Here we examine whether postganglionic sympathetic neurons are able to mobilize Ca2+ from intracellular stores in response to depolarization, independent of Ca2+ influx. We measured changes in cytosolic ΔF/F0 in individual fluo-4 –loaded sympathetic ganglion neurons in response to maintained K+ depolarization in the presence (2 mM) and absence of extracellular Ca2+ ([Ca2+]e). Progressive elevations in extracellular [K+]e caused increasing membrane depolarizations that were of similar magnitude in 0 and 2 mM [Ca2+]e. Peak amplitude of ΔF/F0 transients in 2 mM [Ca2+]e increased in a linear fashion as the membrane become more depolarized. Peak elevations of ΔF/F0 in 0 mM [Ca2+]e were ~5–10% of those evoked at the same membrane potential in 2 mM [Ca2+]e and exhibited an inverse U-shaped dependence on voltage. Both the rise and decay of ΔF/F0 transients in 0 mM [Ca2+]e were slower than those of ΔF/F0 transients evoked in 2 mM [Ca2+]e. Rises in ΔF/F0 evoked by high [K+]e in the absence of extracellular Ca2+ were blocked by thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ ATPase, or the inositol 1,4,5-triphosphate (IP3) receptor antagonists 2-aminoethoxydiphenyl borate and xestospongin C, but not by extracellular Cd2+, the dihydropyridine antagonist nifedipine, or by ryanodine at concentrations that caused depletion of ryanodine-sensitive Ca2+ stores. These results support the notion that postganglionic sympathetic neurons possess the ability to release Ca2+ from IP3-sensitive internal stores in response to membrane depolarization, independent of Ca2+ influx.Recent studies have provided evidence that depolarization in the absence of extracellular Ca The ganglia were then incubated for 20 min at 37°C in a Earle’s Balanced Salt solution  containing 10 U/ml papain , followed by a 30-min incubation in Earle’s Balanced Salt solution supplemented with 1.3 mg/ml type II collagenase (Worthington) and 2.2 mg/ml dispase II . Cells were then dissociated by trituration with a fire-polished glass Pasteur pipette in trituration solution  and plated on poly-D-lysine–coated No.1 circular glass cover slips. The isolated neurons were maintained in 5% CO2-95% O2 at 37°C in trituration solution and used ~24 hours after plating. All procedures were approved by the Indiana University School of Medicine Institutional Animal Care and Use Committee.Sympathetic neurons were prepared in a manner previously described . DBA/J m2+, signal intensities of pixels located inside the neuron soma (excluding the nuclei) were measured, spatially averaged, and background corrected [F(t)]. Background fluorescence was measured as the average of a 40 x 40 pixel cell-free area outside the neuron soma of interest in each frame of every time series. Baseline fluorescence intensity (F0) was determined by averaging F over the 10-s interval preceding the cell’s exposure to elevated [K+] in the extracellular medium ([K+]e), and the time course of normalized fractional dye fluorescence [ΔF/F0(t)] was obtained, where ΔF equals F(t)- F0. Signal correction for bleaching was not necessary.A coverslip containing sympathetic neurons was transferred to a 263-μl recording chamber . Cells were loaded at room temperature with fluo-4 by incubation with the acetoxymethyl (AM) ester form of the dye  at a final concentration of 2 μM in normal Tyrode’s solution. After 20 min, cells were washed several times with dye-free Tyrode’s solution and transferred to an inverted microscope (Axioscope) equipped with a Zeiss x63 1.4 numerical aperture water immersion lens. The microscope was attached to a confocal laser-scanning unit (Zeiss LSM 510). Fluo-4 fluorescence was probed every second by illumination with 488-nm laser light and emission was detected between 500 and 550 nm. The diameter of the pinhole was set to its maximum for all measurements. Images were taken in frame-mode at a pixel density of 512 x 512. Fluorescence signals were digitized at 8-bit resolution and analyzed using Metamorph software . To quantitate amplitude and time course of changes in cytosolic fluo-4 fluorescence, i.e., Ca+] solution  in the presence of 2 mM Ca2+ for 30 s, followed by a 1-min exposure to normal Tyrode’s solution. A second 30-s exposure to the same [K+]e in the absence of extracellular Ca2+ (with 200 μM EGTA added to the extracellular solution) was applied, and then the high [K+]e solution was replaced with Ca2+-free normal Tyrode’s solution. The first and second applications of high [K+]e were separated by 3-min exposures to Ca2+-free normal Tyrode’s solution. Bath solution exchanges were performed via manual injections (~500 μl/s) through the input port of the perfusion chamber. For each consecutive bath fluid exchange in the experimental protocol, the injected volume was 7 ml, corresponding to ~27 times the chamber volume. Solution changes were rapid, based on the fast and steady change in membrane potential achieved when cells were exposed to external solutions with elevated [K+] (see 2+-free Tyrode’s solution contained the following (in mM): 140 NaCl, 5 KCl, 1 MgCl2, 0.2 EGTA, 10 HEPES, and 10 D-glucose (pH 7.4). Assuming a 50 μM total contaminating Ca2+, the free [Ca2+]e was estimated to be ~ 43 nM e, Na+ was adjusted to maintain osmolarity [+]e-elicited Ca2+ response in Ca2+-free bath solution gradually decreased with consecutive [K+]e tests. Therefore, the response to one tandem only of high [K+]e exposure (in 2 and 0 mM [Ca2+]e) was tested in an individual neuron.Cells were exposed to a high [K[K+] see . Nominal ~ 43 nM . For Ca2molarity . Where iP/6 method. Currents and voltages were low-pass filtered at 1 kHz and 5 kHz, respectively, using the built-in four-pole Bessel filter and sampled at 5 and 25 kHz, using a Digidata 1440A, acquired using Clampex10 and analyzed with Clampfit10 . Data collection was started ~2 to 3 min following membrane breakthrough.Whole-cell voltage- and current-clamp recordings were performed at room temperature using a Multiclamp 700B patch-clamp amplifier . Patch pipettes were pulled in a model P-97 puller  from borosilicate glass capillaries and heat-polished prior to use with a Narishige MF-83 microforge . When filled with internal solution, the pipette resistance ranged from 2 to 5 MΩ. During the recording, changes in the bath solution were made by gravity driven perfusion. Pipette capacitance was zeroed on sealing. Whole-cell capacitive transients were compensated by 60–80%. Residual linear capacitive and leak currents were subtracted by the–2+ currents contained (in mM) 120 Cs-Aspartate, 1 MgCl2, 4 MgATP, 0.3 Na2GTP, 10 EGTA and 10 HEPES (pH 7.2), whereas the bath solution was composed of (in mM) 140 TEA-Cl, 2 MgCl2, 2 CaCl2, 10 glucose and 10 HEPES (pH 7.4). For measuring ion currents through voltage-gate Ca2+ channels in the absence of extracellular Ca2+, the bath solution contained (in mM) 140 NaCl, 2 MgCl2, 0.2 EGTA, 10 glucose and 10 HEPES (pH 7.4). For measuring action potentials, the pipette solution contained (in mM) 145 K-Aspartate, 2 MgCl2, 5 HEPES, 5 Na2ATP and 1.1 EGTA (pH 7.2), and the bath solution consisted of (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES and 10 glucose (pH 7.4). For Ca2+-free bath solutions, calcium was omitted and EGTA (0.2 mM) was added. When recording the effects of elevating [K+]e on membrane potential, [Na+] was lowered to maintain osmolarity. Osmolarities of all bath and pipette solutions were adjusted to 310 and 315 mOsm, respectively, using mannitol. Membrane potentials were adjusted for liquid junction potentials unless stated otherwise.Pipette solution for measuring whole-cell CaICa and to determine the ICa. activation curve, currents were evoked by 300-ms pulses ranging from -90 to +60 mV in steps of 5 mV. The interval between voltage steps was 10 s. The peak ICa.L density at each potential was plotted as a function of test voltage to generate the I-V curves. Activation curves were fitted with the following Boltzmann distribution equation: G/Gmax = 1/{1+exp[V1/2 –V)/k]}, where G is the voltage-dependent calcium conductance, Gmax is the maximal calcium conductance, V1/2 is the potential at which activation is half-maximal, V is the membrane potential, and k is the slope. G values were determined by the following equation: G = Imax/(V-ECa), where ECa is the reversal potential.To characterize voltage dependence of peak I/Imax = 1/{1 + exp[(V–V1/2)/k]} + C, where Imax is the peak current elicited after the most hyperpolarized prepulse, V is the preconditioning pulse potential, and C is a constant.To examine steady-state inactivation, the voltage that gave maximal peak current was used for subsequent protocols. Cells were administered a series of prepulses (-90 to 40 mV) lasting 300 ms, from a holding potential of -90 mV, followed, after a 20-ms gap at -90 mV, by a 300-ms depolarization to a voltage eliciting the maximal peak current (0 mV). The interval between conditioning prepulses was 10 s. The resulting curves were normalized and fitted using the following Boltzmann distribution equation: CM = τ/ΔV * I0/(1 –I∞/I0), where CM is membrane capacitance, τ is the time constant of the capacitance current relaxation, I0 is the peak capacitive current determined by single exponential fit and extrapolation to the first sample point after the voltage step ΔV, and I∞ is the amplitude of the steady-state current during the voltage step  has been previously shown by others to produce stable and reproducible membrane depolarization in sympathetic ganglion neurons in culture, increasing intracellular free [Ca2+] in the presence of external Ca2+ [+]e depolarization with fluorescent Ca2+ imaging to examine the possibility that membrane depolarization evokes Ca2+ transients within isolated sympathetic ganglion neurons of adult mice not only in the presence of extracellular Ca2+ but also in its absence. Representative Ca2+ responses of two