinase functions as an allosteric activator of a host of enzymatic proteins [2+-initiated signaling cascade should hold considerable promise for the future of disease-related research [Hyperglycemia has been shown to be one of the most devastating predisposing factors for the onset and development of diabetic cardiomyopathy –4, 24. Rdiabetes , 24, 25.e medium , 24, 25.e medium . Intrigue medium , 28, 29.ondition . Anothern of Akt , in lineproteins . The areresearch .2+ homeostasis against glucose toxicity. Considering the unique role of CaM kinase in regulation of cardiac energy metabolism and survival, understanding the mechanism of action of CaM kinase in myocardial contractile regulation under pathological conditions such as hyperglycemia should be essential in the elucidation of CaM kinase in diabetic complications.In summary, the findings from the present study revealed improved cardiac contractile and intracellular Ca


An exogenous astrocytic Gq G protein-coupled receptor was resistant to scaling, suggesting that the alterations in astrocyte Ca2+ signaling result from changes in activity of the surface mGluRs rather than a change in intracellular G protein signaling molecules. These findings suggest that astrocytes actively detect shifts in neuronal firing rates and adjust their receptor signaling accordingly. This type of long-term plasticity in astrocytes resembles neuronal homeostatic plasticity and might be important to ensure an optimal or expected level of input from neurons.Very little is known about the ability of astrocytic receptors to exhibit plasticity as a result of changes in neuronal activity. Here we provide evidence for bidirectional scaling of astrocytic group I metabotropic glutamate receptor signaling in acute mouse hippocampal slices following long-term changes in neuronal firing rates. Plasticity of astrocytic mGluRs was measured by recording spontaneous and evoked Ca Therefore, recording astrocyte Ca2+ activity provides a very useful tool to measure the timing of astrocytic Gq GPCR activation.A number of studies in vitro suggest that Ca2+ activity can also be used to estimate changes in Gq GPCR expression levels. A number of specific changes in spontaneous and evoked Ca2+ elevations occur following manipulation of Gq GPCR expression levels in cultured cells. These include an increase in the percentage of cells in the population exhibiting spontaneous Ca2+ elevations 2+ response latency to agonist in situ studied the relationship between agonist concentration and astrocytic Ca2+ response characteristics. With increasing agonist concentration, astrocytic Gq GPCR Ca2+ responses change from single-peak, to multi-peak, to long-lasting plateau patterns in situ. Here, we set out to determine if long-term changes in CA3 neuron firing rates lead to long-term plasticity of spontaneous and evoked astrocytic Gq GPCR Ca2+ elevations in CA1 stratum radiatum (s.r.) astrocytes in acute hippocampal slices.Previous studies 2+ elevations in the astrocyte soma and fine processes, here we provide evidence indicating bidirectional, rapid scaling of group I metabotropic glutamate receptor (mGluR) Ca2+ signaling in s.r. astrocytes of the hippocampus in situ following long-term increase or decrease in neuronal firing rates. Calcium responses evoked by stimulating a foreign astrocytic Gq GPCR (the MrgA1R) were unaltered, suggesting that changes in Ca2+ activity are due to changes in surface mGluR activity rather than components of the intracellular Gq GPCR signaling pathway. Taken together, our findings suggest that astrocytes scale their mGluR signaling activity bi-directionally following long-term changes in neuronal action potentials.Based on a comprehensive analysis of evoked and spontaneous astrocyte CaExperiments in this study were specifically approved by the Institutional Animal Care and Use Committee guidelines of the University of California, Riverside, to ensure minimal animal suffering.2+-free saline containing (in mM): 125 NaCl, 2.5 KCl, 3.8 MgCl2, 1.25 NaH2PO4, 26.0 NaHCO3, 25 glucose, and 1.3 ascorbic acid, bubbled with 5% CO2-95% O2, pH 7.4. Subsequently, slices were incubated for 45 minutes at 35°C in standard ACSF containing (in mM): 125 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 MgCl2, 1.25 NaH2PO4, 26.0 NaHCO3, 25 glucose, and 0.1 Trolox, bubbled with 5% CO2-95% O2, pH 7.4, 315 mOsm. Slices were then incubated 4–6 hours at room temperature in either standard ACSF or modified ACSF to manipulate neuronal action potentials.Acute slices were chosen for this study because the majority of the neuronal network and neuron-to-astrocyte communication remains intact in acute slices. It has been estimated that more than 40% of the synapses are still associated with astrocyte processes after recovery from dissection +]o ACSF +1 µM of the Na+ channel blocker tetrodotoxin (TTX). 