However, little is known about the roles of M-MDSCs in acute myeloid leukemia (AML) as opposed to their multiple roles in solid tumors.  Monocytic myeloid-derived suppressor cells (M-MDSCs) characterized with the phenotype of CD14+HLA-DRlow/- by flow cytometry in the peripheral circulating blood of 109 newly diagnosed adult patients with AML and 30 healthy controls (HC). Then, we, respectively, validated the clinic significance of circulating M-MDSCs on the relevance of spectral features for diagnostic stratification, induction therapy response, treatment effect maintenance, and long-term survival in AML.  We examined the frequencies of M-MDSCs identified for CD14+ monocytes  and peripheral blood mononuclear cells (PBMCs) . Elevated circulating M-MDSCs in patients with AML were significantly associated with low complete remission (CR) rate, high relapse/refractory rate, and poor long-term survival, but had no correlation with common clinic risks and cytogenetic molecular risk categories.  Circulating M-MDSC frequencies of AML were significantly higher than those of HC both in CD14 It was demonstrated that circulating M-MDSCs are elevated and associated with poor prognosis in AML, suggesting M-MDSCs might be a prognostic indicator for AML. Recently, a large number of evidences suggest that the proliferation, survival, and drug resistance of leukemic blasts are modulated by the tumor microenvironment (TME), which are composed of a variety of cells, extracellular matrix, and extracellular molecules. Especially, several individual immune cell subsets and immune molecules are also involved in the TME of AML . CD14-poM-MDSCs) –5. M-MDSM-MDSCs) . HoweverM-MDSCs) .So far, only some studies have reported that the frequencies of M-MDSCs are higher in the peripheral blood of AML than that of healthy controls (HC) , 9. ThosThis study included 109 newly diagnosed adult patients with AML and age-/sex-matched 30 healthy adult controls in the Second Hospital of Anhui Medical University between 2013 and 2019. All patients were characterized by their own diagnostic stratification and received standard induction chemotherapy followed by either consolidation therapy or allogeneic hematopoietic stem cell transplantation  in accordance with the Chinese guidelines for diagnosis and treatment of adult AML . The detμL PBMCs was incubated with monoclonal antibodies and analyzed by flow cytometry Cytomics® FC500 , and EXPO 32 Multicomp software was used for data acquisition and analysis. The following monoclonal antibodies were purchased from Beckman Coulter : FITC-labeled CD14 (clone RMO52), PE-labeled HLA-DR (clone Immu-357), and PC5-labeled CD45 (clone J.33). M-MDSCs subsets were stained and identified by the phenotype of CD14+HLA-DRlow/- in CD45++ cells. Isotype-matched controls were included in the experiments and were used to define the cutoff point for positive/negative staining.Peripheral blood mononuclear cells (PBMCs) were stratified on FicollHypaque  and separated by centrifugation for 25 min (500 g). Subsequently, PBMCs were collected and washed with phosphate-buffered saline (PBS). After washing, 100 t test, chi-squared test, and Fisher's exact probability tests were used as appropriate to evaluate the significance of the differences in data between different groups. If variances within groups were not homogeneous, a nonparametric Mann–Whitney test was used. Overall survival (OS) was used and defined as the time from date of diagnosis until the date of death. The prognostic value was evaluated by Kaplan-Meier survival curves. We used healthy adult controls as an indicator to generate the ROC curve and determined the optimal cutoff value based on the Yoden index. The cutoff values of M-MDSCs were 0.5%. More than or equal to the respective cutoff value was defined as the high-frequency group, and less than the respective cutoff value was defined as the low-frequency group. The p value less than 0.05 was considered statistically significant.All statistical analysis was performed with SPSS 16.0 . The Student's + monocytes . In p < 0.05). For follow-up analyses, a total of 62 cases that received standard treatment were included. As shown in p = 0.0440). Together, it was suggested that elevated circulating M-MDSCs at the time of new diagnosis were a prognostic indicator to predict poor outcomes in patients with AML.62 cases (57%) of 109 patients with AML received standard treatment consisting of one or two cycles of induction chemotherapy followed by either consolidation therapy or allo-HSCT; the other 47 cases (43%) only received supportive care because of their old age or poor physical condition. In p < 0.01). However, in Figures p > 0.05). Altogether, these data showed no correlation between circulating M-MDSCs and common clinic risks except for the FAB subtype.In this article, we analyzed the correlation between the frequency of circulating M-MDSCs and common clinical risks in all 109 AML cases. In p > 0.05). Altogether, these data showed no correlation between circulating M-MDSCs and common cytogenetic molecular risk categories.In the present study, 109 newly diagnosed patients with AML were enrolled. In +CD11b+ cells were suggested as defining the immunesuppressive myeloid cells which were functionally distinct from monocytes and neutrophils in mice [+HLA-DRlow/- monocytes have been grouped into a larger class of MDSCs and are commonly referred to as a special subtype of monocytic myeloid-derived suppressor cells (M-MDSCs) [In the late 1990s, Gr1 in mice , 13. The in mice . In huma in mice . So, CD1M-MDSCs) –16.In recent years, great progress has been made in understanding the roles of M-MDSCs in human malignant tumors. Studies show that M-MDSCs are closely related to the tumor progression and treatment effect of patients with solid tumors , 18. For+CD25+ regulatory T cells (Tregs) and inhibitory cytokine IL-35 is a key factor for the proliferation and apoptosis of leukemic blasts [AML is a heterogeneous hematopoietic malignancy that derives from aberrant proliferation and accumulation of leukemic blasts. Immunological disorders of several individual immune cell subsets and immune molecules have been shown to be related to the pathogenesis of AML . In our c blasts , 25.+ monocytes and PBMCs were significantly increased in the AML group. Additionally, no significant difference in the proportion of circulating M-MDSCs was found in the different age and gender groups. So, it was illustrated that circulating M-MDSCs were elevated in AML, which might complicate with a severe immune dysfunction and contribute to patients' overall immune suppression.In this study, we further determined the proportion of M-MDSCs by the analysis of flow cytometry in the peripheral circulating blood of 109 newly diagnosed adult patients with AML and 30 matched HC. Compared with the HC group, we found that circulating M-MDSCs frequencies both in CD14Regarding the clinical values of M-MDSCs, our study showed that circulating M-MDSCs frequencies were significantly higher in the poor outcome patients with AML after the first induction of chemotherapy. The patients with high circulating M-MDSCs frequencies had a significantly shorter survival within 72 months of follow-up. These data were consistent with the same results of the study in solid tumor and lymphoma as well as myeloma , 26, 27.In summary, circulating M-MDSCs were significantly elevated and closely related to the poor outcome of induction therapy in patients with AML. Circulating M-MDSCs could be used as a prognostic indicator to evaluate the survival of patients with AML. Targeting therapies for M-MDSCs might help to improve the host's antitumor immune function, which might be expected to become a new strategy for AML treatment in the future.


Toxocara canis and Anisakis simplex) and the clinical expression of urticaria and to identify possible parasitic molecular markers for improving the diagnosis of unknown urticaria aetiology. The prevalence of Toxocara and Anisakis infestations was evaluated by measuring the levels of specific IgG (sIgG) and IgE (sIgE) antibodies against crude extracts and isolated components from whole larvae of Anisakis simplex  and Toxocara canis  using immunologic and molecular diagnostic methods. A cross-sectional study was performed in a group of 400 individuals. The study group consisted of 95 patients diagnosed with urticaria (55 with chronic urticaria and 40 with acute urticaria). A control group consisted of 305 subjects without urticaria . Statistically significant differences were demonstrated in the seroprevalence of specific IgG and IgE antibodies between the urticaria patients and the healthy general population when isolated ascarid antigens were evaluated. The prevalence of IgG antibodies against Ani s 1, IgE antibodies against TES-120 and IgE antibodies against TES-70 were significantly different between the control individuals  and patients with urticaria. Moreover, the urticaria patient group demonstrated a higher seroprevalence of antibodies (sIgE and sIgG) against Anisakis simplex larva whole extract than the control group but just with statistically diferences when sIgE was evaluated. The presence of IgE and/or IgG antibodies against Ani s 3 (tropomyosin) can help to discriminate between patients with and without urticaria. Both ascarids seem to be associated with urticaria, although in our region, Anisakis seems to have greater involvement than Toxocara in this relationship. Molecular diagnostics can be used to associate urticaria with parasite infestations. Tropomyosin and Ani s 1 were the most relevant markers to demonstrate the association between urticaria and the most relevant Ascarididae parasites in our region.Urticaria remains a major problem in terms of aetiology, investigation, and management, and although parasitic diseases are considered potential causes, the absence of a consistent link between parasitic infections and skin allergy symptoms leads to the need for a deeper study of parameters that support this association. The objectives of this study were to analyse a possible relationship between parasitism by Ascarididae , through the measure of specific IgE and IgG antibodies against crude and molecular antigens of  Urticaria is a disease characterized by the development of wheals (hives), angioedema, or both. The differential diagnosis includes anaphylaxis, autoinflammatory syndromes, and hereditary angioedema. Chronic spontaneous urticaria is defined as the recurrent development of transient wheals (hives), angioedema, or both for >6 weeks due to known or unknown causes . ChronicAlthough parasitic diseases are considered a potential cause of urticaria, the absence of a consistent link between parasitic infections and skin allergic symptoms in clinical investigations stands in contrast to the fact that some parasites are the most potent inducers of Th2 responses that exist in nature ,5. ConseIn more than 90% of chronic spontaneous urticaria cases seen in routine clinical practices, the search for underlying causes is usually unsuccessful . NonetheIt is recommended to collect “fresh stool” samples from patients with unexplained eosinophilia and/or a relevant history of travel abroad, in order to test them for the presence of cysts, ova, or other parasitic forms. Serology is recommended and can play a fundamental role in those parasitic diseases that are exceedingly difficult to diagnose directly, such as toxocariasis or anisakiasis . EpidemiToxocara and Anisakis are highly prevalent [Anisakis sensitization varies widely, ranging from 22% [Anisakis larvae