fluo-4/AM-loaded neurons elicited by consecutive 30-s exposures to 80 mM [K+]e in the presence of 2 mM [Ca2+]e and in Ca2+-free bath solution (with 200 μM EGTA added) are illustrated in +]e in normal Ca2+ gave rise to increases in global fluorescence intensity, which were larger in the nuclei than in the cytosols  tracing is shown in 2+]e to 2 mM, the peak amplitude of the high [K+]e-evoked Ca2+ transient clearly exceeded that of the preceding transient recorded in the absence of external Ca2+, and its time course was indistinguishable from that of a response typically seen during initial exposures to high [K+]e in 2 mM [Ca2+]e. Identical observations were made in 7 cells. These results prove that the neurons remain viable and support the notion that the differences in Ca2+ transient properties between 2 and 0 mM [Ca2+]e are unlikely to result from unspecific effects of the exposure to the Ca2+ free bath solution. At 80 mM [K+]e, the average peak Ca2+ transient amplitude was markedly smaller in the absence than in the presence of external Ca2+ and their average rise and decay times were markedly prolonged compared to Ca2+ transients evoked in normal [Ca2+]e  over a wide range of voltages including those generated by the lowest and highest [K+]e used in the Ca2+ imaging experiments. Short depolarizations evoked Ca2+ currents exhibiting no, or very little, inactivation  increased in a sigmoidal fashion as the membrane potential became more depolarized. This behavior contrasts with the inverse U-shaped dependence of peak ΔF/F0 on voltage in Ca2+-free bath solution , i. e., steady-state Pact = (peak Pact) * (1 –Pinact). Accordingly, we next determined the voltage-dependence of ICa inactivation. Channel inactivation displayed a U-shaped dependence on voltage  on high [K+]e-evoked Ca2+ responses in isolated sympathetic neurons. Neurons were pre-incubated with thapsigargin for 20 min while being loaded with fluo-4/AM and then subjected to Ca2+ imaging in the continued presence of the inhibitor. Representative plots of ΔF/F0 as a function of time are shown in +]e in the presence of extracellular Ca2+ caused an increase in cytosolic [Ca2+]. A second 30-s exposure to 80 mM [K+] in the absence of external Ca2+ did not evoke a significant increase in intracellular [Ca2+]. Overall, only 2 (10.5%) of 19 thapsigargin-treated neurons developed Ca2+ transients during exposure to elevated [K+]e in the absence of extracellular Ca2+ compared to 47 (78%) of 60 non-treated cells  among the two experimental groups  spared the high [K+]e-induced Ca2+ responses both in the presence and absence of extracellular Ca2+ (+]-induced Ca2+ elevations in 0 mM [Ca2+]e between the ryanodine [14 (100%) of 14 cells] and the control group . Exposures to 80 mM [K+]e and caffeine both reversibly elevated cytosolic [Ca2+]. Pretreatment with 20 μM ryanodine abrogated the caffeine response, but did not affect the high [K+]e-induced response, which is consistent with previous studies  or xestospongin C (10 μM) abolished the [K+]e-evoked Ca2+ transient in the absence, but not in the presence, of extracellular Ca2+ (+]e in 0 mM [Ca2+]e induced Ca2+ transients in only 1 (17%) of 6 2-APB–treated and 2 (10.5%) of 16 xestospongin C- treated neurons, compared to 47 (78%) of 60 control neurons . Further, we found no significant differences in resting membrane potential or potentials obtained during the 80-mM [K+]e challenges either in normal [Ca2+]e or Ca2+-free bath solutions (P > 0.05) between control neurons and neurons following IP3 receptor blockade  gating have previously been identified as the mechanism linking membrane depolarization to Ca2+ release from IP3-sensitive stores in skeletal myotubes in the absence of extracellular Ca2+  and the control group e-evoked Ca2+ release in the absence of extracellular Ca2+ does not require L-type Ca2+ channels.Voltage-dependent changes in L-type Calar Ca2+ . If an implitude . The finchannels . Alternate block . We founlar Ca2+ . The lac2+]i increases in the absence of external Ca2+ are triggered by influx of Na+ and/or K+ through open calcium channels under our experimental conditions. Accordingly, we next examined the effect of pharmacologically inhibiting ion flux through high voltage-gated Ca2+ channels on [K+]e-Ca2+ transients in 0 mM [Ca2+]e. Extracellular Cd2+ has been shown previously to potently block ion flux through neuronal, high voltage-gated calcium channels in a concentration-dependent manner [2+ currents in voltage-clamped neurons (using Ba2+ as the charge carrier) confirmed the absence of resolvable inward currents following the addition of 300 μM Cd2+ to the bath solution  ethylendiamine  [2+ (Kd = 10−4.4 M) [+]e-induced changes in peak ΔF/F0 in the presence of 2 mM [Ca2+]e were not significantly different before and after loading with TPEN   is sever0−4.4 M) . The meaith TPEN , suggestal [K+]e . Identic2+ channels conduct Na+ currents in the absence of extracellular Ca2+, we sequentially measured macroscopic currents carried by these channels in 2 and 0 mM [Ca2+]e. Extracellular concentrations of Mg2+ (2 mM) and Na+ (140 mM) remained unchanged. Tetrodotoxin (1 μM) was present in the bath solution throughout the measurements to block voltage-gated Na+ channels. Exemplar whole-cell current traces acquired in response to step depolarizations to +10 mV before and after Ca2+ withdrawal are shown in 2+ resulted in loss of inward currents, indicating that voltage-gated Ca2+ channels do not carry resolvable Na+ currents in the ionic conditions used here. Overall, these results support the notion that depolarization-induced rises in [Ca2+]i in 0 mM [Ca2+]e are not triggered by ion fluxes through high voltage-gated Ca2+ channels.To further examine whether voltage-dependent Ca2+] seen in response to prolonged depolarization in nominally Ca2+-free bath solution cannot be attributed to Ca2+ influx and subsequent Ca2+-induced Ca2+ release. Further, a skeletal muscle excitation-contraction–like mechanism as reported previously for hypothalamic magnocellular neurons, ischemically injured spinal cord white matter, and hippocampal neurons, can be excluded as the mechanism underlying depolarization-induced Ca2+ mobilization [2+], involving Ca2+- influx-independent activation of a G protein-phospholipase C-IP3 receptor pathway. However, the nature of the voltage sensor was not identified in the latter study [Our results indicate that the increase in global cytosolic [Calization –5. On ther study .+]e-evoked Ca2+ mobilization from IP3-sensitive stores as seen in the present study involves an ion influx-dependent, but not strictly depolarization-dependent, mechanism. Specifically, Na+ permeation of voltage-gated Ca2+ channels in the absence of external Ca2+ is one possible pathway linking Ca2+ release to membrane depolarization in our experimental conditions. We think this possibility to be unlikely for several reasons. First, extracellular Cd2+ at concentrations that completely blocked inward currents through voltage-gated Ca2+ channels, did not suppress high [K+]e-[Ca2+]i transients in the absence of extracellular Ca2+. Second, we did not detect resolvable inward currents through voltage-dependent Ca2+ channels in voltage-clamped, depolarized neurons upon removal of extracellular Ca2+. Third, high [K+]e-Ca2+ transients in Ca2+-free bath solutions could be readily evoked in the presence of 1 mM extracellular Mg2+, which corresponds to 4 times the IC50 of Mg2+-induced block of Na+ current through N-type Ca2+ channels i in the absence of external Ca2+ support our conclusion that depolarization–evoked rises in [Ca2+] require functional IP3 receptors.Although 2-APB has been shown previously to inhibit IP neurons , it alsoP) genes . Howeverll types ,34, simi2+ release constitutes a novel mechanism by which adult sympathetic ganglion neurons couple electrical activity to graded rises in intracellular [Ca2+]. The extent to which this mechanism contributes to the increase in [Ca2+] that normally occurs in response to single or repetitive action potentials remains to be quantitated. Eltit and co-workers previously demonstrated that tetanic stimulation of skeletal myotubes in the absence of extracellular Ca2+ gives rise to long-lasting, IP3-generated, slow Ca2+ signals both in the nucleus and cytoplasm e in the present study.Voltage-induced Caytoplasm . It will2+ transient is small compared to that of a transient elicited in normal [Ca2+]e, it is possible that Ca2+ is released into microdomains in which it may exert strong effects on exocytosis and/or Ca2+-sensitive ion channels and enzymes, ultimately altering excitability, energy homeostasis, and transcriptional activity of the neuron. With regard to the latter, the elevations in nuclear fluo-4 fluorescence that were observed to occur concomitantly with those in the cytosol both in 2 and 0 mM [Ca2+]i, may play a role in transcriptional regulation [2+]e often reached saturation using gain settings that were optimized to monitor cytosolic fluorescence, precluding simultaneous measurements in both compartments.Although the magnitude of the depolarization-induced global Cagulation . The mag2+ like those arising as a consequence depolarization-evoked IP3-recpetor stimulation, may enhance the Ca2+ -sensitivity of nearby ryanodine receptors, thereby converting the cytoplasm in an excitable medium capable of producing regenerative Ca2+ responses.Small elevations in cytosolic CaS1 FigA and B: Family of current traces recorded from an isolated sympathetic neuron in the absence (A) and presence of 300 μM CdCl2 in the external solution. Currents were evoked by 200-ms voltage steps, ranging from -70 to +50 mV in 10-mV increments. C: Peak IBa–voltage relationship for the cadmium-sensitive (circles) and–resistant (squares) currents shown in A and B. CdCl2 eliminated all inward currents.(TIF)Click here for additional data file.