3.5 mM [K+]o ACSF (317 mOsm) was used as standard ACSF (+]o ACSF was used (320 mOsm), while 2.5 mM [K+]o ACSF was chosen as the control condition. In 2.5 mM [K+]o, neurons fired APs very infrequently compared to 3.5 mM [K+]o and 5.0 mM [K+]o -driven synaptic transmission, slices were incubated 4–6 hours in 3.5 mM [Kard ACSF . To incrmM [K+]o .2, 10 HEPES, 4 Mg-ATP, 14 phosphocreatine, and 0.2 Na-GTP, pH 7.3 with KOH, 291 mOsm. Slices were incubated 4–6 hr. in either 2.5, 3.5, or 5.0 mM [K+]o ACSF prior to recording in room temperature ACSF of the same composition. Whole-cell patch-clamp was performed using a Multiclamp 700B amplifier and PCLAMP 10.2.014 software . CA3 neurons on average displayed a resting Vm of −61.7±1.6 mV in 2.5 mM [K+]o ACSF (n = 12), −60.4±1.6 mV in 3.5 mM [K+]o ACSF (n = 8) and −50.51±1.68 mV in 5.0 mM [K+]o ACSF (n = 15). Upon attaining the whole-cell configuration, the cell membrane potential, input resistance, and access resistance were recorded. Change in any of these parameters by 20% or more was used as criteria to reject the data from further analysis. Frequency of spontaneous APs in each recording was analyzed using PCLAMP 10.2.014 software.Neuronal recordings were performed as described previously . Pipettes were pulled from borosilicate glass on a Sutter P-97 Flaming/Brown type horizontal micropipette puller and not fire-polished. Pipettes had resistances of 4.6–6 MΩ when filled with a solution containing the following (in mM): 145 K-gluconate, 2 MgCl2+ indicator was performed as described previously 2+ indicator dye using a backpressure, bolus-loading technique Bulk-loading of astrocytes with Ca2+ indicator via whole-cell voltage clamp was performed as described previously 2, 10 HEPES, 10 glucose, 1.185 Mg-ATP, 10.55 phosphocreatine, and 0.1315 mg/ml creatine phosphokinase, pH 7.3 by KOH, and had resistances of 7–8 MΩ. Also included in the pipette solution was 200 µM of the cell-impermeant version of OGB-1 (Invitrogen). The osmolality of this solution was 300 mOsm. Upon attaining the whole-cell configuration, the resting membrane potential of the astrocyte was recorded in current clamp mode. Astrocytes displayed a resting Vm of −77.8±1.2 mV in 2.5 mM [K+]o ACSF, −73.5±0.7 mV in 3.5 mM [K+]o ACSF and −69.7±1.5 mV in 5.0 mM [K+]o ACSF, with average input resistances of <10 MΩ. Then astrocytes were voltage-clamped at a holding potential of −90 mV from which a voltage-step protocol was initiated to verify the passive electrical properties of the astrocyte. A test pulse of −5 mV was included after each voltage step in order to monitor changes in access resistance. Astrocyte current signals were low-pass filtered at 2 kHz and digitized at 100 kHz via a Digidata 1440 . Astrocytes were voltage-clamped for no longer than 3 min., at which time the patch pipette was gently retracted from the cell. A smooth, stable off-cell and formation of an outside-out patch was a strong indicator of minimal damage to the cell membrane during the patch clamp procedure. Then the astrocytes were allowed to rest for 10 min. prior to initiation of image acquisition. In some experiments, astrocytes were patch clamped a second time with 200 µM Alexa Fluor 568 hydrazide dye (Invitrogen) to determine the amount of the astrocyte filled by OGB-1.Filling of astrocytes with cell-impermeant Ca2+ activity. The green Ca2+ indicators  were excited using a 488 nm argon laser, and emission was collected using a 503–548 bandpass filter. Single plane, 512×512 pixel x-y-t scans of 2.19 µm optical section thickness were used to record astrocyte Ca2+ activity. The pixel size was 12-bit, 0.276 µm/pixel. The scan interval was 1.2 s and data were collected over the course of 40 min. Individual astrocytes in CA1 stratum radiatum were identified first by their location, size, and morphology. In