is very common. In addition, Kolkhir et al. and Gracia-Bara et al. [Anisakis is one of the most common parasites detected in adult patients with chronic spontaneous urticaria.In Spain, the ubiquitous ascarids belonging to the genera revalent . Spain arevalent . The prefrom 22%  to 75% [from 22% , dependia et al. ,11 foundToxocara prevalence in Spain reaches different values, ranging from 1 to more than 40% [Toxocara prevalence of 3.6% in Barcelona. Moreover, they found important differences between subjects with eosinophilia (14%) and a control group (1%) [The reported than 40% –14. Thesoup (1%) .Toxocara infections. Despite of this, data on the high seroprevalence of toxocariasis among immigrants from Latin America to Western countries  are very scarce [Factors that have been associated with an increased prevalence of toxocariasis include low socioeconomic level and poor environmental hygiene , which ay scarce –20.On the other hand, the results regarding whether parasite infections have a protective or predictive role for allergy and asthma are controversial . It seemThere are many reports that recommend including the serodiagnosis of ascarids in the standard working diagnostic methodology, mainly in those patients who present with idiopathic chronic urticaria ,24, withSince these parasitoses are silent in most cases, conducting these types of studies in a routine manner would allow a solid database based on evidence on which more efficient protocols for diagnosis could be proposed, while also highlighting the importance of establishing control programmes for these zoonoses and determining their role in the development of allergic manifestations ,14,26.Toxocara and Anisakis) might be involved in the development of urticaria and how the isolated antigenic/allergenic components of both genera could be associated with urticaria status. The application of component-resolved diagnosis could help define some diagnostic markers for urticaria caused by Ascarididae infections or exposure.Within this context, the aim of this study was to evaluate whether the two most common ascarids in Spain  and IgE (sIgE) antibodies against crude and isolated antigens/allergens using molecular diagnostic methods concretely Component-resolved diagnosis (CRD)  in compaThe Consorci Sanitari de Terrassa's Clinical Research Ethics Committee approved the study called: “Urticaria and silent parasitism by Ascaridoidea” to be carried out at the Terrassa’s Hospital. All participating subjects signed an informed consent to collect a blood sample and publish the results anonymously.A total of 2,798 adult patients (≥16 years old) who presented for the first time at the Allergy Service of Terrassa Hospital (Barcelona) during a 24-month period (from June 2009 to May 2011), were asked to participate in this study. Among them, 277 individuals who met the following inclusion criteria were enrolled.Case population: Adult subjects (≥16 years old) presenting with urticaria.Control populations: Adult subjects (≥16 years old) sensitized to common pneumoallergens and presenting with allergy symptoms, such as rhinitis and/or asthma.A group of 123 serum samples from the healthy general population  was also included. None of the subjects presented allergy or urticaria symptoms, according to a personal history questionnaire filled in before the blood draw.Clinical and epidemiological data on each subject were obtained using questionnaires designed for this purpose. The questionnaire covered age, sex, nationality, living conditions, health status, previous and current allergic manifestations, geophagia, and the presence of gastrointestinal and cutaneous symptoms, including chronic or acute urticaria, itching, and atopic dermatitis.Other related questions, such as those regarding personal hygiene, drug use, weight loss, travel history during the last 5 years, and contact with animals, were also included in the questionnaire. The questionnaire was completed by the doctor who attended to the patients at the Allergy Service and urticaria was diagnosed following the EAACI/GA2LEN/EDF/WAO Guideline .A written informed consent for diagnostic procedures and questionnaires was obtained from all patients. The study protocol was approved by the Clinical Investigation Ethics Committee of Terrassa Health Trust (Barcelona).Dermatophagoides pteronyssinus, Dermatophagoides farinae), moulds , dog and cat epithelia and pollens .All patients underwent skin prick testing  with a sA 5-mL serum aliquot was obtained from each patient for immunological and biochemical analyses. The samples were identified according to standard Spanish rules for biomedical investigations and stored at -40°C until analysis. Complete blood count, serum biochemistry tests (including the level of eosinophil cationic protein), and laboratory determination of total and specific serum IgE levels, when indicated, were performed.Salsola allergen extract is not included in the ImmunoCAP Phadiatop panel, the specific IgE antibody against this allergenic source was measured in all subjects separately with an ImmunoCAP specific IgE assay .Atopy was analysed using the ImmunoCAP Phadiatop  assay , a solidQuantification of the sIgG antibodies anti-Toxocara canis L2 excretory-secretory whole antigens (WATES). Quantification of the specific IgG antibodies against T. canis was performed by ELISA  according to the manufacturer’s instructions [T. canis L2 larvae. The cut-off value was > 0.90 mg/ml.ructions . The proQuantification of the sIgE antibodies anti-Toxocara canis L2 excretory-secretory whole antigens (WATES). Quantification of the specific IgE antibodies against T. canis was performed by FEIA . Briefly, biotinylated WATES antigens were coupled to streptavidin-activated ImmunoCAPs according to the method of Rodrigo et al. [A/L.o et al. . The FEIQuantification of the sIgG and sIgE antibodies anti-Anisakis simplex larva whole antigens. Quantification of specific IgE and IgG antibodies against A. simplex larva crude antigenic extract was carried out by FEIA  [A/L according to the manufacturer’s instructions, and for sIgG, the cut-off value was 4.3 mg/ml calculated by ROC analysis since there are no reference normal values for sIgG.ic, USA) ,33. The Quantification of sIgG and sIgE against the Anisakis simplex isolated allergens Ani s 1, Ani s 3 and Ani s 7. Quantification of specific IgG and IgE antibodies against A. simplex recombinant allergens (Ani s 1 and Ani s 7) was performed by ELISA  [, Spain) ,35,36. TA. simplex recombinant allergen Ani s 3 was performed by FEIA . The cut-off value for sIgE was > 0.10 kUA/L and for sIgG was > 4.4 mg/ml.Quantification of specific IgG and IgE antibodies against the Determination of the levels of sIgG and sIgE against the TES-120, TES-70, TES-32 and TES-26 specific antigens for diagnosis of Toxocara. Levels of sIgG and sIgE against TES-L2 antigenic extracts were analysed by immunoblotting . For determining sIgG content, the manufacture´s indications were followed. To determine sIgE content, the protocol was modified according to the standard sIgE immunoblotting method used in our laboratories [ratories . BrieflyAll immunological tests were performed blindly by the study group, with samples identified only by patient codes. The tests were developed by the same investigator at the Parasitology and Immunoallergy Laboratory of the Lascaray Research Center in Vitoria.Quantitative variables (results of specific IgE and IgG content) were requalified into qualitative variables. The values of the variables that were equal to or greater than the cut-off point were considered positive and the values lower than the cut-off were considered negative. Therefore, 2 qualitative variables were always compared.Qualitative variables are expressed as absolute frequencies and percentages, and quantitative variables are expressed as the mean ± standard deviation. Comparisons between qualitative variables were performed using the Pearson chi-square test and Fisher’s exact test with 2x2 tables. Statistical significance was accepted at p≤0.05.Anisakis-specific IgG antibodies was performed using GraphPad Prism version 7.03 for Windows. To calculate the cut-off value for the specific anti-Anisakis IgG value measured by FEIA ImmunoCAP, a receiver operating characteristic (ROC) curve was constructed.All data of interest were entered into an Excel 2003 for Windows database  by a single investigator. Statistical analysis was then performed in IBM SPSS Statistics for Windows, version 21.0 . Analysis of Anisakis IgG level measured by FEIA (ImmunoCAP) was not previously established. Therefore, in order to establish a cut-off value that would allow the calculation of the seroprevalence of this antibody class in the study population, the distribution of this value was analysed in all three groups of the study: healthy general population, patients with urticaria, and patients without urticaria.Reference normal values for the specific anti-Anisakis IgG values in these three groups. The mean values (mg/ml) and their corresponding standard deviations were 3.32 ± 1.96 in the healthy general population, 5.10 ± 10.20 in patients with urticaria, and 3.22 ± 2.30 in patients without urticaria. Statistically significant differences were found between the urticaria patient group and the control groups: subjects without urticaria (p = 0.008) and the healthy general population (p = 0.039).Anisakis whole larval antigen was obtained by ROC analysis   . The meaToxocara and Anisakis sensitization in the studied population. The analysis was conducted against the Anisakis and Toxocara larva whole antigens as well as against the isolated antigenic components Ani s1, Ani s 3 and Ani s 7 and TES-120, TES-70, TES-32 and TES-26 .Anisakis and/or Toxocara antigens) was 22.70% in the healthy general population, 39% in patients suffering from allergy without urticaria and 53.60% in patients with urticaria. The highest seroprevalence was observed in patients with acute urticaria (60%).The seroprevalence of ascarid sensitization . The seroprevalence of both antibodies in the healthy general population was very low  and slightly higher in the allergic control group .Among the urticaria patients, the highest prevalence was demonstrated for IgG and IgE antibodies against Anisakis and/or Toxocara whole or isolated antigens could be used as markers in the diagnosis of urticaria caused by Ascarididae infections/exposure. With this result in mind, a statistical analysis (Pearson chi-square and Fisher exact test) was performed, and P values were obtained among the different groups included in the study . No statistically significant differences were demonstrated between allergic patients without urticaria (control group) and the individuals with urticaria. Nonetheless, differences between allergic patients without urticaria and patients with acute urticaria were significant (p = 0.021).When both antibody isotypes (IgG and/or IgE) against the whole or isolated larval antigen from the ascarids Anisakis larval whole extract were able to discriminate urticaria patients (chronic and acute) from the subjects without urticaria . Moreover, specific IgG antibodies against Anisakis larval whole extract were able to discriminate patients with chronic urticaria from the healthy general population (p = 0.003). On the other hand, none of the antibody isotypes against Toxocara larval whole antigens were able to discriminate between the controls and the urticaria (chronic or acute) patients.When each antibody isotype was analysed regarding the antigenic source , specific IgE antibodies