It functions as a disulfide-tethered homodimer composed of three main domains, the Venus Flytrap module, cysteine-rich domain, and seven-helix transmembrane region. Here, we present the crystal structures of the entire extracellular domain of CaSR in the resting and active conformations. We provide direct evidence that L-amino acids are agonists of the receptor. In the active structure, L-Trp occupies the orthosteric agonist-binding site at the interdomain cleft and is primarily responsible for inducing extracellular domain closure to initiate receptor activation. Our structures reveal multiple binding sites for Ca2+ and PO43- ions. Both ions are crucial for structural integrity of the receptor. While Ca2+ ions stabilize the active state, PO43- ions reinforce the inactive conformation. The activation mechanism of CaSR involves the formation of a novel dimer interface between subunits.Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor (GPCR) that maintains extracellular CaDOI:http://dx.doi.org/10.7554/eLife.13662.001 Calcium ions regulate many processes in the human body. The calcium-sensing receptor, called CaSR, is responsible for maintaining a stable level of calcium ions in the blood. This receptor can detect small changes in the concentration of calcium ions, and activates signalling events within the cell to restore the level of calcium ions back to normal. Abnormal activity of this receptor is associated with severe diseases in humansCaSR is found in the surface membrane of cells and belongs to a family of proteins called G-protein coupled receptors. Much of the protein extends out of the cell and interacts with calcium ions, phosphate ions and certain other molecules such as amino acids. However, it was not well understood how these small molecules bind to CaSR and how this activates the receptor.Geng et al. have now used a technique called X-ray crystallography to view the three-dimensional structure of the exterior domain of CaSR in its resting state and active state. These structures revealed that, contrary to expectations, calcium ions are not the main activator of the receptor. Instead, Geng et al. found that CaSR adopts an inactive state in the absence or presence of calcium ions, while the active state only forms when an amino acid is bound.Furthermore investigation showed that calcium ions are needed to stabilise the active form, while phosphate ions keep the inactive form stable. Geng et al. also identified the shape changes that must occur as CaSR transitions from its inactive to its active state. In particular, an amino acid binding to the exterior domain causes it to close like a venus flytrap, which is a crucial step in activating the receptor.Taken together, the findings show that the amino acids and calcium ions act jointly to fully activate CaSR. The next steps are to determine the structure of the entire receptor with and without its small molecule partners and to use these structures to design drugs that can alter CaSR’s activity in order to treat human diseases.DOI:http://dx.doi.org/10.7554/eLife.13662.002 It sensand bone . Abnormaisorders . Loss-ofisorders . CaSR alelopment , nutrienelopment , and regelopment .q/11, Gi/o, G12/13 and Gs proteins . Within this family, CaSR and metabotropic glutamate receptors (mGluRs) function as disulfide-linked homodimers , while Grodimers . Ligand rodimers . In addirodimers . This CRtivation . HoweverB receptor  of mGluRs  and GABAreceptor , 2012, areceptor . Here, w2+, and form II in the presence of 10 mM L-Trp and 10 mM Ca2+ , and the interdomain cleft is empty. In addition, each protomer structure contains one Ca42- ions .2+ is bound at four novel sites in the CaSR ECD structure, including one at the homodimer interface. Each CaSR ECD molecule also contains two PO43- ions . Surprisingly, the ligand-binding cleft of each protomer is solely occupied by an L-Trp molecule. Ca43- ions .Agonist binding induces large conformational changes within the CaSR ECD homodimer. First, the VFT module of each protomer undergoes domain closure. Alignment based on the LB1 domains showed that the LB2 domains of inactive and active structures are related by a 29° rotation . Second,The inactive structure of CaSR ECD shows that subunit association in the resting state is primarily mediated by the LB1 domains . This di2 of solvent-accessible surface area, and can be divided into two regions. Site I is located at the center of LB1 domain and is flanked on either side by the two symmetric parts of site II  of each protomer . A dimerructures . For mGlructures  . The disease-causing mutation L159P renders the receptor less sensitive to Ca2+  . This Ca2+ ion bridges the end of helix G in LB2 with a loop in CR domain. An abundance of disease-related mutations are found at these dimer interaction sites, including (1) R172G and D215G at site III, (2) R227L and R227Q at site IV, and (3) G557E at site V  that transverses the domain . First, t site V . All thet site V    . The CR-site VI)   .In the active structure, the amino acid L-Trp is bound at the interdomain cleft of the VFT module, in agreement with previous mutational data , 2002 F. L-Trp f3H]-L-Trp in the absence and presence of Ca2+. Isotope dilution of [3H]-L-Trp with nonradioactive L-Trp led to a displacement of [3H]-L-Trp in a concentration-dependent manner. The addition of 2 mM Ca2+ increased the amount of L-Trp bound to CaSR ECD at any given concentration, suggesting that extracellular Ca2+ enhances L-Trp binding, possibly by affecting the L-Trp-binding affinity and kinetics of CaSR. The half-maximal inhibitory concentration of L-Trp  was approximately 2 mM regardless of whether the experiment was performed in the absence (2.11 ± 0.72 mM) or presence (2.04 ± 0.10 mM) of 2 mM Ca2+. Nevertheless, the binding affinity of L-Trp to CaSR ECD with and without Ca2+ may still differ as it depends on the concentration and binding affinity of the radiolabeled ligand. Further studies are needed to characterize the effect of Ca2+ on L-amino acid binding at the orthosteric agonist site of CaSR.We measured the direct interaction between L-Trp and CaSR ECD by scintillation proximity assay (SPA)  Figure . CaSR EC2+ mobilization through CaSR  of 0.12 ± 0.06 mM when extracellular Ca2+ was present at 2.5 mM. The efficacy and potency of L-Trp decreased at lower concentrations of Ca2+ that are within the physiological range (L-Trp EC50 = 0.75 ± 0.51 mM at 1.5 mM Ca2+), consistent with previous proposal that multiple amino acids need to act in concert to control the function of CaSR  (2+ ion (2+-binding sites observed in the CaSR ECD structures has been reported previously.We identified four distinct Carotomer) . The ina . The structural configuration of site 1 is similar in the inactive and active structures even though it is only occupied in the active state  or indirectly through water molecules. Alignment of the inactive and active structures in this region showed a small conformational change , indicating that these are high-occupancy sites. The Ca2+ ions at sites 3 (7.3 – 9.0 σ) and 4 (5.8 σ) have weaker anomalous peaks, which suggest low occupancy and possibly low affinity. The Ca2+ ion at site 4 has the weakest peak compared with other sites, which is consistent with the site being occupied only at elevated Ca2+ concentration for receptor activation.The CaWe identified a total of four anion-binding sites in the inactive and active CaSR ECD structures based on anomalous difference maps (1–4 or 1'-4’ in the second protomer) . Sites 142- ions in the inactive state and PO43- ions in the active state given their respective presence in the crystallization reagents. It is also possible that endogenous anions other than SO42- and PO43- are bound at these sites.In the inactive structure, electron densities revealed that anions were bound at sites 1–3 . In the The anion at site 1 is coordinated by the guanidine group of R62 and backbone nitrogen of Y63 . In the The anion bound at site 2 is held in place by multiple hydrogen bonds with the side chains of R66, R69, W70, and S417, and main chains of R415, I416, and S417 . The strAnion-binding site 3 is adjacent to site 2. The bound anion is also coordinated by R66 and additionally by T412 . In the The anion at site 4 is coordinated by H192, T195, K225 and R520 in the active structure . In the 2+-dependent receptor response and found that the presence of SO42- slightly decreased receptor activity, increasing the EC50 of Ca2+ by approximately 25%  It binds at the interdomain crevice of the VFT module, the canonical agonist-binding site for class C GPCRs  (2+ ion is found at the putative orthosteric agonist-binding site to induce domain closure. (4) Mutations of L-Trp-binding residues completely blocked Ca2+-induced IP accumulation and intracellular Ca2+ mobilization (2+-mediated receptor response. (6) L-Trp directly activates CaSR-mediated intracellular Ca2+ mobilization in the presence of extracellular Ca2+.Our structural analyses of CaSR ECD provide direct evidence that amino acids are agonists of CaSR, and they act concertedly with Ca C GPCRs . (2) L-T analogs . The res analogs . For exa analogs  and GABAr (S130) , respectlization , indicat2+ ion fulfills at least three functional roles. First, it maintains the structural integrity of CaSR, as manifested by the importance of Ca2+-binding site 2 for receptor function. Second, it is directly involved in receptor activation. Specifically, the Ca2+ ion at site 4 stabilizes the unique homodimer interface between membrane-proximal LB2 and CR domains in the active state. Third, Ca2+ enhances L-Trp binding to CaSR ECD, possibly by reinforcing the L-Trp bound and active conformation of the receptor. Furthermore, the actions of Ca2+ and amino acids on CaSR are inter-dependent. While the presence of extracellular Ca2+ above a threshold level is required for amino-acid-mediated CaSR activation, amino acids increase the sensitivity of the receptor toward Ca2+. Taken together, we conclude that amino acids and Ca2+ ions are indeed co-agonists of CaSR, acting jointly to trigger receptor activation.On the other hand, Ca2+ ion activate the receptor on its own as demonstrated in various cell-based functional assays? It is possible that amino acids are present in cell culture media and extracellular fluid at sufficient concentration to prime the receptor to respond to increasing concentrations of Ca2+. We have obtained crystals of CaSR ECD in the absence of any additional amino acids. Nevertheless, the structure showed a stretch of continuous density in the interdomain cleft that may belong to an endogenous ligand  An L-amino acid closes the groove in the extracellular VFT module, thereby inducing the formation of a novel homodimer interface between subunits. (3) Ca2+ ions stabilize the active state by enhancing homodimer interactions between membrane-proximal domains to fully activate the receptor. The combination of agonist-induced VFT closure and specific association of membrane-proximal CR domains in CaSR will likely lead to rearrangement of the transmembrane domains for receptor activation.In summary, activation of CaSR involves an intricate interplay of amino acids, Caer GPCRs , CaSR exer GPCRs . (1) CaSB receptor. Second, receptor activation requires the association of membrane-proximal domains. For CaSR, this involves the formation of a novel homodimer interface between the LB2 and CR domains. For GABAB