bulk-loaded slices astrocytes were further identified by their sequestering of Ca2+ indicator and characteristic long-duration Ca2+ responses compared to neurons 2+ responses. Recordings were completed within 40 minutes to limit any possible TTX-induced scaling effects on control slices. All imaging was performed using 0.5% laser power to limit the possibility of photosensitivity induced spontaneous Ca2+ oscillations 2+ elevation during 10 to 15 minutes baseline recording were counted as spontaneously active cells.An Olympus Fluoview 1000 confocal microscope, software, and LUMPlanFl 60×/1.10NA water immersion objective  were used to record astrocytic Ca2+ responses in astrocytes. Agonist was applied not for a specific duration, but until a clear response was evident. The reason for this was twofold: First, often different agonist concentrations were used. Lower agonist concentrations require longer wash-in times to reach sufficient tissue concentration to bind a sufficient number of receptors to evoke a response. Second, the experimental manipulations (TTX or elevated [K+]o) clearly affected response latency to a fixed concentration of agonist, presumably because the functional expression of the receptors had changed. All recordings were performed in 1 µM TTX to eliminate neuronally-driven astrocyte Ca2+ elevations that may result from agonist activation of neuronal group I mGluRs 2+ returned to baseline in-between DHPG applications to limit receptor desensitization of group I mGluRs. A Gq GPCR agonist cocktail consisting of 10 µM each of histamine, carbachol, and ATP to stimulate astrocytic histamine H1, muscarinic acetylcholine, and purinergic receptors, respectively, was applied for two main purposes: 1) as a positive control for viable astrocytes that did not respond to DHPG; and 2) to determine the specificity of changes to group I mGluRs vs. other astrocytic Gq GPCRs. Cells which failed to respond to agonist cocktail were excluded from subsequent analysis.Drugs were bath-applied using an electronic valve controller . The group I mGluR agonist (RS)-3,5-DHPG  or the group I and II agonist trans-ACPD (50 µM) was applied to evaluate evoked group I mGluR Ca0) within each ROI represented increases in Ca2+ concentration 0 values were scored as Ca2+ elevations if the peak amplitude was greater than two standard deviations above the mean of baseline fluorescence for at least two sample points. For patch clamp experiments, within each astrocyte, microdomains were identified as compartments of astrocyte processes that exhibited spontaneous Ca2+ elevations independently from other parts of the cell. It is well-documented that the smallest astrocyte processes are too small to be resolved by confocal or 2-photon microscopy 2+ activity was clearly resolved in ROIs over “bulk” fine processes that appeared as a cloud-like fuzz at the astrocyte periphery. Microdomains occurring at the same time were scored as separate events if intervening ROIs did not exhibit Ca2+ elevations. Within each microdomain, the ROI that corresponded to the “initiation site” of the spontaneous or evoked Ca2+ transient was identified based on the earliest onset among all ROIs in the same microdomain, and was later used to represent this microdomain for further analysis. Spontaneous Ca2+ transients covering ≥75% of the planar area of the astrocyte were defined as “whole cell” Ca2+ elevations and analyzed separately.Slices or single astrocytes from bolus-loading and patch clamp experiments were given a numeric code and analyzed blindly. Square-shaped regions of interest (ROIs) sized to match the cell soma (for bulk-loading and bolus-loading experiments) or fixed at 3.5 µm/side  were placed over the astrocyte soma and/or processes using Olympus Fluoview 1000 software. Increases in average fluorescence intensity normalized to baseline fluorescence . Because the frequency of spontaneous Ca2+ transients observed in astrocyte processes may be affected by the area of each astrocyte that can be imaged, frequency was normalized to the astrocyte area prior to comparison between groups. Rise time was defined as the difference between the response onset and the time corresponding to the peak amplitude (0 to 100%). For agonist-evoked responses, latency was calculated as the time between initiation of agonist perfusion to the