against Although statistically significant differences in the seroprevalence of specific IgE and IgG antibodies against Ani s 1 between urticaria patients and the healthy general population were demonstrated, only the specific IgG antibody level was able to discriminate these groups (p = 0.028). No statistically significant differences were demonstrated between the prevalence of both antibodies in urticaria patients and in the allergic patient controls.Toxocara isolated antigens explored as urticaria markers, TES-70 and TES-120 were able to discriminate between acute urticaria patients and the healthy general population via specific IgE antibody levels .Furthermore, both IgE and IgG antibodies against Ani s 3 were able to discriminate the chronic and acute urticaria patients from the healthy general population and from the allergic patient controls (p<0.001). These results, together with the seroprevalence data, make Ani s 3 a candidate diagnostic marker of urticaria of unknown origin. In the case of Ani s 7, seroprevalence data for specific IgG or specific IgE did not reveal significant differences. Neither IgG nor IgE antibodies were able to discriminate between the studied groups. Among the Several authors agree that parasite infections are relatively common in patients with urticaria, but a causal link is difficult to detect. Such infections should only be regarded as the underlying cause of urticaria when treatment with anti-parasitc drugs leads both to the eradication of the parasites and the remission of urticaria.Although routine screening for parasitic infections in patients with urticaria is not recommended, some authors ,41 suggeIn Europe, mainly in southern countries, anisakiasis  and toxoToxocara ES-L2 antigens or their recombinant components are the main tool for the diagnosis of toxocariasis [However, to date, serological assays using cariasis . Studiescariasis ,44, but cariasis –47.Toxocara L2-ES whole extract was 3% in the healthy general population and 5.50% in allergic subjects without urticaria. When specific IgE antibodies were measured, the prevalence in the control and allergic groups was 6.50% and 7.10%, respectively. No statistically significant differences were found between these groups regardless of the isotype of immunoglobulin measured. These results agree with the data obtained by other authors in studies performed in similar populations and in the same country (Spain) [In the present study, the seroprevalence of IgG antibodies against  (Spain) ,15,44. TUndoubtedly, the seroprevalence data measured by specific IgE and by specific IgG antibodies are more appropriate and more reflective of the real-world prevalence than data for a single parameter. In the literature, the prevalence of these parasitic infections, have been reported mainly through the use of only one parameter, i.e., specific IgG antibodies against toxocariasis or specific IgE antibodies against anisakiasis ,48,49.Both IgE and IgG antibodies play important roles not only as key parameters in immunological diagnoses but also as factors in several aspects of immunity, including protective strategies , and in Toxocara, the evaluation of both specific antibodies isotypes to ES-L2 whole antigens, did not show statistically significant differences among the studied groups, demonstrating similar toxocariasis low prevalence in all groups.About Toxocara, the seroprevalence of anisakiasis in Spain is quite high because is one of the European countries with the highest levels of fish consumption [Anisakis antigens ranges from 0.40 to 27.40% [Anisakis larval whole extracts is documented in the range from 11.70% to 50% and between 50% to 100% when the allergy is exclusively associated to fish or seafood [Unlike sumption ,51–55. Io 27.40% . Among o seafood .Anisakis in atopic subjects and found very similar results to those reported herein  [Anisakis seroprevalence according to specific IgE measurements in atopic patients and in patients with chronic urticaria.The results obtained in this work showed a 4% seroprevalence value for specific IgE antibodies in the healthy general population and 15% in allergic subjects without urticaria. This last group included patients with respiratory allergies, with mites the main cause of sensitization (95%). Lin et al. (2014)  and Cabactively) ,56. In Ictively)  found a ctively)  also fouAnisakis larval whole extracts were observed between the healthy general population (4%) and patients with urticaria (33.70%). The group of allergic patients also showed significant differences from the subjects with urticaria (p = 0.001).In the present study, the most significant differences in IgE antibodies against Anisakis antigens had not yet been defined. In our study, the establishment of normal values of specific IgG to Anisakis allowed to discriminate between individuals without urticaria and patients with urticaria, with special mention to the patients with the chronic urticaria. However, differences among all groups were clearly significant when the specific IgE antibodies against Anisakis larval whole extract were measured.The use of specific IgG antibodies to assess the anisakiasis seroprevalence and its possible association with other diseases, such as urticaria , Crohn’sNevertheless, the overall seropositivity, including both immunoglobulin isotypes against both ascarid whole antigens, was clearly higher in all studied groups and statistical significant differences were found between the healthy general population and patients with chronic or acute urticaria.Anisakis and in Toxocara [In recent decades, several isolated components from parasitic sources have emerged for diagnostic use. Antigens/allergens with diagnostic and prognostic value have been identified both in Toxocara ,50,60.Anisakis components are used mainly in the molecular diagnosis of the allergy to Anisakis, and Toxocara components have been used mainly as tools for toxocariasis seroprevalence studies [Several authors report that  studies ,50,60. F studies –50,60–63In these cases, specific IgG against Ani s 1, specific IgE against TES-120 and specific IgE against TES-70 were useful and effective tools to discriminate the healthy general population from patients with urticaria.A. simplex and patients with acute urticaria who are merely sensitized to A. simplex [Data on anti-Ani s 1 specific IgE from the literature are restricted to mainly healthy subjects or allergic patients, and the prevalence shown in these studies is similar to that obtained in the present investigation ,64. Mart simplex .Anisakis tropomyosin was revealed as a very potent discriminative urticaria marker. Both specific IgE and specific IgG antibodies against this component were able to discriminate groups of individuals without urticaria from patients with urticaria. Moreover, the specific anti-tropomyosin IgG antibodies were also able to discriminate between the healthy population and patients with chronic or acute urticaria while specific anti-Ani s 3 IgE was able to discriminate the healthy population and allergic population from chronic urticaria patients. These results indicate that tropomyosin could be an interesting marker of urticaria associated with ascarid infections in our region.The results obtained in this report, demonstrated that If we accept that tropomyosin-specific IgE but not IgG antibodies play an important role in the sensitization of allergic patients, the use of both parameters could be interesting to differentiate sensitization from actual parasitic infection.The results obtained in this work using diagnosis by components based on the Ani s 1, TES-120 and TES-70 antigens, strongly suggest that the molecular diagnosis is a useful tool for discriminating the healthy population from urticaria patients in our region.A. simplex in subjects exposed due to fish-eating habits [Immunological cross reactions among different ascarids and among ascarids and mites or crustaceans have been demonstrated by several authors –69. On tg habits ,70. Addig habits . ImmunolToxocara or Anisakis is highly irrelevant. Therefore, only the cross-reactive antigens/allergens shared not only with other helminths but also with mites or crustaceans should be taken into account when establishing the relationship between parasitism by ascarids and urticaria. In recent years, with the use of isolated components, the concept of cross-reactivity has been reviewed, and the concept of species-specific components and cross-reactive antigens/allergens is becoming more accurate and much better delimited [In our area, polyparasitism by different nematodes or the incidence of nematodes other than elimited ,71,72.Anisakis demonstrated that Ani s 1 is the immunodominant specific allergen, and the quantification of IgE specific to Ani s 1 is able to discriminate between asymptomatic and allergic patients [Investigations of Spanish and Italian patients allergic to patients ,63. Sevepatients ,74. Yamapatients .Anisakis results using mainly specific immunodominant components through IgE quantification.Although cross-reactivity can be a limitation to establish the association between the studied ascarids and urticaria, mainly when larval crude extracts are used, the differences in prevalence among the urticaria patients and the reference groups found in this study are relevant enough to alleviate this limitation. This point is even more evident if we take into account the Anisakis and urticaria. Cross-reactions with mite allergens or shellfish allergens could also be a limitation to explain this association. The results obtained in this work show that the prevalence of tropomyosin for the allergy patients was significant lower than the obtained for the urticaria group. The differences were very significant and more so if we consider that 40% of the allergic patients were sensitized to house dust mites. A similar concept could be applied to the limitations due to shellfish allergy. Nwaru and colleagues reported a very low prevalence of shellfish allergy in Europe, with values from 0.06% and 0.10% [Surprisingly, in our country, the panallergen tropomyosin is the component that is most significant for studying the association between ascarids, represented mainly by nd 0.10% .Toxocara and Anisakis seem to be involved in the association between parasitism and urticaria, in our region, the genus Anisakis appears to be the most important member of this taxonomic group regarding this association. The prevalence of anisakiasis was higher than that of toxocariasis, as was expected, as dietary habits determine the high prevalence of anisakiasis in this population.Although both Anisakis larva crude extract, Ani s 1 and tropomyosin could be considered markers for parasite-caused urticaria. The low prevalence of Toxocara found in this study and its significance regarding the healthy general population and urticaria patients make this species a smaller factor, but it should not be excluded as a possible cause of urticaria.Despite the apparent limitations to establish the ascarid-urticaria association due to mite and shellfish sensitization or other helminth infections in our region, the results obtained in this work, where helminth poly-parasitism is very scarce, strongly suggest that specific IgE and IgG antibodies against The use of different antibodies isotypes and the component resolved diagnosis concept are useful to establish the association between parasite immunologic results and patients with different clinic status as allergy, acute urticaria and chronic urticaria.S1 Checklist(DOCX)Click here for additional data file.