receptor, which lacks the CR region, agonist leads to the formation of a large heterodimer interface between the LB2 domains  was cloned into the pFBDM vector  for exprMutants of CaSR ECD were generated in which either two or three N-linked glycosylation sites were eliminated. Both mutant constructs contained the mutations N386Q and S402N, while one also had the additional mutation N468Q. A Flag tag was engineered at the C-terminus of each construct to facilitate affinity purification. In agreement with previous studies of full-length CaSR in mammalian cells , we foun2. To isolate the L-Trp-bound receptor, purified CaSR ECD protein was washed extensively with a solution containing 20 mM Tris, pH 8.0, 10 mM L-Trp, and 10 mM CaCl2 prior to crystallization. This would facilitate the complete saturation of L-Trp- and Ca2+-binding sites within the receptor.Wild-type and mutant CaSR ECD were secreted from sf9 insect cells infected with the corresponding recombinant CaSR ECD virus. The CaSR ECD protein was isolated from cell supernatant by anti-Flag antibody (M2) affinity chromatography, and eluted with 100 µg/ml Flag peptide in 50 mM Tris, pH 7.5 and 150 mM NaCl. The protein was further purified by gel filtration chromatography  in 20 mM Tris, pH 8.0 and 150 mM NaCl to remove aggregates. Finally, the CaSR ECD protein was applied to an ion exchange column  in 20 mM Tris, pH 8.0 and eluted using a linear salt gradient from 0 to 1 M NaCl. All purification procedures were performed in the presence of 10 mM CaCl2, which is within the normal range of Ca2+ concentrations in plasma.We applied an enzymatic deglycosylation procedure to wild-type receptor and the CaSR ECD mutant with two glycosylation-site mutations. Each construct was expressed in sf9 cells in the presence of the N-glycosylation processing inhibitor kifunensine (1 mg/L) to produce high mannose glycoproteins that were sensitive to enzymatic cleavage by endoglycosidase H (Endo H). The cell supernatant was applied to an M2 anti-Flag antibody affinity column, and the bound CaSR ECD protein was eluted with 100 µg/ml Flag peptide in 50 mM Tris, pH 7.5 and 150 mM NaCl. Well-folded CaSR ECD was separated from aggregates by gel filtration chromatography in 20 mM Tris, pH 8.0, and 150 mM NaCl. The CaSR ECD protein was subsequently digested overnight with Endo H in 50 mM Na Citrate, pH 5.5. The partially deglycosylated CaSR ECD protein was further purified by ion exchange chromatography in 20 mM Tris, pH8.0 using a linear salt gradient from 0 to 1 M NaCl. The protein purification and enzymatic deglycosylation procedures were performed either in the absence or presence of 2 mM CaClWild-type CaSR ECD did not form well-diffracting crystals possibly because the presence of flexible and heterogeneous carbohydrates on the protein surface interfered with crystallization.2SO4, 100 mM Tris pH 8.5 in the absence and presence of 2 mM CaCl2. Protein crystallization was achieved by hanging drop vapor diffusion method using 24-well VDX plates . The CaSR ECD crystals were flash-cooled with liquid nitrogen in a cryoprotecting solution containing 3.0 M Li2SO4, 100 mM Tris pH 8.5, and the same concentration of CaCl2 as used for crystallization. These CaSR ECD crystals have the space group F222 with one molecule per asymmetric unit (form I).The CaSR ECD mutant carrying two glycosylation-site mutations formed diffracting crystals after enzymatic deglycosylation. The crystals were obtained at 20°C in 1.5 M Li2. The crystals were obtained using hanging drop vapor diffusion technique. These crystals were flash-cooled with liquid nitrogen in a cryoprotecting solution containing 0.9 M Na Citrate, 100 mM Tris, pH 8.0, 10 mM CaCl2, and 20% glycerol. (2) Crystals of the L-Trp-bound CaSR ECD mutant were grown at 20°C in 1.6 M NaH2PO4, 0.4 M K2HPO4, 100 mM Na2HPO4/citric acid pH 4.2, 10 mM CaCl2, and 10 mM L-Trp. The crystals were obtained by sitting drop vapor diffusion method using 24-well Cryschem plates (Hampton Research). These crystals were flashed-cooled with liquid nitrogen in a cryoprotecting solution containing 20% glycerol and all other components of the crystallization solution. The crystals obtained from both conditions belong to space group C2 and have two molecules per asymmetric unit (form II).The CaSR ECD mutant carrying three glycosylation-site mutations crystallized under two different conditions. (1) In the absence of additional amino acids, CaSR ECD formed the best diffracting crystals at 20°C in 0.9 M Na Citrate, 100 mM Tris, pH 8.0, and 10 mM CaCl2+. This native dataset was used to solve the inactive-state CaSR ECD structure. Form I crystals grown in the absence of additional Ca2+ diffracted to much lower resolution than those obtained in the presence of 2 mM Ca2+ and were therefore not used for structural determination.Diffraction data were collected at the 24ID-C and 24ID-E beamlines of Advanced Photon Source (APS). To minimize radiation damage at low energy, a native dataset was collected at the energy of Se-K edge (λ=0.9792 Å) for a single form I crystal of CaSR ECD grown in the presence of 2 mM Ca2+. These data sets were integrated individually by XDS  for L-Trp-bound form II crystals of CaSR ECD, each from a single crystal. The four data sets were processed individually by XDS  and CCP42+ ions, and two PO43- ions. The Ca2+ and PO43- ions were identified by anomalous difference Fourier maps calculated using data collected at a wavelength of 1.7712 Å. We modeled PO43- as the anions in the CaSR ECD structure because they were major components of the crystallization solution. The anomalous scattering of Ca2+ at 1.75 Å has been used successfully to identify the Ca2+-binding sites in a voltage-gated calcium channel  axis was identified from the self-rotation function. Polyalanine models generated from the individual LB1 and LB2 domains of mGluR3 ECD structure   attached to Asn261, Asn287, Asn446, Asn468, Asn488, Asn541, and Asn594. In addition, each protomer had one CaMolecular replacement searches were carried out using PHASER . Model bFull-length human CaSR was cloned into a pcDNA3.1(+) vector  for expression in human embryonic kidney (HEK) 293 cells. A Flag tag was inserted after the signal peptide of CaSR. Mutants of CaSR were constructed using the QuikChange mutagenesis system .HEK293 T/17 cells (ATCC) were transfected by Lipofectamine 2000 (Life Technologies) with wild-type or mutant CaSR plasmids. Cells permeabilized with 0.5% Triton X100 were used to determine the total expression level of CaSR in transfected cells. Untreated cells were used to determine the cell surface expression level of the receptor. The amount of surface protein detected for each construct was normalized to that found in the total cell lysate.The cells were blocked with 5% milk, and then incubated with mouse anti-Flag M1 antibody  as the primary antibody to measure CaSR expression. Donkey anti-mouse IRDye 800-labeled antibody  was used as the secondary antibody. Fluorescent signals were measured with an Odyssey Infrared Imager (LiCor). The results of three independent experiments were used for statistical analysis.Most of the mutants were expressed on the cell surface at levels comparable to that of the wild-type receptor. The exceptions were R66H, R69E, I81M, T100I, N102I, and S417L, which reduced the surface expression of the mutant receptors to approximately 70–75% of the wild-type level.1), a degradation product of inositol 1,4,5-triphosphate (IP3) that is stable in the presence of LiCl. Briefly, HEK293 T/17 cells were transiently transfected with wild-type or mutant full-length CaSR plasmids. The cells were stimulated with increasing concentrations of Ca2+ two days post transfection in a buffer containing 10 mM HEPES pH 7.4, 0.5 mM MgCl2, 4.2 mM KCl, 146 mM NaCl, 5.5 mM glucose, and 50 mM LiCl. The reaction mixture was then incubated with an IP1 analog coupled to a d2 fluorophore (acceptor) and an anti-IP1 monoclonal antibody labeled with Eu Cryptate (donor). The IP1 produced by cells upon activation of CaSR competes with IP1 coupled to the dye d2 for binding to the anti-IP1 antibody. The resulting FRET signal is inversely proportional to the concentration of IP1 in the sample. The fluorescence data was acquired at 620 and 665 nm using an EnVision plate reader  after laser excitation at 320 nm. The FRET signal was calculated as the fluorescence ratio (665 nm/620 nm). Basal activity was determined in the absence of Ca2+ stimulation. The percent stimulation of each receptor mutant was calculated based on the wild-type response obtained under the same condition. Data analysis was performed using the non-linear regression algorithms in Prism . Data points represent average ± s.e.m. of triplicate measurements.Measurement of inositol phosphate (IP) accumulation was carried out using the homogenous time-resolved fluorescence (HTRF) IP-one Tb kit . This assay quantifies the accumulation of inositol 1-monophosphate (IP2+-stimulated IP accumulation was determined using SO42- instead of PO43- because of the modest solubility of CaHPO4 (1.5 mM), the predominant form of calcium phosphate salt at physiological pH (7.4). Specifically, dose-dependent Ca2+-induced IP accumulation was measured in the absence and presence of 10 mM Li2SO4 in the reaction buffer.The effect of anion on Ca2+ mobilization was performed using a FLIPR Fluorescent Imaging Plate Reader (FLIPR) Calcium Assay kit (Molecular Devices). Briefly, HEK293 cells were transiently transfected with wild-type or mutant full-length CaSR plasmids and cultured overnight. The cells were incubated in a loading medium containing 50% Opti-MEM, 50% Ca2+-free Hank’s balanced salt solution, 2.5% fetal bovine serum, 20 mM HEPES pH 7.4, 2.5 mM probenecid and 2 µM fluorescent Ca2+ indicator Fluo-4 AM (Life Technologies) for 1 hr, and then placed into the FLIPR. CaCl2  was added at 10 s, and changes in fluorescence were monitored over a period of 250 s following excitation at a wavelength of 488 nm and detection at 510–560 nm. Data analysis was performed using the non-linear regression algorithms in Prism (GraphPad Software). Data points represent average ± s.e.m. of triplicate measurements.Measurement of intracellular Ca2+, HEK293 cells were transiently transfected with wild-type CaSR plasmid and cultured for 48 hr. The cells were washed three times with an assay buffer  to remove any endogenously bound ligand, and pre-incubated with 10 mM L-Trp for 20 min before stimulation with extracellular Ca2+.To measure the potentiating effect of L-Trp on the response of CaSR to extracellular Ca2, 20 mM HEPES-NaOH, pH 7.45) that contained 1 mM CaCl2, 0.8 mM NaH2PO4 and 1 mg/ml BSA for 1.5 hr at 37°C. The cells were washed and stored in a fura2-AM-free loading solution prior to experiments. Fura2-loaded HEK-CaSR cells were transferred into a perifusion chamber, placed in the light path of a Zeiss Axiovert fluorescence microscope , and perifused with PSS containing various concentrations of Ca2+ and L-Trp.HEK293 cells that stably expressed the CaSR (HEK-CaSR cells) were cultured on coverslips in 24-well plates, and loaded in the dark with fura2-AM (5 µM) in physiological saline solution , using alternating wavelengths of 340 and 380 nm at 0.5 s intervals, and imaged at 510 nm. For each data set, regions of interest corresponding to the locations of 10 individual cells were selected and digital images were captured using an AxioCam camera