peak of the response. Analysis of rise time was chosen in addition to response latency due to long wash-in time of agonists in hippocampal slices vs. cultured cells, which might obscure potential significant differences in response latency.The amplitude, frequency, and kinetics of spontaneous Gq GPCR signaling events and agonist-evoked mGluR responses were determined for each microdomain. The average baseline fluorescence intensity was calculated by averaging 30 seconds of stable baseline immediately preceding each Ca2+ responses, specific response patterns were grouped into 3 categories: “single peak”, “multipeak”, or “plateau”. Single peak responses were defined as a response with only one data point within ±10% of the peak value. Plateau responses maintained the peak amplitude for at least 3.6 seconds, and usually for much longer. Therefore, the duration of single peak responses was much shorter compared to plateau Ca2+ responses. Multi-peak responses were defined as a minimum of at least 2 single peaks in rapid succession, with ≤10.8 s gap between each single peak. In addition, multipeak responses were defined only when the fluorescence intensity fell below 50% amplitude after the first peak prior to rising again for secondary peaks. If the second peak occurred before the first peak decayed by at least 50%, this event was defined as a plateau response. Cells or microdomains that did not produce Ca2+ elevations to group I mGluR agonist were grouped as non-responders (NR) as long as these cells were deemed viable based on responses to agonist cocktail. Analysis of agonist-evoked response duration and area was performed only for plateau responses.For agonist-evoked Cat-test was used for statistical comparison of means between the control and treated groups. Pearson’s chi-square test was used for comparison of the Ca2+ activity patterns between control and treatment groups. Fisher’s exact 2-tail test was used for comparison of percentages of specific Ca2+ activity patterns between control and treatment groups. Significance is described at the *0.05, **0.01, or ***0.001 levels.Analyses were performed using number of astrocytes as ‘n’ for bulk-loading and bolus loading experiments, and number of microdomains for patch-clamp experiments. Error bars represent S.E.M. in all figures. Student’s independent + concentrations ([K+]o) on AP frequency of CA3 pyramidal neurons , we measured the effect of different extracellular K neurons . CA3 neu n = 12;  and onlyd n = 8;  and fired n = 8; . This did n = 8; . However n = 15;  and a si n = 15; . Based oin vitro have provided evidence indicating how changes in Gq GPCR expression levels correlate to specific changes in spontaneous and agonist-evoked Ca2+ elevations 2+ activity was recorded in the soma of stratum radiatum (s.r.) astrocytes in acute mouse hippocampal slices bulk-loaded with the Ca2+ indicator Calcium Green-1 AM +]o ACSF or 3.5 mM [K+]o ACSF plus 1 µM TTX   . There w. 68.2%)  or duratp<0.001; . These r2+ response, with lower concentrations leading to single-peak or oscillating (“multipeak”) responses while higher concentrations produce long-lasting plateau responses 2+ signaling of astrocytic group I mGluRs increases after long-term blockade of neuron-to-astrocyte receptor signaling.Previous studies have also revealed a dose-dependent effect of group I mGluR agonist concentration on the pattern of the Ca2+ indicator. Astrocytes taking up the Ca2+ indicator tend to be very near the slice surface, where astrocytes and their synaptic associations may be compromised during slice preparation. To address this concern we loaded s.r. astrocytes with OGB-1 AM Ca2+ indicator dye using a more recently developed backpressure, bolus-loading protocol 2+ indicator 40–75 µm into the slice, we obtained improved resolution and signal-to-noise in deeper, healthier tissue compared to conventional bulk-loading protocols. Overlay of the SR-101 and OGB-1 AM signals was used to identify bolus-loaded cells as astrocytes , and TTX incubated slices  . Rise time of the spontaneous Ca2+ transients for TTX-treated astrocytes was significantly faster compared to the control group (The percentage of spontaneously active astrocytes bolus-loaded with Ca slices) . The incol group , p<0.01.2+ elevations remained consistent between the two groups . We first tested astrocytes