In recent years, genetics, physiology, and structural biology have advanced into the molecular details of the sensory physiology of auditory hair cells. Inner hair cells (IHCs) and outer hair cells (OHCs) mediate two key functions: active amplification and non-linear compression of cochlear vibrations by OHCs and sound encoding by IHCs at their afferent synapses with the spiral ganglion neurons. OHCs and IHCs share some molecular physiology, e.g. mechanotransduction at the apical hair bundles, ribbon-type presynaptic active zones, and ionic conductances in the basolateral membrane. Unique features enabling their specific function include prestin-based electromotility of OHCs and indefatigable transmitter release at the highest known rates by ribbon-type IHC active zones. Despite their compact morphology, the molecular machineries that either generate electrical signals or are driven by these signals are essentially all segregated into local subcellular structures. This review provides a brief account on recent insights into the molecular physiology of cochlear hair cells with a specific focus on organization into membrane domains. The organ of Corti inside the cochlea forms the sensory organ of our sense of hearing. It houses two types of mechanosensory hair cells: three rows of outer hair cells  and one row of inner hair cells  span the entire length of the basilar membrane. Sound-induced vibrations of the tympanic membrane from approximately one Å are sufficient to elicit hair cell responses while vibrations of hundreds of nm are required to saturate that response. The ensuing receptor potential (RP) drives their key output function: active micromechanics (OHCs) and synaptic sound encoding (IHCs). OHCs and IHCs are highly specialized for operating at the fast pace inherent to auditory signaling. This applies to both the unique motor process of OHCs and the temporal bandwidth of signal transmission at the specialized ribbon synapses in IHCs. A sophisticated set of ion channels enables operation of these processes in the kHz range (OHCs) and hundreds of Hz range (IHCs). Both show afferent and efferent innervation but to different extents and serving different functions. Efferent control by medial olivocochlear projections is more prominent at OHCs, where an unconventional cholinergic mechanism inhibits electromotility (eM). Afferent synaptic transmission of IHCs to myelinated type I spiral ganglion neurons  mediates cochlear sound encoding, while OHCs do not seem to contribute to sound encoding but instead signal to unmyelinated type II SGNs, likely to report cochlear damage. Once dysfunctional or lost, OHCs and IHCs cannot be replaced and sensory hearing loss results.1, indications for which had previously also been reported for hair cells of the avian auditory papilla2. OHCs do not seem to engage in such direct coupling1 but interact mechanically3. More generally, neighboring hair cells face a common mechanical input resulting from the sound-driven traveling wave. At a subcellular level, it has become increasingly clear that essentially all of the various membrane specializations that generate and shape their electrical signals (i.e. RPs) on the one hand and the effectors that drive the cell’s output in response to the RP on the other hand are assembled into spatially confined membrane nanodomains rather than evenly distributed across the isoelectric membrane.Mammalian hair cells are small and compact cells and therefore are also understood to be electrically compact, i.e. isoelectric. Nevertheless, recently, it has been demonstrated that IHCs of the rodent cochlea can undergo macromolecular and low-resistance electrical coupling to form functional mini-syncytia4. Equally important, basolateral channels, including low-voltage activated Ca2+ channels, are essential for shaping the RP in terms of both amplitude dynamics and timing.The higher-level principle governing the subcellular organization of hair cells is their nature as epithelial cells, imposing structural and functional polarization of the cell surface into apical and basolateral membranes. In all hair cells, the mechanoelectrical transduction (MET) machinery that provides the stimulus-modulated transducer conductance is found at the apical pole. Opposing the depolarizing apical MET conductance, basolateral potassium conductances establish the hair cell’s negative resting potential, whereby the balance between apical and basolateral conductances defines its actual value estimated as –40 and –55 mV in OHCs and IHCs, respectivelyFinally, the effectors of the RP are also built into the basolateral membrane region. This is where functional divergence between IHCs and OHCs is fundamental: in the basolateral membrane of the IHCs, ribbon-type active zones (AZs) transmit temporal and amplitude information contained in the RP with unprecedented precision to the postsynaptic auditory neurons. In OHCs, in contrast, the RP primarily drives ultrafast cell length changes, termed eM, by a machinery that occupies large parts of the basolateral membrane domain.2+ or to avoid cross-talk of intracellular signals, in other cases the underlying subcellular physiology remains unknown or speculative. Here we attempt to describe recent progress in understanding the subcellular compartmentalization of membrane physiology in cochlear hair cells, the molecular composition of identified domains, and their functional relevance.In recent years, the nanoscale organization of several functional modules and their molecular constituents (and their interactions) has been resolved. While in some cases the physiological meaning of compartmentalization is well understood, e.g. to provide nanoscale coupling by the diffusible messenger CaA supramolecular MET complex is located at the tips of all but the tallest row of stereocilia, thus defining a highly specialized and spatially extremely confined domain  that als6, lipoma HMGIC fusion partner-like 5 [LHFPL5]8, transmembrane inner ear [TMIE]10, calcium‐ and integrin‐binding protein 2 [CIB2]11, and protocadherin 15 [PCDH15]12) and membrane lipids such as phosphatidylinositol-4,5-bisphosphate (PIP2)14 (17.Recent data show that the auditory MET complex involves intricate interactions among a set of proteins (i.e. transmembrane channel-like protein isoform-1/2 [TMC1/2](PIP2)14 . Adding 26. While TMC2 is only transiently expressed in auditory hair cells during early postnatal development, TMC1 expression rises gradually and is essential for adult auditory MET25. Evidence for TMC proteins forming the MET pore comes, among others, from chemical modification of introduced cysteine residues, which altered ion selectivity of the MET currents27. Homology modeling of TMC structure with TMEM16 predicts two separate pores in a dimeric TMC complex, located at the margin of each subunit25. Interestingly, this model would see the pore “open” to the surrounding lipid bilayer laterally, rather than fully surrounded by protein. However, the TMC architecture might be complemented by TMIE, closing the pore towards the lipid bilayer10. Direct evidence that TMC1/2 proteins can form mechanosensitive ion channels was recently shown when orthologues of human TMC1/2 were successfully purified and reconstituted in liposomes and their mechanosensitivity demonstrated26. This study used CmTMC1  and MuTMC2 , which possess 72% and 66% sequence identity to human TMCs, respectively. Verification of mechano-gating for mammalian TMC1 as well as further evidence in support of the current model of TMC1 dimers with two independent pores25 will require future work. Of note, while quasi-simultaneous28 opening of both pores seems possible, their simultaneous closure is harder to envision. A temporally linked but separated closing of two pores, consistent with stochastic single pore properties, has not been observed directly in previous single channel recordings30.TMC1/2 proteins constitute a core component of the MET complex and are now thought to contribute to its pore-forming subunits2+-driven process33 that modulates the MET channel’s resting open probability and thus its dynamic current/stimulus relationship36. Ca2+ was thought to interact either with the MET channel itself38 or act on myosin motors40 that slip or climb along the stereocilia actin core, thus controlling tip link tension. However, data showed that MET channels are present only at the tip of stereocilia and the presumed myosin “adaptation” motors at the upper tip link insertion point, making a modulation of those motors by Ca2+ entering through the MET channel unlikely.Traditionally and primarily based on work in turtle and bullfrog hair cells, adaptation of the MET current was thought to be an exclusively Ca43, recently, three different mechanisms controlling the MET channel resting open probability were proposed: i) a resting tension-generating, myosin-based process45  an effect of extracellular Ca2+ that interacts with the lipid bilayer and modulates membrane stiffness44.After some controversial data on the matterrocess45 , ii) a f2+-dependent and Ca2+-independent models for adaptation came from comparison of the different stimulus methods used in previous studies. Where a fluid jet stimulates the hair bundle with a steady force (force clamp), a stiff probe stimulates the hair bundle with a constant displacement (displacement clamp). These two stimulation paradigms can either obscure or reveal Ca2+-dependent and -independent processes46. These data also suggested that the “natural” stimuli detected by IHCs and OHCs are different: the free-standing IHCs are driven by force (as with the fluid jet), while the OHCs are stimulated by displacement prescribed by the overlying tectorial membrane, which OHC hair bundles are connected to (as with the stiff probe)42.Reconciliation of Ca2+ ions, the lipid bilayer, its organization, and its nanodomain properties are most certainly important for normal MET channel function. Close functional interaction of ion channel complexes with their lipid environment is a well-established concept60 . Such speed is the underlying assumption for many models of cochlear amplification, where eM amplifies cochlear vibration in a cycle-by-cycle manner. However, a series of recent detailed analyses of prestin and eM kinetics challenged this view, reporting lowpass behavior with corner frequencies at around 20 kHz, well below the upper frequency limit of hearing in many mammals, which obviously urges a reconsideration of the mechanism of cochlear amplification73.In the OHC, the RP drives eM, which is mediated by prestin SLC26A5, , a membe74. This model was soon confirmed by the first experimental crystallographic structure of a bacterial SLC26 homolog75. Subsequent structural and biochemical analysis revealed that SLC26 proteins, including prestin, are dimers76. Finally, the first experimental structure of a eukaryotic homolog, mouse SLC26A9, recently uncovered additional details, particularly on substrate binding and unexpected involvement of the conserved intracellular C-terminus (STAS domain) in the dimerization of SLC26 proteins77. Together, all of these structural findings suggest that SLC26 transporters may act by an elevator mechanism, in which a