controlled by Stallion SB.4.1.0 PC software .2+ mobilization data consisted of excitation ratios (F340/F380) plotted against time (min). Ratio data were integrated, expressed as integrated response units (IRUs), and corrected for baseline (PSS containing 0.5 mM Ca2+). Concentration-dependent response data were fitted to the equation:Single-cell intracellular Caa = maximum response; b = basal response; C = activator concentration; e = EC50 in mM; and n = Hill co-efficient. Estimates of curve-fitting parameters were obtained using Prism (GraphPad Software).in which: 2 and amino acids. The protein was incubated in 100 mM NaCitrate, pH 5.5 overnight to remove any endogenously bound ligand. The CaSR ECD protein was then separated from any free ligand by gel filtration chromatography in 20 mM HEPES, pH 8.0 and 150 mM NaCl.Wild-type CaSR ECD protein was purified by anti-Flag M2 antibody affinity chromatography and gel filtration chromatography in the absence of additional CaCl3H]Trp (1 Ci/mmol) to 600 ng of purified CaSR ECD was measured with the scintillation proximity assay (SPA) using 1.25 mg/mL Yttrium silicate (YSi) protein A SPA beads  in conjunction with 0.125 ng/mL of anti-Flag M2 antibody in 20 mM HEPES, pH 8.0 and 150 mM NaCl at 4°C. Increasing concentrations of non-radioactive L-Trp were added to compete for receptor binding in the presence of 0 mM or 2 mM CaCl2. Each reaction was also performed in the presence of 60 mM non-radioactive L-Trp for background correction. The reactions were allowed to proceed for 1 hr to reach equilibrium. The plates were then counted in a Microbeta counter . Data were analyzed using the non-linear regression algorithms in Prism (GraphPad).Binding of 0.1 mM L-eLife. Your revised article has been favorably evaluated by Randy Schekman (Senior Editor), a Reviewing Editor, and one reviewer.Thank you for resubmitting your work entitled "Structural mechanism of ligand activation in human calcium-sensing receptor" for further consideration at The manuscript has been improved but there is a remaining issue that needs to be addressed before acceptance, as outlined below:1) In the point by point response to Reviewer #3, comment #3, the authors stated the following:2+ did not affect the binding affinity of L-Trp, indicating that Ca2+ion is not directly involved in L-Trp recognition. This is consistent with our structural observation that no Ca2+ ion is found at the L-Trp-binding site. Nevertheless, Ca2+ ion increased the amount of L-Trp bound to CaSR ECD at any given concentration, indicating that extracellular Ca2+ enhances the L-Trp-binding site occupancy, possibly by stabilizing the L-Trp-bound and active conformation of CaSR.""The presence of 2 mM CaThe authors should clarify what they mean by this. If calcium stabilized the Trp-bound state it would presumably be seen as an increase in Trp affinity. An increase in amount of radio-Trp that is bound that does not shift the affinity curve would seem to suggest adding to circulation Trp binding sites (i.e. receptors) that were previously not available (from an unavailable/inactive pool?). Or is there another possible explanation? Or does this raise a caveat about the experiment? This should be addressed in the text.Reviewer #1:2+ on L-Trp activity (and vice versa).I believe the revised version of the manuscript "Structural mechanism of ligand activation in human calcium-sensing receptor" is addressing the essential revisions that were raised during the first reviewing process. In particular, the authors have addressed the allosteric effect of Ca Essential revisions:50 in cell-based assays using different ionic conditions (such as no Ca2+ to 10 mM Ca2+).1) Evaluate the L-Trp EC2+ concentrations by monitoring changes in CaSR-mediated intracellular Ca2+ response in single cells. In agreement with previous findings, L-Trp can activate CaSR when the extracellular Ca2+ concentration is above the threshold level of 0.5 mM. The effect of L-Trp on CaSR was concentration-dependent, with an apparent EC50 of 0.12 ± 0.06 mM when extracellular Ca2+ was present at 2.5 mM. The efficacy of L-Trp decreased at lower concentrations of Ca2+ that are within the physiological range (L-Trp EC50 = 0.75 ± 0.51 mM at 1.5 mM Ca2+), consistent with previous proposal that multiple amino acids need to act in concert to control the function of CaSR. The new data are presented in Following the referees’ suggestion, we have evaluated the L-Trp EC50 at different Ca43- on the response of CaSR to Ca2+, compare responses (IP accumulation) to extracellular Ca2+ in the presence and absence of PO43-, in wt and mutants of the anion binding sites.2) To demonstrate the inhibitory effect of PO2+ in the absence and presence of the anion SO42-. We chose SO42- to substitute for PO43- because CaSO4 is more soluble than CaHPO4, the predominant form of calcium phosphate salt at physiological pH. At pH 7.4, approximately 61.3% of the phosphate ions exist in the form of HPO42- and 38.7% are in the form of H2PO4-. The solubility of CaHPO4 and Ca(H2PO4)2 are 1.5 mM and 71 mM, respectively. The solubility of CaSO4 is 15 mM.Following the referees’ suggestion, we have measured the response of CaSR to extracellular Ca2+ was slightly lower in the presence of than in the absence SO42-. This confirms that anions have a negative allosteric effect on the receptor. The new data are presented in As predicted by the referee, and consistent with our structural observation, we found that the response of CaSR to Ca42- on mutants of the anion binding-sites. Our structures revealed that the anion-binding residues R62 at site 1 and R66 at site 3 form hydrogen bonds across the interdomain cleft in the absence of bound anions to stabilize the closed conformation of the receptor. We found that the effect of SO42- on Ca2+-induced activation of the R62M mutant was similar to that of the wild-type receptor, while the presence of SO42- had little effect on the activity of the R66H mutant. However, we cannot conclude which anion-binding sites are involved in mediating the inhibitory effect of anions because these mutations are expected to have both positive and negative effects on the receptor function.We have also examined the effects of SOA) The mutations R62M and R66H are expected to eliminate the inhibitory effect of anions by preventing their binding to the receptor.B) These same mutations would also disrupt the formation of hydrogen bonds across the interdomain cleft by R62 and R66, thereby eliminating the interactions that favor receptor activation.C) Residue R66 is involved in the binding of anion at both sites 2 and 3. Since anion-binding site 2 appears to be crucial for structural integrity of the receptor, disruption of this site by the R66H mutation would negatively affect receptor function.2+, compare responses (IP accumulation) to extracellular Ca2+ in the presence and absence of L-Trp, in wt and mutants of the L-tryptophan binding sites.3) To demonstrate the potentiating effect of L-Trp on the response to Ca2+. During the course of our study, we found that it was very difficult to measure the functional effect of L-Trp through IP accumulation. Instead, we monitored the effect of L-Trp on CaSR-mediated intracellular Ca2+ mobilization. As predicted by the referee, Ca2+-induced receptor activation is higher in the presence of L-Trp than in the absence. The new data are presented in Following the referee’s suggestion, we have measured the potentiating effect of L-Trp on the response of wild-type CaSR to extracellular Ca2+-dependent receptor response in the absence of any amino acid. Therefore, these mutants would not be suitable for the study of the L-Trp effect. For the two mutants that displayed partial receptor activity (S147A and Y218S), we found that they did not respond to 10 mM L-Trp in the presence of 2 mM extracellular Ca2+.We have shown using IP accumulation measurements that most alanine mutants of the L-Trp- binding site abolished CaReviewer #1:2+ and PO43- ions act respectively as positive and negative allosteric modulators of the CaSR receptor activity by modulating, at least in part, the binding of the orthosteric agonist L-Trp. One key experiment would thus be to evaluate the L-Trp EC50 in cell-based assays using different ionic conditions (such as no Ca2+ to 10 mM Ca2+).I think it is tempting to speculate that the CaAlthough this might strengthen the manuscript, I do not believe these data would be critical for publication.2+ concentrations by monitoring changes in CaSR-mediated intracellular Ca2+ response in single cells. In agreement with previous findings, L-Trp can activate CaSR when the extracellular Ca2+ concentration is above the threshold level of 0.5 mM. The effect of L-Trp on CaSR was concentration-dependent, with an apparent EC50 of 0.12 ± 0.06 mM when extracellular Ca2+ was present at 2.5 mM. The efficacy of L-Trp decreased at lower concentrations of Ca2+ that are within the physiological range (L-Trp EC50 = 0.75 ± 0.51 mM at 1.5 mM Ca2+), consistent with previous proposal that multiple amino acids need to act in concert to control the function of CaSR. The new data are presented in Following the referee’s suggestion, we have evaluated the L-Trp EC50 at different CaReviewer #2:I highly evaluate the scientific merits and the general impact of the paper. I judge it is worthy for publication, but only after satisfactory revisions. I have four major comments which require attention.43-1) Experiments to demonstrate the inhibitory effect of PO43- ions promote the inactive configuration" is rather speculative from the two reasons in the following.The conclusion in the Discussion that " PO43- promotes the inactivated conformation. The authors showed mutants of the PO43- binding sites have lower IP accumulation in response to extracellular Ca2+. I think the data are not sufficient. To demonstrate the inhibitory effect of PO43- on the response of CaSR to extracellular Ca2+ conclusively, I would like to request to present data comparing the responses (IP accumulation) to extracellular Ca2+ in the presence and absence of PO43-, in wt and mutants of the anion binding sites. The expected result is lower response to Ca2+ in the presence of PO43- than in the absence, not in mutants but in wt.A) There are no direct evidence which shows PO2+ in the absence and presence of the anion SO42-. We chose SO42- to substitute for PO43- because CaSO4 is more soluble than CaHPO4, the predominant form of calcium phosphate salt at physiological pH. At pH 7.4, approximately 61.3% of the phosphate ions exist in the form of HPO42- and 38.7% are in the form of H2PO4-. The solubility of CaHPO4 and Ca(H2PO4)2 are 1.5 mM and 71 mM, respectively. The solubility of CaSO4 is 15 mM.Following the referee’s suggestion, we have measured the response of CaSR to extracellular Ca2+ was slightly lower in the presence of than in the absence SO42-. This confirms that anions have a negative allosteric effect on the receptor. The new data are presented in As predicted by the referee, and consistent with our structural observation, we found that the response of CaSR to Ca42- on mutants of the anion binding-sites. Our structures revealed that the anion-binding residues R62 at site 1 and R66 at site 3 form hydrogen bonds across the interdomain cleft in the absence of bound anions to stabilize the closed conformation of the receptor. We found that the effect of SO42- on Ca2+-induced activation