bolus-loaded with Ca2+ indicator for their spontaneous somatic Gq GPCR activity and responsivity to DHPG  compared to control (59.1% ±12.4%) (2+ elevations was slower in astrocytes incubated in 5.0 mM [K+]o compared to controls (2+ activity (data not shown), consistent with findings after long-term blockade of neuronal APs in TTX.If the observed changes in astrocytic Gq GPCR activity occur as a direct consequence of a reduction in neuronal APs, then the effect should be reversible when neuronal AP frequency is significantly elevated. To test this, we compared spontaneous and evoked astrocyte Ca to DHPG . In 5.0  ±12.4%) , p<0.05.controls . There w+]o incubated slices. At 30 and 50 µM DHPG, the rise time of evoked Ca2+ elevations in astrocytes incubated in 5.0 mM [K+]o was significantly slower compared to control slices . There was no difference in rise time of Ca2+ responses to agonist cocktail , suggesting that the effect is primarily due to changes in group I mGluRs. As the concentration of DHPG increased, the percentage of responding astrocytes increased, as expected . At all DHPG concentrations tested, 5.0 mM [K+]o treated astrocytes were less likely to respond, and the pattern of responses shifted toward the weaker, single peak phenotype compared to astrocytes incubated in 2.5 mM [K+]o ACSF. There was no difference in the amplitude or latency of DHPG- or agonist cocktail-evoked somatic Ca2+ responses in 5.0 mM [K+]o treated vs. control astrocytes (data not shown).Three successive concentrations of DHPG  were applied to compare evoked group I mGluR responses between control and 5.0 mM [K+]o treatment occurs in microdomains, OGB-1 Ca2+ indicator dye was delivered into astrocytes via patch clamp. All astrocytes in both conditions exhibited spontaneous Ca2+ transients at least once in microdomain(s). In control ACSF, 16.7% of astrocytes had spontaneous somatic Ca2+ transients, as opposed to only 9.1% of astrocytes in 5.0 mM [K+]o ACSF . However, as expected, the DHPG-evoked rise time was significantly slower following incubation in 5 mM [K+]o ACSF compared to the control group , suggesting that group I mGluRs are the main astrocytic receptors affected by changes in basal neuronal firing rates . Almost all microdomains that exhibited spontaneous Ca2+ transients also responded to agonist cocktail in control conditions. However, only a certain percentage of microdomains responded to 50 µM DHPG after 5.0 mM [K+]o incubation  failed to respond to 50 µM DHPG . This is a very surprising finding that also suggests reduced group I mGluR evoked Ca2+ signaling after long-term increase in neuronal APs. Among microdomains that responded after incubation in 5 mM [K+]o ACSF, there was a significant shift toward the weaker single-peak phenotype compared to control cells  hippocampal astrocytes are driven almost exclusively by Gq GPCR-mediated release from internal stores 2+ transients. Data were replicated using three different, complementary approaches to load astrocytes with Ca2+ indicator. After 4–6 hour blockade of action potentials in acute hippocampal slices, we observed the following changes in astrocyte Ca2+ signaling: 1) an increased percentage of astrocytes exhibiting spontaneous Ca2+ elevations; 2) a faster rise time of spontaneous Ca2+ transients; 3) increased response probability to the group I mGluR agonist DHPG; and, 4) a faster rise time (and shorter response latency in microdomains) of evoked mGluR Ca2+ responses. These changes were observed in both astrocytic somata and microdomains. Furthermore, 4–6 hr of increased CA3 neuron firing rates in 5 mM [K+]o ACSF led to a decrease in the percentage of spontaneously active astrocytes and a significantly slower rise time of both spontaneous Ca2+ transients and DHPG-evoked Ca2+ responses. In agreement with previous reports of all-or-none astrocytic Gq GPCR responses upon achieving a threshold agonist concentration 2+ elevations.It is well-documented that neuronally-released transmitter can stimulate astrocytic Gq GPCRs 2+ signaling match well with previous studies which have directly assessed the effects of manipulating Gq GPCR expression levels on evoked Ca2+ activity. For example, Wang and Thompson +]o. Shao and McCarthy 2+ responses, and found that higher α1 Gq GPCR expression levels correlated directly to an increased percentage of