mobile domain (core or transport domain) binds the substrate and delivers it to the other side of the membrane by a rotational–translational movement, with the other main domain (gate or scaffold domain) acting as a static scaffold. It will be exciting to see if the eM mechanism of prestin can also be understood in the framework of such an elevator mechanism.Recent advances at the structural level are beginning to shed light on the molecular mechanisms underlying eM. Combined homology modeling, molecular dynamics simulations, and functional analysis clarified the membrane architecture and domain organization of the prestin protomer, showing a 7+7 inverted repeat architecture, and organization into two major opposing domains, tentatively termed gate and core domains78, whereas the basal area of the membrane is largely devoid of prestin . The major OHC current is known as IK,n for its unusually negative voltage range of activation, resulting in constitutive activity at physiological membrane voltages. The underlying K+ channels contain the pore-forming KCNQ4 subunit (Kv7.4)86. KCNQ4 proteins localize to the basal cap of the OHC86. This localization appears straightforward in avoiding interference with the high prestin density required in the lateral domain. Moreover, a recent report suggests that clustering of KCNQ4 may enforce cooperativity that alters functional properties87. In particular, clustering was shown to speed up gating kinetics and contribute to shifting the voltage range of activation, which in OHCs is much more negative than of heterologous KCNQ4 channels, a puzzle so far unexplained.2+-activated K+ channels. These three channel types constitute the postsynaptic ion channel complement of cholinergic olivocochlear inhibitory synapses and mediate unusual inhibitory postsynaptic potentials by an indirect ionotropic mechanism: the hair cell’s ionotropic α9/α10 cholinergic receptors are highly Ca2+-permeable channels88. Ca2+ influx rapidly activates colocalized Ca2+-activated K+ channels, resulting in net outward current and hence hyperpolarization. For a long time, it has been known that small-conductance SK2 channels mediate the outward current, and accordingly their localization is confined to the subsynaptic membrane, outlining this functional domain90  is well understood. Large-conductance BK channels underlie IK,f97, and KCNQ4 (Kv7.4) channels mediate IK,n98. However, the channel(s) underlying the slowly activating K+ current IK,s remained unknown. Dierich et al.95 combined expression data with pharmacology and biophysical characterization to dissect IK,s and found that it is mediated by Kv1.8, Kv11.1, and Kv12.1 channels. Modeling IHC RPs indicated that the complex K+ channel complement helps to ensure temporally precise signal encoding over a wide dynamic range95.97 , too far away to supply BK clusters with Ca2+ signals. Two papers now reveal a molecular mechanism that elegantly reconciles both issues: IHCs express LRRC52 as a BK-interacting protein101. LRRC52 acts as a gating modifier, strongly shifting the voltage sensitivity into a range that matches the hair cell’s physiological membrane potential even in the absence of elevated Ca2+101, a property previously determined for BK channels in IHCs102. Moreover, LRRC52 is responsible for clustering BK at the cell’s neck region, where basolateral Ca2+ signaling is out of reach101.Under the assumption that the IHC is electrically compact, there is no obvious reason for spatial segregation of these channels, and an electrical point model was sufficient to produce all the properties of recorded RPs. Nevertheless, BK distribution is highly clustered into patches close to the neck region of IHCs97 . This is+ channel populations in the same cell? Lower-resolution immunohistochemistry is consistent with predominant localization of KCNQ4 to the upper part of the lateral membrane98. Similarly, Kv11.1 immunoreactivity is mainly found in the upper half of the lateral membrane95.What about localization of the other K+ channel entities into the upper region of the IHC remains elusive; a possible rationale may be to keep large K+ outward fluxes away from the synapses, where they could depolarize the postsynaptic endings, interfering with precise signal encoding103.The physiological meaning of molecular sequestration of BK into distinct clusters and together with the other K109. Hallmarks of the hair cell synapses are i) electron-dense synaptic ribbons, which provide AZ scaffolding, supply synaptic vesicles (SVs) and require the core component RIBEYE111 and the anchoring scaffold bassoon115, ii) exocytosis at high rates and with utmost temporal precision that is sustained over long periods of time by efficient endocytic SV recycling and SV replenishment to the large readily releasable pool of SVs124, and iii) heterogeneity of presynaptic structure and function that likely enables IHCs to decompose sound intensity information into different neural channels129.During the last few years, substantial progress has been made toward the understanding of hair cell synaptic transmitter release, and much has been covered in elaborate reviews108). For example, the CaV1.3-based132 presynaptic Ca2+ channel complex is active already at the IHC resting potential at the subset of synapses that likely encode soft sounds and inactivates fairly little to reliable signal sound intensity. Beyond splicing of the pore-forming CaV1.3α subunit133, choice of the auxiliary CaVβ and CaVα2δ subunits136, interacting proteins such as Ca2+-binding proteins138, harmonin139, RIMs140, and RIM-BPs120 shape the precise biophysical properties of the channels at a given AZ 127 of transmitter release of the basolateral IHC membrane, while estimates of the extrasynaptic channel density are much lower (<10 channels per µm2 in mature IHCs)141. This compartmentalization of Ca2+ influx requires the action of multidomain proteins of the AZ: their deletion generally reduces the number of Ca2+ channels available for triggering SV release121 and can also alter the Ca2+ channel topography127. In the case of bassoon deletion, Ca2+ channels do not properly cluster127. Interestingly, even deletion of RIBEYE, not suspected to directly interact with Ca2+ channels, altered the topography and function of the Ca2+ channels110. The multidomain proteins of the AZ and the “superscaffolding” synaptic ribbon are likely to stabilize the Ca2+ channel cluster that faces massive membrane turnover due to exocytosis and endocytosis at the AZ and periactive zone membrane domains, respectively. So far, estimates of Ca2+ channel mobility have not been obtained for hair cell AZs, but studies in cultured neurons imply that it might be substantial142.CaV1.3-based Ca2+ channels of hair cells have been assessed by cell-attached recordings149 as well as by non-stationary variance analysis151. There is consensus on the average total number of activatable Ca2+ channels per AZ (100) but likely method-related partial discrepancies for estimates of open probability and single channel current. Interestingly, evidence for cooperative gating, revealed for heterologously expressed CaV1.3 channels152, has not been reported thus far. Recently, the presynaptic complement Ca2+ channel was revisited using Ca2+ imaging154, and a range of 20–300 channels per AZ was estimated127. Such massive differences in Ca2+ channel clustering are likely to impact their biophysical properties and the coupling of Ca2+ influx to SV release. Indeed, recently, it was shown that AZs vary in the control of exocytosis by Ca2+ influx155. However, the expectation that membrane nanodomains with a high density of Ca2+ channels might present an active local depolarization exceeding the overall hair cell potential seems not to be supported: the voltage dependence of activation does not correlate positively with the maximal Ca2+ influx126. A negative feedback to presynaptic Ca2+ influx is mediated by the release of protons during SV exocytosis that partially inhibit the Ca2+ channels under physiological pH buffering156, and a potential modulation of the IHC Ca2+ channel by lipids158 remains to be elucidated. Despite the heterogeneity of the control of exocytosis by Ca2+ influx among hair cell AZs, the voltage dependence of Ca2+ influx generally predicts that of SV release155. Site-specific trafficking of components of the Ca2+ channel complex might involve adapter proteins such as Gipc3126, and differences in the capacity of tethering multidomain proteins available at the AZ121 likely contribute to the heterogeneous Ca2+ channel complement. Recently, several candidate mechanisms for setting up AZ heterogeneity have been proposed: planar cell polarity signaling159, instruction by postsynaptic neurons160, and efferent olivocochlear signaling161. Finally, from the perspective of sound coding in the auditory system, it will be of utmost importance to elucidate how properties of the presynaptic AZs relate to the functional166 and molecular167 diversity of SGNs.Single channel properties of CaIn summary, the auditory hair cells provide an impressive model system for compartmentalization of electrical signaling in compact cells. Already the mechanosensory hair bundle shows different compartments and sub-domains which are essential for the reception and processing of mechanical stimuli. This is likely true for the largest (5 µm) and smallest (<500 nm) stereocilia. Although our technological means are insufficient, as of yet, to directly investigate the interplay of single lipid molecules or rafts with different proteins, pharmacological and genetic manipulations show a close interaction between the cell membrane and proteins, where alterations in either can cause deafness. Understanding these interactions not only at the tip region (MET-complex) but also along the shaft and in the ankles of stereocilia will be a crucial and painstaking endeavor.2+ sources and Ca2+-driven effectors provide the obvious rationale for tight clustering of their components into precisely defined domains. Furthermore, this example also shows how the cellular domain architecture translates into functional separation of events even in a compact cell. Thus, in prehearing IHCs, transient efferent innervation results in interspersed afferent and efferent synapses, both driven by Ca2+ signals. Although the distances between both structures are in the low μm range, Ca2+ signals produced at efferent sites fail to elicit vesicular release at nearby afferent presynapses, demonstrating that the functional architecture associated with each domain and the efficient cellular Ca2+ handling ensure functional isolation168.For some functional units, particularly afferent and efferent synapses, co-localization of Ca2+ channels is established, potentially by varying the topography of release sites and Ca2+ channels or simply by domain overlap in the case of large Ca2+ channel clusters. The fascinating question of how hair cells establish subcellular heterogeneity among their Ca2+ channel clusters and, more generally, the membrane nanodomains of the AZs remains to be elucidated.Moreover, detailed studies of the molecular components of the IHC ribbon synapse showcase how subcellular membrane domains are assembled. Nevertheless, it will be an important task for the future to elucidate how differential control of SV release by CaA similarly stringent analysis of the makeup of the other basolateral domains, in particular the OHC lateral membrane, is still outstanding and promises to reveal important insights into the micro- and nano-mechanics of electromotility.170 and promise to provide insights into the role of lipids in hair cell function.Finally, in addition to the sophisticated assemblies of membrane proteins and membrane-associated proteins, local differences in lipid composition across the various domains are likely to contribute to their identity and functionality. Tools for tracking and manipulating individual lipid species in living cells have been developed in recent years