of the R62M mutant was similar to that of the wild-type receptor, while the presence of SO42- had little effect on the activity of the R66H mutant. However, we cannot conclude which anion-binding sites are involved in mediating the inhibitory effect of anions because these mutations are expected to have both positive and negative effects on the receptor function.We have also examined the effects of SOA) The mutations R62M and R66H are expected to eliminate the inhibitory effect of anions by preventing their binding to the receptor.B) These same mutations would also disrupt the formation of hydrogen bonds across the interdomain cleft by R62 and R66, thereby eliminating the interactions that favor receptor activation.Residue R66 is involved in the binding of anion at both sites 2 and 3. Since anion-binding site 2 appears to be crucial for structural integrity of the receptor, disruption of this site by the R66H mutation would negatively affect receptor function.43- or SO43-, because there are no anions in the crystallization solution other than PO43- or SO43-. Is there a possibility that carryover endogenous anion(s) other than PO43- is bound to the anion binding site? (similarly to the case of the putative carryover of endogenous amino acid(s) at the L-tryptophan binding site when crystalized in the absence of L-tryptophan).B) The authors assume that the electron density represents the presence of PO43- or SO42- is bound at the anion-binding sites. We have added a sentence in the text to mention the caveat.We agree with the referee that it is possible carryover endogenous anion(s) other than PO2) Experiments to demonstrate the potentiating effect of L-tryptophan2+ conclusively, please present data comparing the responses (IP accumulation) to extracellular Ca2+ in the presence and absence of L-tryptophan, in wt and mutants of the L-tryptophan binding sites. The expected result is higher response to Ca2+ in the presence of L-tryptophan than in the absence, not in mutants but in wt.I have similar comments to the case of anion effect. To demonstrate the potentiating effect of L-tryptophan on the response of CaSR to extracellular Ca2+. As predicted by the referee, Ca2+-induced receptor activation is higher in the presence of L-Trp than in the absence. The new data are presented in Following the referee’s suggestion, we have measured the potentiating effect of L-Trp on the response of wild-type CaSR to extracellular Ca2+-dependent receptor response in the absence of any amino acid. Therefore, these mutants would not be suitable for the study of the L-Trp effect. For the two mutants that displayed partial receptor activity (S147A and Y218S), we found that they did not respond to 10 mM L-Trp in the presence of 2 mM extracellular Ca2+.We have shown using IP accumulation measurements that most alanine mutants of the L-Trp- binding site abolished Ca3) Comparison with the mGluR and GABABRThe authors discussed differences and similarities of the CaSR structure to those of mGluR and GABABR. However, it is hard to follow the explanations in the text, without structure images for comparison.A) The authors mentioned the degree of rotational change of two LB1 interface in CaSR with GABAB and mGluR upon activation . In Following the referee’s recommendation, we have added images of the two states of mGluR1 and GABAB receptor comparing the rotated angle of the LB1-LB1 interface in each receptor system .B) The authors described an overall similarity of VFT cleft structure of CaSR and mGluR. They also wrote the tryptophan binding site of CaSR corresponds to the glutamate binding site of mGluR . To clarify these points, please present a superimposed image of the VFT cleft of CaSR and mGluR, in e.g. We agree with the referee and have added a superimposed image of the agonist-binding cleft of CaSR and mGluR1 in 4) Gd3+ binding siteIn the crystal structure of mGluR1, a binding site for Gd3+ was identified at the dimer interface . I would like to have explanations whether or not a similar structure was identified in CaSR.3+ ion in the mGluR1 structure is coordinated by two acid residues, E238 and D242 from each subunit. The corresponding residues in the CaSR structure are R220 and E224. The basic residue R220 would not be compatible with the binding of a cation to CaSR at this location.We think the referee’s question is very interesting, and have superimposed the structures of CaSR and mGluR to compare this region of the dimer interface. As shown in Reviewer #3:Geng et al. reported the crystal structures of entire extracellular domain of calcium sensing receptor of calcium free form and loaded forms in the presence of calcium and PO43- ions. This work is of importance for providing insights for the activation mechanism via dimer interface between subunits. There are several major concerns that need be addressed:1) The crystal structures were determined for CaSR with mutations of either two (N386Q and S402N) or three (N468Q) N-glycosylation sites. However, no study has been carried out on the effects of these mutations on expression and function of CaSR. It is important to provide evidence with experimental data that mutations at these glycosylation sites do not result in alteration of the function and expression.2+-induced IP accumulation was statistically the same for the wild-type and mutant receptors, indicating that these glycosylation mutations did not alter CaSR function . Following the referee’s suggestion, we have also generated two full-length CaSR constructs for functional analysis in mammalian cells, one carrying the two glycosylation-site mutations N386Q and S402N, and the other carrying the additional mutation N468Q. We found that Cafunction .On the other hand, previous studies of full-length CaSR in mammalian cells indicate that disruption of the glycosylation sites impairs proper processing and expression of the receptor at the cell surface . In agreement with these findings, we found that expression of CaSR ECD in insect cells was essentially eliminated when more than three glycosylation sites were mutated. After testing mutations at each of the potential N-linked glycosylation sites individually and in various combinations, we chose the constructs reported in this study to maximize the reduction of carbohydrate content while maintaining a sufficient expression level for structural analyses.2) Since mutations could lead to reduction in cell surface expression due to trafficking, it is encouraged to provide confocal images to ensure that the observed change in intracellular IP activity is not due to reduction of protein expression levels on cell surface.We appreciate the referee’s suggestion. Unfortunately, we do not have any experience with confocal imaging. We did measure the cell surface expression levels of wild-type and mutant CaSR by on-cell western analysis, which was described in the Methods section. Most of the mutants in our study were expressed on the cell surface at levels comparable to that of the wild- type receptor. The exceptions were R66H, R69E, I81M, T100I, N102I, and S417L, which reduced the surface expression of the mutant receptors to approximately 70-75% of the wild-type level. Nevertheless, the reduction in the protein expression level on cell surface could not account for the much greater decrease in intracellular IP accumulation for each of these mutants. We therefore concluded that the mutations affected receptor function.3) The identification of Trp binding site is very important for the activation mechanism. It will be very helpful to report Trp binding affinity of the ECD of CaSR.3H]-Trp. In the absence of Ca2+, the binding of [3H]-L-Trp to CaSR ECD was inhibited by nonradioactive L-Trp with a half-maximal inhibitory concentration (IC50) of 2.1 ± 0.7 mM. The presence of 2 mM Ca2+ did not affect the binding affinity of L-Trp, indicating that Ca2+ ion is not directly involved in L-Trp recognition. This is consistent with our structural observation that no Ca2+ ion is found at the L-Trp-binding site. Nevertheless, Ca2+ ion increased the amount of L-Trp bound to CaSR ECD at any given concentration, indicating that extracellular Ca2+ enhances the L-Trp-binding site occupancy, possibly by stabilizing the L-Trp-bound and active conformation of CaSR.We agree with the referee, and have determined the binding affinity of L-Trp for CaSR ECD by scintillation proximity assay using radiolabeled  = 4.2pH = -log[H+] = 1 x 10-4.2 M ≈ 0.1 mM/[H3PO4])pK-3) = 4.2 - log([H2PO4-]/[H3PO4])-log(7.5 x 102PO4-]/[H3PO4] = 102.1[H2PO4- <--> H+ + HPO42-Ha2 = pH - log([HPO42-]/[H2PO4-])pK-8) = 4.2 - log([HPO42-]/[H2PO4-])-log(6.2 x 102PO4-]/[HPO42-] = 103[H42- <--> H+ + PO43-HPOa3 = pH - log([PO43-]/[HPO42-])pK-13) = 4.2 - log([PO43-]/[HPO42-])-log, a Reviewing Editor, and one reviewer.The manuscript has been improved but there is a remaining issue that needs to be addressed before acceptance, as outlined below:1) In the point by point response to Reviewer #3, comment #3, the authors stated the following:2+ did not affect the binding affinity of L-Trp, indicating that Ca2+ ion is not directly involved in L-Trp recognition. This is consistent with our structural observation that no Ca2+ ion is found at the L-Trp-binding site. Nevertheless, Ca2+ ion increased the amount of L-Trp bound to CaSR ECD at any given concentration, indicating that extracellular Ca2+ enhances the L-Trp-binding site occupancy, possibly by stabilizing the L-Trp-bound and active conformation of CaSR.""The presence of 2 mM CaThe authors should clarify what they mean by this. If calcium stabilized the Trp-bound state it would presumably be seen as an increase in Trp affinity. An increase in amount of radio-Trp that is bound that does not shift the affinity curve would seem to suggest adding to circulation Trp binding sites (i.e. receptors) that were previously not available (from an unavailable/inactive pool?). Or is there another possible explanation? Or does this raise a caveat about the experiment? This should be addressed in the text.3H]-L-Trp binding competition studies alone that the binding affinity of L-Trp is the same with or without Ca2+. Given our structural observation that Ca2+ stabilizes the active conformation of the receptor, we agree that Ca2+ would most likely enhance the binding affinity of L-Trp since the interaction of L-Trp with an active receptor is expected to be more extensive than with an inactive receptor. In an active receptor, L-Trp would contact both the LB1 and LB2 domains. L-Trp may still bind to the open cleft in an inactive receptor, but its interaction with the receptor will be limited to the LB1 domain, which would result in lower affinity. The binding of glutamate to an open cleft has previously been observed for mGluR1 .We think referee #3 has raised a valid criticism. As pointed out by referee #1 below, we cannot conclude based on our [3H]-L-Trp required would be ≥10 mM, and a relatively low specific radioactivity of the commercially available radio-labeled ligand may preclude such experiments. Our competition experiments in the current study were performed at 0.1 mM [3H]-L-Trp, a concentration that is about 20-fold below the determined half-maximal inhibitory concentration (IC50) of non-labeled L-Trp for replacing radiolabeled L-Trp.Unfortunately, as indicated by referee #1, it would be difficult for us to measure the binding affinity of L-Trp in a saturation binding experiment since the concentration of [2+ may affect the on- and off-rate of L-Trp binding to CaSR ECD. Specifically, L-Trp may have a slower off-rate when bound to the active conformation. Therefore, the higher amount of L-Trp binding observed with Ca2+ may reflect the different L-Trp binding affinity and kinetics exhibited by the inactive and active receptors. We have modified our text to include these explanations and to indicate that additional studies would be needed to characterize the role of L-amino acid binding to the orthosteric agonist site of CaSR.In addition, the presence of Ca