astrocytes in the population responding to agonist. Furthermore, phenylephrine-evoked Ca2+ responses had a shorter response latency as expression levels increased. Similar effects were observed in the present study for astrocytic DHPG-evoked responses after 4–6 hr incubation of hippocampal slices in TTX.The observed changes in Ca2+ signaling may be driven by changes in intracellular signaling molecules or the surface receptors. Belair and colleagues 2+ responses in perisynaptic Schwann cells (PSCs) of the frog neuromuscular junction after chronic blockade of postsynaptic nAchRs. Although these researchers did not attempt to differentiate among multiple potential mechanisms of modifications in evoked PSC Ca2+ responses, they did observe changes in muscarinic PSC Ca2+ response amplitudes, suggestive of a greater amount of Ca2+ released from internal stores in these cells 2+ events, an indication that coupling to intracellular mechanisms of Ca2+ release is not altered by long-term manipulation of neuronal firing rates in our study. To explore this issue more deeply, we performed experiments using hippocampal slices from mice in which astrocytes express a foreign Gq GPCR (the MrgA1R) vs. coupling to intracellular mechanisms of Ca2+ release. In MrgA1R+ slices incubated in TTX, agonist-evoked MrgA1R Ca2+ elevations were no different from those in MrgA1R+ slices incubated in control ACSF. However, evoked group I mGluR responses in the same MrgA1R-expressing astrocytes were significantly faster. As previous evidence suggests that MrgA1 receptors and endogenous group I mGluRs engage the same Gq GPCR signaling molecules 2+ signaling mainly reflect changes in the surface group I mGluRs.Because metabotropic receptors are coupled to intracellular signaling cascades, alterations in Ca2+ responses is that the pattern is graded from single peak, to oscillatory (“multipeak”), to plateau as the agonist concentration is increased 2+ responses shifted significantly from single peak toward plateau response patterns. This effect was also bi-directional: After increasing CA3 neuronal firing rates in 5 mM [K+]o ACSF, the pattern of astrocytic Ca2+ responses to DHPG shifted toward single peak responses. The robust effect on response pattern further suggests that the signaling activity of astrocytic group I mGluRs scales up or down following long-term changes in neuronal firing rates. GPCR/Ca2+ response patterns may represent distinct “codes” dictating a unique cellular response, conferring a single GPCR subtype with the ability to control various cellular outcomes A characteristic of astrocytic Gq GPCR evoked Ca2+ activity remains mysterious both in terms of its underlying mechanisms as well as its physiological purpose. While it is clear that these events require IP3R-mediated Ca2+ release from internal stores 2+ activity in situ appear to exhibit constitutive Gq GPCR activity. Nett et al. 2+ oscillations occur independent of neuronal activity. Furthermore, previous studies in vitro established that the amount of constitutive activity is proportional to GPCR expression levels vs. ligand-mediated receptor activation. Because astrocytes express a wide variety of Gq GPCR subtypes 2+ activity 2+ transients following manipulation of neuronal firing rates.Spontaneous astrocytic CaPlasticity of astrocytic mGluRs has been demonstrated previously in neurological disorders. For example, mGluRs have been shown to change in reactive astrocytes in models of epilepsy in vitro and in situ. Although this is a non-physiological approach, there is evidence to support homeostatic ‘scaling up’ of neuronal receptors following sensory deprivation. For example, Desai et al. in vitro and in situ appears to be an appropriate model of compensatory changes based on the effects of sensory deprivation in vivo.TTX has been commonly used to induce neuronal homeostatic scaling + and glutamate to control neuronal excitability 2+ signaling patterns may code for changes in gene transcription The physiological significance of the observed homeostatic-like plasticity of astrocytic mGluRs remains to be determined. An overarching function of astrocytes is homeostasis of the synaptic microenvironment to provide optimal conditions for synaptic signaling. Neuron-to-astrocyte receptor communication may be important for regulating cerebral blood flow