SOCE is unusual in requiring interaction between proteins in different membranes. STIM1, when it senses loss of ER Ca2+, unfurls domains that interact with Orai1 PM Ca2+ channels. The stoichiometry of the SOCE complex is contentious, but it determines the regulation and functional consequences of SOCE. We show that native complexes are likely to comprise a single Orai1 channel and a few STIM1 dimers, too few to cluster Orai1 channels. We suggest that SOCE may be digitally regulated by local ER depletion, and that local SOCE-evoked Ca2+ fluxes are small enough to allow substantial intracellular redistribution of Ca2+ through ER tunnels.Store-operated Ca 2+ concentration regulate diverse cellular activities and are usually evoked by opening of Ca2+ channels in intracellular Ca2+ stores and the plasma membrane (PM). For the many signals that evoke formation of inositol 1,4,5-trisphosphate (IP3), IP3 receptors coordinate the contributions of these two Ca2+ sources by mediating Ca2+ release from the endoplasmic reticulum (ER). Loss of Ca2+ from the ER then activates store-operated Ca2+ entry (SOCE) by causing dimers of STIM1 to cluster and unfurl cytosolic domains that interact with the PM Ca2+ channel, Orai1, causing its pore to open. The relative concentrations of STIM1 and Orai1 are important, but most analyses of their interactions use overexpressed proteins that perturb the stoichiometry. We tagged endogenous STIM1 with EGFP using CRISPR/Cas9. SOCE evoked by loss of ER Ca2+ was unaffected by the tag. Step-photobleaching analysis of cells with empty Ca2+ stores revealed an average of 14.5 STIM1 molecules within each sub-PM punctum. The fluorescence intensity distributions of immunostained Orai1 puncta were minimally affected by store depletion, and similar for Orai1 colocalized with STIM1 puncta or remote from them. We conclude that each native SOCE complex is likely to include only a few STIM1 dimers associated with a single Orai1 channel. Our results, demonstrating that STIM1 does not assemble clusters of interacting Orai channels, suggest mechanisms for digital regulation of SOCE by local depletion of the ER.Increases in cytosolic Ca In animal cells, the many receptors that stimulate formation of inositol 1,4,5-trisphosphate (IP3) provide coordinated access to both Ca2+ sources (3 stimulates the opening of IP3 receptors (IP3R), which are large Ca2+-permeable channels expressed mostly within ER membranes. IP3 thereby triggers Ca2+ release from the ER (2+ is provided by store-operated Ca2+ entry (SOCE), which is activated by loss of Ca2+ from the ER. The reduction in ER free-Ca2+ concentration causes Ca2+ to dissociate from the luminal Ca2+-binding sites of stromal interaction molecule 1 (STIM1), a dimeric protein embedded in ER membranes. This loss of Ca2+ causes STIM1 to unfurl cytosolic domains that interact with the PM Ca2+ channel, Orai1, causing its pore to open and Ca2+ to flow into the cell through the SOCE pathway (a), or that each dimer interacts with only a single C-terminal tail leaving the remaining STIM1 subunit free to cross-link with a different Orai1 channel (12 STIM1 molecules around a single Orai1 channel) (b) (see references in ref. c) and to explain the variable stoichiometry of Orai1 to STIM1 at MCS ., 5.2+ si pathway –10. The  pathway –13. Orai pathway A. Compet pathway B, a, or channel) B, b . In-gel fluorescence, Western blotting with an anti-GFP antibody, and knockdown of STIM1 expression using short hairpin RNA (shRNA) confirmed that STIM1-EGFP was the only EGFP-tagged protein expressed in the STIM1-EGFP HeLa cells . Western blot (WB) analyses revealed that 52 ± 8% of the expressed STIM1 was tagged with EGFP , and with our HeLa cells having two copies of chromosome 11 on which the STIM1 gene is located . The Ca2+ signals evoked in Ca2+-free Hepes-buffered saline (HBS) by cyclopiazonic acid (CPA), which reversibly inhibits the ER Ca2+ pump and thereby unmasks an underlying Ca2+ leak , were also unaffected by the gene editing , reflecting the activity of SOCE. The amplitude of this SOCE, whether evoked by maximal or submaximal depletion of Ca2+ stores, was indistinguishable in the two cell lines . The unchanged response to submaximal stimulation of SOCE in the edited cells indicates that STIM1-EGFP is functional. The amplitude of the sustained increase in [Ca2+]c evoked by histamine, which probably reports the activity of SOCE, was also indistinguishable in the two cell lines, though smaller than the SOCE evoked by CPA . These results, demonstrating that SOCE is normal in STIM1-EGFP HeLa cells, justify use of these cells for optical analyses of STIM1 redistribution after store depletion.We used CRISPR/Cas9 to attach EGFP to endogenous STIM1 in HeLa cells C and conith EGFP G and H.  located . The ove located H, but thgh IP3Rs , was ind editing J. This o2+-free HBS to irreversibly inhibit the ER Ca2+ pump caused accumulation of STIM1-EGFP puncta within the TIRF field. The accumulation was 50% complete by ∼2 min, complete within ∼4 min, and stable thereafter for at least 15 min  . The accumulation of STIM1-EGFP beneath the PM was due to increases in both the number of puncta and their average fluorescence intensity . In step-photobleaching analyses using TIRFM, the amplitude of the final bleaching event was indistinguishable for STIM1-EGFP puncta in stimulated and unstimulated cells , suggesting that the puncta were all similarly located in the TIRF field. The brighter puncta in cells with empty stores must therefore contain more STIM1-EGFP molecules. These results confirm that loss of Ca2+ from the ER causes STIM1-EGFP to form puncta beneath the PM that are both larger and more abundant than in cells with replete Ca2+ stores.We used total internal reflection fluorescence microscopy (TIRFM) to interrogate cytoplasm immediately beneath the PM and showed that STIM1-EGFP formed puncta within the ER A. Additit 15 min B–D. The E and F) , 17, alt2+ stores and activate SOCE . We also confirmed that the conditions used to identify cells before photobleaching caused negligible prebleaching . In cells with empty Ca2+ stores, the average number of fluorophores per punctum was 5.82 ± 0.31 . Not all puncta resolve the final bleaching step required to estimate the fluorophore content. However, the fluorescence intensity distributions and mean intensities of all puncta and those amenable to step-photobleaching analysis  were indistinguishable . This confirms that our bleaching analysis was unbiased. EGFP tags are detected as fluorescence with ∼80% efficiency .The fixation required for step-photobleaching analyses has been reported to empty Caate SOCE , 19, butficiency , suggestSI Appendix, Fig. S2), and it is confirmed by additional evidence using STIM1 immunostaining to compare the distribution of all STIM1 with that of STIM1-EGFP. The results demonstrate that puncta identified by the STIM1 antibody, which recognizes native and tagged STIM1 , were also labeled with EGFP, and that for each punctum the intensities of EGFP and antibody fluorescence were tightly correlated . Finally, we expressed mCh-Orai1 in STIM1-EGFP HeLa cells and, although the results were variable, we confirmed that in cells with empty Ca2+ stores, STIM1 and STIM1-EGFP were coimmunoprecipitated with mCh-Orai1 and in the same ratio as their expression in the cell lysates . In store-depleted cells, most puncta (99.5%) contain at least 6 STIM1 molecules, but only 15.6% contain more than 20 STIM1 and only 2.2% have more than 30 . Nevertheless, our results suggest a significant colocalization STIM1 puncta with mCh-MAPPER in control and thapsigargin-treated STIM1-EGFP HeLa cells. Under both conditions, ∼50% of STIM1 puncta colocalize with mCh-MAPPER . There are, of course, more STIM1 puncta near the PM of cells with empty Ca2+ stores .MAPPER is a fluorescent MCS reporter derived from STIM1 . We used+ stores D and so 2+ stores . We conclude that store depletion causes only a modest (∼50%) increase in the average number of STIM1 molecules within a punctum (from ∼10 to ∼15). The conclusion is substantiated by analyses of WT HeLa cells with immunostained STIM1 puncta, where store depletion caused their average fluorescence intensity to increase by ∼55% . Hence, most native puncta in cells with empty stores contain enough STIM1 to activate no more than two Orai1 channels if each channel binds three STIM1 dimers (a), and only one Orai1 channel if each subunit binds its own STIM1 dimer (b).The mean fluorescence intensity of puncta after store depletion is 138% of that in cells with full Ca+ stores G, sugges1 dimers B, a, andM1 dimer B, b.2+ channels associated with a STIM1 punctum using TIRFM after immunostaining for Orai1 before and after store depletion. We reasoned that the distribution of fluorescence intensities for each immunolabeled spot before store depletion likely reports antibody binding to a single hexameric Orai1 channel, while that determined for Orai1 colocalized with STIM1 after store depletion reports the number of Orai1 channels associated with a SOCE complex. We confirmed both the specificity of the antibody (Ab) by demonstrating loss of immunostaining after treatment with siRNA to Orai1, and the ability of the Ab to resolve increases in the number of Orai1 within an immunolabeled spot .We estimated the number of Orai1 Ca2+ stores , but a significant fraction of Orai1 (14.5 ± 0.4%) colocalized with STIM1 in cells with empty stores  . After store depletion, the fraction of STIM1 puncta colocalized with both MAPPER and Orai1 significantly increased   or empty Ca, n = 6) G. We sug, n = 6) H.2+ stores, each SOCE complex typically comprises about seven STIM1 dimers and a single Orai1 channel. Whether dimeric STIM1 binds to one or two subunits of a hexameric Orai1 channel is unresolved , consistent with electrophysiological evidence indicating a lack of Ca2+-mediated interactions between native Orai1 channels (2+-release-activated channel) currents are unusually large, comparison of whole-cell SOCE currents with the number of ER-PM MCS suggests about five open Orai1 channels per MCS  is comparable to the number of STIM1 puncta in store-depleted cells (∼400/cell), whether identified by immunostaining  or as STIM1-EGFP . There are considerably more Orai1 channels .In HeLa cells, the number of MCS identified by MAPPER in the TIRF field , although as discussed we may underestimate the number of MCS in store-depleted cells . These observations suggest the presence of ∼100 MCS populated by both STIM1 and Orai1 in the TIRF field, suggesting ∼200 active junctions, each with a single active Orai1 channel, in a maximally activated HeLa cell. This is sufficient to account for the increase in [Ca2+]c evoked by store depletion . The modest rate of Ca2+ flux into each SOCE junction through a single Orai1 channel    , may nevffectors .b). We speculate that assembly of native STIM1 puncta around Orai1 after store depletion is driven largely by recruitment of STIM1 dimers already resident within the MCS, allowing each MCS to function as an autonomous digital regulator of SOCE (The increase in size of STIM1 puncta after store depletion is modest, from about five to about seven dimers G, a chan of SOCE  Fig. 4HHb. We spSTIM1 gene in HeLa cells. Appropriate tagging was verified using WB, in-gel fluorescence, and sequencing of genomic DNA. Immunocytochemistry, immunoprecipitation, and measurements of changes in [Ca2+]c evoked by SOCE were used to confirm that the EGFP tag did not perturb STIM1 function. TIRFM and step-photobleaching analyses were used to establish numbers of STIM1 molecules within the puncta formed after depletion of intracellular Ca2+ stores. Immunostaining with a validated antibody was used to examine the composition of Orai1 puncta. Details of the methods are provided in SI Appendix.CRISPR/Cas9 was used to attach EGFP to a single copy of the Supplementary File