Theoretical considerations demonstrate that the different recovery kinetics can be modelled by simulating changes in the Ca2+-buffering capacity of the outer segment. Importantly, the Ca2+-buffer action preserves the fast response rising phase, when the Ca2+-dependent negative feedback is activated by the light-induced decline in intracellular Ca2+.The phototransduction enzymatic cascade in cones is less understood than in rods, and the zebrafish is an ideal model with which to investigate vertebrate and human vision. Therefore, here, for the first time, the zebrafish green cone photoresponse is characterized also to obtain a firm basis for evaluating how it is modulated by exogenous molecules. To this aim, a powerful method was developed to obtain long-lasting recordings with low access resistance, employing pressure-polished patch pipettes. This method also enabled fast, efficient delivery of molecules via a perfusion system coupled with pulled quartz or plastic perfusion tubes, inserted very close to the enlarged pipette tip. Sub-saturating flashes elicited responses in different cells with similar rising phase kinetics but with very different recovery kinetics, suggesting the existence of physiologically distinct cones having different Ca G-protein-coupled enzyme cascades are ubiquitous signalling systems: they transduce physical or chemical stimuli, triggered by photon absorption or ligand binding , into intracellular messages that elicit adaptive responses , 2. The Vertebrate visual phototransduction (reviewed in –14) is oHere we present a new method for the ex vivo study of a G-protein coupled enzymatic cascade, the ultimate goal being to up- and down-regulate a photoreceptor protein and, in parallel, measure how the light response is affected. An obvious strategy for realizing this is, for instance, to deliver the specific protein itself, or its monoclonal antibody, during whole-cell recording of photoresponses, here performed for the first time on green-sensitive cones of zebrafish. This animal has attained increasing interest in recent years as a model for the investigation of vertebrate and even human vision . Moreove2+ dynamics. The Ca2+-buffering capacity of the outer segment strongly regulates this dynamics: importantly, a minimal model fitting the photoresponses demonstrates that this Ca2+-buffer prevents the slow-down that can occur in the response rising phase when a negative feedback (whatever its nature) regulating receptor sensitivity is activated during the light response. Finally, the extremely fast rising phase of the response to supersaturating flashes is consistent with the hypothesis that rhodopsin may interact with transducin in the dark by forming preassembled complexes , 0.6 MgCl2, 0.6 MgSO4, 1.5 CaCl2, 10 glucose . The Petri dish containing the head was transferred in a fully darkened box equipped with infrared LEDs (wavelength: 940 nm) and a high definition webcam that had its infrared filter removed and was connected to an external monitor. After inserting the hands through the two light-tight holes of the box, the cornea of each eye was focussed on the monitor screen and it was cut with a razor blade piece. Each retina was separated from the pigment epithelium by sucking it gently into the tip of a 200 μl pipette, which was also used to mechanically dissociate the photoreceptors by triturating the retina. An aliquot (~2 ml) of the Ringer solution containing the photoreceptors was transferred to the recording chamber placed on the microscope  stage. The preparation was illuminated with a cluster of ultra-bright infrared LED (900 nm) and focused on a fast digital camera  coupled to the microscope.A healthy zebrafish was dark-adapted for 3 h and sacrificed in dim red light (generated by LEDs with wavelength of 660 nm) by cranial concussion followed by decapitation and pithing. The head was swiftly immersed in a Petri dish containing Ringer solution of the following composition (in mM): 115 NaCl, 3 KCl, 10 HEPES free acid [Ra), and the membrane resistance; the holding potential was always -40 mV. Whole-cell recordings were low-pass filtered at 2 Khz by an eight-pole Butterworth filter , sampled at 19.2 kHz/16 bits by a A/D board  controlled by Clampex software (Molecular Devices), and analyzed with Clampfit (Molecular Devices), Sigmaplot  and Mathcad  software. To reject as much noise as possible, each photoresponse shown in the figures was the average of three or more responses to the same flash; this average response was then further filtered by using a local smoothing routine implemented in the commercially available program Sigmaplot. The routine parameters were set to obtain low-pass filtering of 30 Hz . Traces longer than 2 s were just low-pass filtered at 20 Hz with the Gaussian filter of the pClamp routine, since they illustrate only slow aspects of the photoresponses: their huge number of points made it impractical to use the Sigmaplot smoothing routine on desktop computers.Light responses of zebrafish cones were recorded using the whole-cell configuration of the patch-clamp technique, under dark-adapted condition at room temperature (20–22°C). Pipettes were usually sealed on the cone outer segment, unless specified otherwise; details of the patch-clamp apparatus is described elsewhere , 31. TheResults are given as means±SEM.2, 1 GTP, 5 ATP, 5 HEPES . All chemicals were purchased from Sigma . The intracellular dialysis of zebrafish cones was performed by using pressure-polished pipette and an intrapipette perfusion system (Patch pipettes were filled with an intracellular solution containing (in mM): 40 KCl, 70 K-Asp, 5 MgCln system . Besidesn system , allowinn system .xyz micromanipulator and coupled to the binocular port of the inverted microscope . The LED was aligned so as to have its light spot automatically centered and in focus on the zebrafish cone, when the latter, illuminated with the cluster of ultra-bright infrared LEDs, was viewed in sharp focus through a 60x objective on the microscope camera . Enough space was left between the trichromatic LED and the binocular port to host neutral density filters, when required; the trichromatic LED positioning and measurement of its spot size were performed with the following procedure. First, a particular subdivision of the grid of a calibrated microscope slide was focused and centered on the inverted microscope 60x objective, by using the microscope infrared LED illumination. Then a straight microscope, mounted on a second xyz micromanipulator, was aligned to the inverted one, and it was moved so its 60x objective was focused and centered on the same grid subdivision using the trichromatic LED as a light source. The LED spot, observed with the straight microscope, was finally focused and centered with its xyz manipulator on the grid subdivision, and its geometry measured (it resulted a uniform disk of 300 μm of diameter). The microscope slide was then replaced by the sensor  of a power/energy meter , and the light power produced by the green and the red section of the trichromatic LED was measured at all the voltages used in the experiments. Voltages, neutral density filters, and light stimulus duration were matched so as i) to have a wide range of light intensities spanning more than 5 order of magnitude, with currents always falling in the green and red LED linear range (1–50 mA) and ii) to have the same light intensity of red and green LED (the blue one was not used). The duration of all flashes up to 1.77·104 photons/μm2 of intensity was 1 ms, above this intensity it was 10 ms.Light stimuli (flashes in the dark and flashes on a background of light) were designed using the voltage protocols of Clampex software. One of the two analogical outputs of the Digidata board was connected to a calibrated voltage-to-current converter driving an ultra-bright trichromatic LED (with center wavelength red/green/blue of 627.5±8/525±10/467.5±8 nm), that had its plastic lens replaced with a frosted filter. The LED was mounted on a miniaturized http://rsb.info.nih.gov/ij/) and was 15.3±3.9 μm2 (n = 20). Using the ratio between the geometrical rod outer segment area and the collecting area of ~0.062 calculated by Baylor et al., 1979, it can be roughly estimated that the zebrafish cone collecting area is ~1 μm2. If this line of reasoning proves to be correct, then the numerical values of the flash intensities reported throughout the paper (in photons/μm2) are also the number of photons absorbed by the cone.To estimate the physical collecting area of the zebrafish cone outer segment , the quantum efficiency of photoisomerization and the specific axial rhodopsin density must be known . Moreovea and a), following the injection of lucifer yellow into the cell. In principle, many factors influence such dynamics, for example cell geometry, where the pipette is sealed on the cell, the effective pressure applied inside the pipette perfusion tube, the position of this tube inside the pipette, pipette geometry and access resistance (Ra). Despite all these factors, fluorescence grew with similar kinetics in all cells examined: in the regions close to the pipette, fluorescence dynamics were described by the sum of a fast (parameters with the subscript f) exponential component and a slower (subscript s) one:Besides bright-field viewing of the cells, the camera and its controlling software  were also employed for fluorescence imaging experiments; the excitation light was generated by a monochromator  coupled to the epifluorescence port of the microscope via an optical fiber. Solution loading in the cell was checked by using lucifer yellow  progressively decreased (i.e. it was “low-pass filtered”) as the distance between the pipette (i.e. the point of dye injection) and the integration region was larger. Eventually, the fluorescence increased in the distal regions  with a slow monoexponential kinetics . As an example, The fast component amplitude (i.e. kinetics . TherefoRa~6.7±1.1 MΩ range: 2.0–13.0 MΩ n = 36) recordings and efficient cytosolic incorporation of exogenous molecules. Long lasting recordings , chcordings  with precordings  were obt3 photons/μm2 in intensity suppressed ~40% of the dark current  but it did not respond to a red flash , were delivered in triplets in repeated sequences. The small drift in the baseline was due to small changes in the current flowing through the inner segment channels rather than in changes in the light sensitive (or dark) current flowing through the cGMP channels, because the amount of current suppressed by saturating flashes (with intensities ≥1.8·104 photons/μm2) was the same  from the beginning to the end of these recordings. Responses to the same flash intensities were averaged and smoothed (see below and 5 photons/(μm2·sec), lasting 8 s) were occasionally delivered during this recording  