Today, major efforts are devoted toward optimizing red-emitting [red fluorescent protein (RFP)-based] GECIs (R-GECI), as these provide several advantages over GFP-based reporters, for instance, increased imaging depth, reduced photodamage by longer imaging wavelengths and, in principle, are better suited for use with prevalent blue-absorbing optogenetic tools . However, excessive fluorescence from intersecting neighboring cells in very dense tissues, notably the brain, hinders the ability to collect signals from single cells and their processes. This challenge can be addressed by photoactivatable (PA) fluorescent proteins that can be rendered fluorescent on demand by user-defined targeted light. This allows activation and, thereby, collection of fluorescent signals exclusively from desired cells and their processes, while leaving all neighboring cells in the dark . Nevertheless, there are no PA R-GECIs. Here, we sought to develop PA-R-GECIs. To do so, we initially explored a recently discovered phenomenon of Ca2+-independent increases in fluorescence  in an emerging R-GECI, which has led us to rationally engineer several functional PA-R-GECIs. We also take advantage of our findings to quickly engineer a novel PA-RFP, namely, PA-mRuby3.Emerging genetically-encoded Ca Whereas other methods can address some of these challenges by sparsely labeling single neurons , wide dynamic-range and is suitable for two-photon imaging, to name a few (2+-independent—increases in fluorescence (here denoted artifacts) when illuminated with near-UV-to-green light  that would be amenable to rational mutagenesis so as to convert the transient photoconversion into stable photoactivation. We first examined the sequences of various PA-FPs, explicitly PA-GFP, PA-mCherry, and PA-TagRFP , in addiWe therefore deemed it worthy to focus on this residue in R-GECO1  . Importantly, whereas PA-GFP required two additional mutations for photoactivation, specifically phenylalanine preceding the chromophore and a serine within it  could be detected when imaged at 561 nm , likely due to very different expression levels in different HEK293 cells, rather than resting Ca2+-levels . Importantly, we deem visible basal fluorescence as a highly useful feature for detecting cells that express the clone prior photoactivation. This eliminates the need to introduce additional non-PA optical markers. In fact, basal fluorescence is so low in PA-GFP, and also in PA-GCaMP, therefore requiring imaging with low intensity 405 nm  for spotting positive cells; to then target for photoactivation , as we could not determine a priori which mutation would endow the best performances in photoactivation and in Ca2+-responsivity. Though most of the variants did not express at all , despite the residue’s high degree of homology to isoleucine as is originally found in R-GECO1wt, lost its direct 405 nm-induced red emission (-I78L) failed to respond to Ca2+  and expressed it in HeLa cells. Notably, we did not expect this clone to display any photoactivation based on observations with PA-GFP in which T203H enables photoactivation, whereas H203T cancels it. Counterintuitively, R-GECO1T exhibited the largest extent of photoactivation of all clones tested, thus far, in HeLa cells  by short bouts of 405 nm illumination , as well as in media with or without Ca2+ . This translated into much higher degrees of photoactivation. More specifically, both standard- and Ca2+-free media enabled cells to undergo ∼8  and ∼7 fold  increases in fluorescence , even to lower extents than seen in HeLa cells  when illuminated with 561 nm  in HeLa cells (though to lower extent than R-GECO1T), but with significantly larger Ca2+-responses (ΔF/F = 23.80 ± 2.83)  . It alsotivation  and 561-tion   . Neverth ± 0.13) . We brie ± 0.13) . RCaMP1h ± 0.13) , whereas ± 0.13) .T exhibited the largest extent of photoactivation with contrast as high as ∼100 fold in HEK293 cells   reduces the effective range for photoactivation, while maintaining the same total fluorescence .We began engineering H115T mutants because, so far, substitution to a threonine yielded best performances. We also made a H115I substitution because we were curious to see the potential involvement of this in the transient photoactivation seen in all R-GECOs, including RCaMP1h. Surprisingly, all six clones exhibited robust and stable photoactivation; much larger than most of the responses obtained for the different R-GECO variants . Notably n = 96) . Howeveresponses . In genes tested . We hypoence see . Lastly,adiation . Thus, oF/F = 13.63 ± 1.37, n = 29) in HeLa cells . In addition, these display low to moderate basal florescence  in mApple or mPlum-based probes, namely, R-GECO1 and -1.2, respectively, resulted in generating functional and PA probes. More specifically, PA-R-GECO1 and -1.2 express efficiently at 37°C in HEK293, HeLa cells, as well as in cultured primary neurons, undergo robust 405 nm-induced photoactivation and readily report on Cace e.g., . Importace e.g., . We alsoeric RFP .2+-independent fluorescence that disappears within half a second. Such long-lived isomerization of the red chromophore to an anionic fluorescent state  , akin to GCaMPs, RCaMPs (RCaMP1h and jRCaMPa or b) belong to a second class in which the increase in fluorescence depends on both Ca2+-induced shift in fluorescence and in the quantum yield of the excitable state. Thus, in the case of R-GECI, it can be assumed that mutations around the chromophore that enhance photoactivation limit the subsequent increase in absorption following Ca2+-binding. Conversely, studies of R-GECO’s unique photoconversion behavior show that short wavelengths (405 and 488 nm) shift the absorbance of the calcium-bound population of R-GECO1 to the right therefore decreasing its fluorescence (by up to 40%) when illuminated with its optimal imaging wavelength   . Howeverum yield . In fact 561 nm) . Thus, i by Ca2+ .2+-performance  and the extent of photoactivation or Caformance . This isanalysis . Future T and -1.2T, display basal fluorescence that can be imaged by very low 561 nm laser powers. This should therefore allow to detect the probes in vivo by use of two-photon imaging  by additional methods, such as directed evolution, and expect to see the emergence of new PA-R-GECIs in the very near future.The ability to generate numerous PA-GECIs, by incorporation of a single point mutation at position 203 (GFP numbering), suggests that this mechanism is not restricted to a handful of RFP and should therefore be transferrable to other R-GECIs (and likely other RFPs). We anticipate that the PA-GECIs developed here will serve as a starting point for the development of additional or enhanced versions . Point mutations were introduced by PCR, saturating position I78  by all other amino acids: I78L, I78T, I78N, I78H, I78A, I78Y, I78Q, I78P, I78C, I78K, I78G, I78F, I78D, I78E, I78S, I78R, I78V, I78W  and transfected with 0.5–1 μg plasmid DNA with the use of ViaFect  for overnight incubation in Dulbecco‘s modified Eagle’s medium (DMEM) with 5–10% fetal bovine serum (FBS) at 37°C and 5% CO2. Primary neuronal hippocampal neurons were collected as previously described (in vitro (DIV), neurons were transfected with 0.5–1 μg DNA of the different variants by the calcium-phosphate transfection method . This lens transmits identically to the one used for power measurements of the 405 nm laser line and it illuminates an area on the sample of 0.2 μm2. The power density (PD) under these conditions is ∼106 W/cm2. The AOTF transmission is calibrated to be linear so that 1% laser power slider is equivalent to 10 KW/cm2 @ 405 nm. Cells were imaged in a standard imaging solution containing (in mM): 138 NaCl, 1.5 KCl, 1.2 MgCl2, 2.5 CaCl2, 10 D-glucose, 10 HEPES, pH 7.4. Ca2+-imaging was performed for several minutes with low intensity 561 nm to avoid bleach. To test functionality, we applied histamine  or additional Ca2+  when using HeLa or HEK293 cells, as previously described  and a Ca2+-ionophore; ionomycine (10 μM). This treatment induces Ca2+-release and promotes Ca2+-depletion from cells. For saturating Ca2+, we incubated cells with extracellular Ca2+ (2 mM) and ionomycine (10 μM) . We monitored emission at 8 nm intervals. Excitation of R-GECIs was performed by 405 and 561 nm lasers. Brief pulses of 405 nm . The output power of the 405 nm laser line was ∼3.4 mW when using an air objective (10x/0.45). It is measured at 100% power slider without laser blinking . Our experiments were always performed by uni-directional scanning, hence the power is corrected by a factor of ∼0.5, hence ∼ 1.7 mW. Our experiments were conducted using a water immersion objective lens 20x  with a focal spot diameter of 0.5 μm (D = 1.2 escribed . For ima (10 μM) . Image a (10 μM) ; 15 s.F/F) was calculated by (Ft–F0)/F0, where Ft is measured fluorescence  at a given time t and F0 is initial baseline fluorescence, typically calculated from averaging the 10 first images. ΔF/F = 1 describes an increase by 100%, equivalent to twofold increase in fluorescence. Owing to easily observable basal fluorescence (see post hoc Tukey) where relevant and relationship between Ca2+-performance and extent of photoactivation was tested by Spearman correlation using SigmaPlot 11.Change in fluorescence .WH and SB designed, performed, and analyzed the experiments and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Via three receptors known to date, including estrogen receptors α (ERα) and β (ERβ) and the G protein-coupled estrogen receptor 1 , E2 regulates numerous calcium-dependent activities in cardiovascular tissues. Nevertheless, effects of E2 and its receptors on components of the calcium signaling machinery (CSM), the underlying mechanisms, and the linked