of recording. Responses in 3 to 104 photons/μm2. This feature was observed clearly in ~40% of the recordings (n = 25), while others had a faster recovery with a single kinetic component, . In fact, a continuum of behavior in between these two recovery types :I0 is the half-saturating intensity and m is Hill coefficient. This equation fitted the data point at low intensities but not at higher ones , or vice-versa . The best fit was given by the exponential saturation equation:I0 = 3·103 photons/μm2 (red curve).Besides these differences in recovery phase, all green-sensitive cones had similar photoresponse rising time and sensitivity, with a threshold of ~104 photons/μm2) − but it slightly altered the rising phase of stronger flashes at early times. Since this resulted in an underestimation of the delay between the flash delivery time and onset of the response (inset), the analysis of the responses to supersaturating flashes were performed on unsmoothed traces .In order to reduce the background noise, photoresponses were averaged and further processed with a smoothing routine see . This opresponse , inset, observed . The linblack bars, black traces, green) and at the end (red) of the recording were almost identical. The average flash responses in the presence of a background of light were smaller and faster in respect to the ones recorded without any adapting light (5 photons/(μm2·s) suppressed 63±9% of the dark current  and reduced the sensitivity as follows: a flash delivering 4.89·103 photons/μm2 in the dark suppressed ~70±4% of the current , while a flash twice as strong delivered on the background suppressed ~45±12% of the current  to be quickly inactivated. Therefore, the difference between the two recovery phases may originate from different intracellular Ca2+ dynamics and this would be detected by several Ca2+ sensor proteins regulating the phototransduction cascade at various levels  described by the Poisson equation [As and τ are constants . This equation is intended to fit the smallest average response to a flash  is quite accurate for the fast responses  (red trace) is always faster than the experimental responses . These slow responses can be fitted by a different expression for xs(t) but, for the sake of simplicity, the fitting illustrated in red trace) is good enough for the argument that follows. Let’s assume that the photoresponse to a strong stimulus is described by the same equation and same τ as the response to the dimmest stimulus, but with a larger As. Let’s also assume that this “larger” photoresponse is the “input” of a system regulated through a negative feedback mechanism (H be the gain and G the amount of “regulated photoresponse” (or “output”), xo(t), subtracted (i.e. negatively “feedbacked”) from the input. The more intense the light absorbed by the photoreceptor, the larger the decline in intracellular Ca2+ and, consequently, the larger parameter G will be. A Ca2+ buffer introduces a delay, Δ, between the time in which an intracellular Ca2+ change takes place and the time in which the Ca2+ sensor proteins detect it. Let xoΔ(t) be the delayed output that is now subtracted from the input, as a consequence of the delay Δ introduced in the feedback path by the buffer [Consider a system that produces a “photoresponse” equation :xs(t)=Aechanism  aimed ate buffer . It follxo(t) (brown traces) is just scaled down when compared to the un-regulated response, xs(t) (red traces), if:In the absence of any delay, the amplitude of the regulated responses, black dotted trace), and one fast and recovering with a single component kinetics (black dotted trace), it is necessary to set Δ≠0. This accelerates the theoretical photoresponse recovery which exhibits a dual (green trace) or single kinetics component (green trace), depending upon the values of τ and Δ. The former is constrained by the fit to the two responses to the dimmest flash , as observed in other cone species, where Δ was experimentally increased by incorporating into the cone millimolar amounts of the fast Ca2+ buffer BAPTA [G and/or Δ are increased; the frequency of the oscillations become smaller as Δ is made larger. It is important to emphasize that the dramatic changes in the photoresponse recovery waveform, produced by Δ, is independent by the nature of the feedback (i.e. which element of the transduction system is affected by the decline in intracellular Ca2+). Notably, the rising phase of the delayed regulated response is faster than the one with no delay . In general, for a receptor to have a large operative range, its sensitivity must be progressively reduced as stimulus intensity is increased, and this requires the presence of a negative feedback. However, negative feedback compresses the entire response while, instead, a delay provides the same initial fast rising phase as the unregulated response but avoids the later response saturation. The advantage of this strategy is obvious: the survival of an animal is ultimately determined by the speed with which its photoreceptors detect a predator in dim or in strong light. It is possible that, in general, the response kinetics of any signal transduction cascade, regulated through a negative feedback by one or more intracellular messengers, is tuned by the capacity of cytosol to buffer this/these messenger/s, always ensuring a fast response onset for a wide range of stimulus intensities.To fit two representative experimental photoresponses of  to the sst flash  while ther BAPTA . The osc2+ concentration, strongly indicating that these two recovery types were generated by two different delays (Δ) in Ca2+ feedback. On the basis of the present findings, it may be that the intrinsic Ca2+-buffering capacity of the zebrafish cone was somewhat modified during these whole-cell recordings , transforming a dual component response recovery into a single component one or vice-versa. However, even in recordings lasting more than 20 min, the stability of the response waveform rules out any significant alteration in the concentration of any of the key components involved in shaping the photoresponse  but there was no change in the response waveform within this period of time. Finally, the recordings obtained with a pipette solution from which the Ca2+ contamination (~1 μM) had been removed using 25 μM of BAPTA ethane-N,N,N',N'-tetraacetic acid, K+ salt) were indistinguishable from the ones obtained in the absence of this Ca2+ chelator. This demonstrates that the strong cooperative Ca2+ regulation of guanylate cyclase, the influx of Ca2+ through the light-regulated channels, and the efflux of Ca2+ through the exchanger come together to form a powerful system to clamp the intracellular Ca2+ concentration (and the dark current) to its physiological value. Indeed, to contrast this system and finally affect the photoresponse recovery kinetics, the photoreceptor outer segment must be dialyzed with hundreds of micromoles of BAPTA or of Ca2+ [2+ dynamics and that this is primarily regulated by the Ca2+ buffering capacity. It is then possible that the photoreceptor Ca2+ dynamics is regulated at one or more stages by a still unknown mechanism. In different low vertebrate photoreceptor cells, the latter could be locked at different levels of activation, thus giving the observed differences in the recovery phase. With evolution, this mechanism could, in turn, be regulated in the same cell by specific physiological requirements, giving a different response to the same stimulus, as observed by Cao and colleagues.Interestingly, the damped oscillations of the recovery phase are thought to be typical of primate cones. However, as pointed out by Cao and colleagues , this is of Ca2+ , 21. All


Increased plasma levels of C-reactive protein (CRP) are closely associated with cardiovascular diseases, but whether CRP is directly involved in the pathogenesis of atherosclerosis is still under debate. Many controversial and contradictory results using transgenic mice and rabbits have been published but it is also unclear whether CRP lowering can be used for the treatment of atherosclerosis. In the current study, we examined the effects of the rabbit CRP antisense oligonucleotides (ASO) on the development of atherosclerosis in WHHL rabbits. CRP ASO treatment led to a significant reduction of plasma CRP levels; however, both aortic and coronary atherosclerotic lesions were not significantly changed compared to those of control WHHL rabbits. These results suggest that inhibition of plasma CRP does not affect the development of atherosclerosis in WHHL rabbits. The e models . In our e models . In addie models  and rabbe models . To exam in vivo studies, CRP ASOs were dissolved in saline solution and intravenously injected into WHHL rabbits through ear veins (60 mg/Kg BW/week) twice a week for 16 weeks. Control mismatched oligonucleotides were injected in the same way as CRP ASOs.Watanabe heritable hyperlipidemic (WHHL) rabbits  were bren = 9 for each group, containing 5 males and 4 females). These WHHL rabbits started to develop aortic atherosclerosis, which represents the so-called early-stage lesions, such as fatty streaks in humans [n = 10 for each group) were used. These old WHHL rabbits developed advanced lesions, which are similar to human atherosclerotic plaques  [To examine whether rabbit CRP ASO administration could affect the development of atherosclerosis, we designed and performed two experiments. For the first experiment, we used young WHHL rabbits aged 3~4 months (n humans . Therefooulders) . In thisPlasma total cholesterol (TC), triglycerides (TG), and HDL-cholesterol (HDL-C) were determined using Wako assay kits . For the analysis of lipoprotein profiles, plasma was resolved by electrophoresis in 1% agarose universal gels , and then the gels were stained with Fat Red 7B . Plasma  t-test. In all cases, statistical significance was set at P < 0.05.All values are expressed as the mean ± SD and statistical significance was determined using Student'sP = 0.06). Reduced plasma levels of CRP were consistent with lower hepatic expression of CRP mRNA, as shown by RT-PCR analysis  and TG  than controls, (P < 0.01) and chylomicron remnant contents (P < 0.05)  . To elucn lipase . The bas en face lesion areas in all parts compared to those of controls. We further compared the microscopic lesion size and histological features. Quantitative analysis of the microscopic lesions and macrophage and smooth muscle cell contents along with CRP immunoreactive protein deposition did not reveal any significant differences between CRP ASO-treated WHHL rabbits and controls  is not only a predictor but also a potential risk factor of cardiovascular events . SeveralIt should be pointed out, however, that the current results cannot rule out the possibility that CRP may be involved in other inflammatory diseases.Unexpectedly, we found that CRP ASO treatment elevated plasma lipids in WHHL rabbits due to enhancement of apoB-containing particle production. CRP ASO-induced lipid raising effect was not found in human clinical trials using CRP ASOs. It is currently unknown whether elevated plasma lipids are caused by CRP inhibition or CRP ASOs themselves (such as off-targeting effects).In conclusion, we found that CRP lowering does not have significant influence on the initiation and progression of atherosclerosis in WHHL rabbits; thus, CRP may not be a therapeutic target for the treatment of atherosclerosis.