functional impact are only beginning to be elucidated. A picture is emerging of the reciprocality between estrogen biology and Ca2+ signaling. Therein, E2 and GPER, via both E2-dependent and E2-independent actions, moderate Ca2+-dependent activities; in turn, ERα and GPER are regulated by Ca2+ at the receptor level and downstream signaling via a feedforward loop. This article reviews current understanding of the effects of E2 and its receptors on the cardiovascular CSM and vice versa with a focus on mechanisms and combined functional impact. An overview of the main CSM components in cardiovascular tissues will be first provided, followed by a brief review of estrogen receptors and their Ca2+-dependent regulation. The effects of estrogenic agonists to stimulate acute Ca2+ signals will then be reviewed. Subsequently, E2-dependent and E2-independent effects of GPER on components of the Ca2+ signals triggered by other stimuli will be discussed. Finally, a case study will illustrate how the many mechanisms are coordinated to moderate Ca2+-dependent activities in the cardiovascular system.17β-Estradiol (E In this section, key CSM components in cardiovascular tissues will be briefly described to facilitate review of the relevant effects and mechanisms of estrogenic agonists and receptors.The CSM herein refers to proteins responsible for the generation or sequestration of intracellular Ca2+ store in cardiomyocytes, vascular smooth muscle cells (VSMCs) (2+ and mitochondria house ~25% (2+ reservoirs (2+ reaches 5 × 10−4 M in the ER/SR and lysosomes and 1.3–2.5 × 10−4 M between the trans-Golgi and cis-Golgi (2+ in response to inositol-triphosphate receptor (IP3R) and ryanodine receptor (RyR) stimulation  is the main Ca (VSMCs) , 2, and use ~25% . The Goluse ~25% , 5 and lservoirs , 7. Ca2+is-Golgi . The medmulation . Crossta+ fluxes . In neonparallel , and mitllel (2+ .2+-ATPases (SERCAs) are the key Ca2+ uptake mechanisms. For each ATP hydrolyzed, they pump 2 Ca2+ ions into the ER/SR in exchange for less than four H+ ions  mediates Ca2+ uptake into the Golgi with nanomolar affinity for Ca2+. Unlike the SERCA, Ca2+ transport by SPCA is not associated with counter transport of H+. In the medial Golgi, both SERCA and SPCA participate in Ca2+ uptake (The secretory pathway Ca+ uptake .2+ uptake is mediated by the voltage-dependent anion channel (VDAC) and the mitochondrial Ca2+ uniporter (MCU). VDACs are non-selective anion channels in the open state yet in the “closed” state permit influxes of cations such as K+, Na+, and Ca2+ into the mitochondria  and the essential MCU regulator . IP3R2 predominates in the cardiomyocytes  are the main SR Ca2+ release channels  stimulates Ca2+-induced Ca2+ release (CICR) via RyR2, contributing to myocardial contraction. In VSMCs, RyR2 predominates in the aorta and pulmonary and cerebral arteries, while RyR3 is the only isoform in basilar arteries   , 50. SOI (SOICR) .2+ store depletion triggers Ca2+ influx   activity and proliferation  , maintai (LTCCs) –67, and  (LTCCs) –71. Myoc (LTCCs) –76. In V animals . In ECs,feration –82.2+ channels (VDCCs) are the hallmark of tissue excitability and are present in cardiomyocytes and VSMCs, but not ECs. In cardiomyocytes, LTCCs are located mostly in transverse T tubules in apposition to RyR2s  prevail for Ca2+ extrusion in non-excitable tissues while the Na+-Ca2+ exchanger (NCX) is more important in excitable cells. SERCA2a, NCX, and PMCA sequester, respectively, ~70, 28, and 2% of cytosolic Ca2+ in cardiomyocytes . At low Ca2+, a C-terminal autoinhibitory domain binds to two cytosolic loops and inhibits pump activity. Increased Ca2+ promotes CaM–PMCA interaction, which removes inhibition and activates Ca2+ efflux , assumes an active conformation to bind estrogen-responsive elements (EREs) in the promoters of target genes, modulating their transcription  value of ~3 × 10−7 M  most membrane-bound [35S]GTPγ-S from cells overexpressing GPER and treated with E2 coimmunoprecipitate with Gαs  E2 promotes GPER-dependent cyclic adenosine monophosphate (cAMP) production (i/o association are results that pertussis toxin prevents (1) E2-induced, GPER-mediated ERK1/2 phosphorylation in cells transfected with GPER  GPEoduction . Supportith GPER , 157; s in ECs .GPER is robustly expressed in cardiovascular tissues –136. In 2+-CaM complexes. In VSMCs and ECs, GPER coimmunoprecipitates with CaM in a constitutive association that is promoted by treatment with E2, G-1, or receptor-independent stimulation of Ca2+ entry  of 1.3 × 10−7-5 × 10−6 M, values within the physiological Ca2+ range  triggers 45Ca2+ uptake that is inhibited by LTCC antagonists  induces Ca2+ store release and entry, yet only the former is required to activate mitogen-activated protein kinase (MAPK) (−6 M) also triggered Ca2+ signals in these cells. In ECs, E2 (10−10-10−9 M) triggers Ca2+ store release and entry, effects not affected by ERα/ERβ inhibitor tamoxifen  . Interesamoxifen , 174. Thing GPER , 159. La/− cells , 176.2 (10−11-10−9 M) potentiates ICa,L in neurons and HEK293 cells overexpressing the α1C subunit; nifedipine displaces membrane E2 binding; and E2's effect is reduced by a dihydropyridine-insensitive LTCC mutant, indicating that E2 binds to the dihydropyridine-binding site  Gβγ activates IP3Rs by interacting with the IP3-binding sites  stimulates very small Ca2+ signals (<10−7 M)  . One canted eNOS , 190, woin VSMCs  would bein VSMCs . Howeverin VSMCs , 192–194in VSMCs , 195–198SMCs  inhibits norepinephrine- and phenylephrine-induced arterial constriction in the presence of extracellular Ca2+ but not that induced in Ca2+-free medium (1 adrenoceptor agonists can activate both (−8-10−6 M) suppresses SOCE induced by thapsigargin or bradykinin  animals  and combined ERα/ERβ antagonists/GPER agonists  on ICa,L in cardiomyocytes from both WT and ERα−/−/ERβ−/− animals, as reviewed in  inhibits nifedipine-sensitive Ca2+ spikes in LTCC-expressing A7R5 SMCs, an effect prevented by GPER antagonist G-15 (10−6 M)  ; these c(10−6 M) , 211. Colaxation .How E2inhibits electrically induced VDCE is still unknown. Hypothetically, at high levels, E2 binding to the dihydropyridine-binding site on LTCC -mediated potentiation of VDCE, recent evidence suggests that GPER may be an intrinsic component of β1AR activation. Thus, G-1 inhibits isoproterenol-induced increases in left ventricle (LV) pressure, heart rate, ectopic contractions, ICa,L, LTCC phosphorylation, and total myocardial Ca2+ signal, while the GPER inhibitor G-36 promotes ISO-induced Ca2+ signal and LTCC phosphorylation  does not affect the Vmax of SR vesicle Ca2+ uptake in canine LV tissue   . During activity . These sse modes . However−3 M) inhibits mitochondrial 45Ca2+ uptake (2+ retention capacity (mCRC), a combination of mitochondrial Ca2+ uptake, total mitochondrial Ca2+-binding sites, and mitochondrial Ca2+ release mechanisms, is a determinant of the protective role of the mitochondria during cytoplasmic Ca2+ overload. E2 (4 × 10−8 M) increases myocardial mCRC following ischemia–reperfusion, an effect abolished by genetic deletion of GPER but not of ERα or ERβ; mechanistically, this effect seems to involve PKC-dependent, MAPK-dependent phosphorylation of glycogen synthase kinase (GSK)-3β, leading to inhibition of the mitochondrial permeability transition pore (2 (10−8 M) inhibits high Ca2+-induced cytochrome c release from myocardial mitochondria (2 (10−8 M) treatment inhibits mitochondrial Ca2+ uptake, an effect abolished by the ERα/ERβ antagonist ICI182,780 (10−8 M)  . The mec unknown .2-dependent and E2-independent mechanisms (E2-dependent mechanisms are evidenced by the effects of G-1 (10−8-10−6 M) and E2 (1–5 × 10−9 M) to inhibit PMCA-mediated efflux in primary ECs without affecting PMCA expression levels and to promote PMCA phosphorylation at Tyr1176 (2 treatment (E2-independent mechanisms are indicated by the findings that (1) GPER constitutively interacts with PMCA4b via the anchoring action of PSD-95 at their C-terminal PDZ-binding motifs; (2) overexpression of GPER decreases PMCA activity; (3) GPER knockdown promotes PMCA activity; and (4) PSD-95 knockdown or truncation of the PDZ-binding motif on GPER releases GPER–PMCA association and promotes PMCA activity  upregulates total CaM by around 7-fold and free Ca2+-CaM by ~15-fold in primary ECs. Data obtained using specific estrogen receptor agonists, gene silencing, and receptor overexpression indicate that GPER, but not ERα or ERβ, mediates this effect. Thus, the GPER agonist G-1 (10−9-10−7 M), but not the ERα agonist propyl pyrazole triol (PPT) (3 × 10−10-2 × 10−7 M) or the ERβ agonist diarylpropionitrile (DPN) (10−10-5 × 10−8 M), increases CaM expression; GPER knockdown reduces the effect of E2 to upregulate CaM; and E2 upregulates CaM in SKBR3 cells that express only GPER and not ERα or ERβ . Functionally, E2 upregulates CaM and promotes the PMCA–CaM interaction; however, the predicted stimulatory effect on Ca2+ extrusion is masked by E2-induced inhibitory phosphorylation at Tyr1176 of PMCA  and raising troughs , collectively confining tissues in a narrower yet more sustained operating range of Ca2+. Also, GPER-mediated increases in CaM expression and CaM network linkage improve Ca2+ signal transduction efficiency. Considering the Ca2+ sensitivity of Ca2+-dependent proteins in this context, one can predict that those with low Ca2+ sensitivity (requiring high Ca2+ for activation) are more likely to be affected by the inhibition of Ca2+ signal generation. On the other hand, proteins with high Ca2+ sensitivity (requiring low Ca2+ for activation) are more likely to be promoted by the inhibition of Ca2+ removal and less affected by the suppression of Ca2+ signal generation  values ~1.8 × 10−7 and 4 × 10−7 M  values to ~0.7 × 10−7 and 1.3 × 10−7 M, thus rendering the synthase active at resting cytoplasmic Ca2+ (2 and GPER (1) prolong endothelial cytoplasmic Ca2+ signal by inhibiting Ca2+ efflux  inc+ efflux . When we+ efflux , 190, eNwith G-1 . Importa of SOCE , due obvsitivity . Thus, v2+-dependent estrogen receptors—ERα and GPER—how does the presence of one influence the effects of the other on Ca2+ signaling? A complex relationship is predicted to exist in which ERα transcriptional activities affect the expression of certain Ca2+ signaling proteins but are themselves influenced by the amplitudes and dynamics of Ca2+ signals limited by GPER activation and the availability of CaM that is promoted by GPER action (2+ signaling.Considering the two CaR action . In turnR action , 127, thR action , 127. Th2+-dependent activities is becoming evident. Considering the impact of estrogen and its receptors on Ca2+signaling, E2, and in many cases, GPER exert inhibitory effects on many components of the CSM in cardiovascular tissues, from Ca2+ store release and uptake (2+ entry (2+ removal mechanisms (Considering the impact of Ca2+signaling on estrogen biology, both ERα and GPER are strongly regulated by direct Ca2+-dependent interactions with CaM. These interactions serve to stabilize receptor dimerization and enhance subsequent transcriptional activities  ore of ERα , 170]. Aproduced . Recipro of eNOS , 3 (170, of eNOS .2+ overload, and reducing vascular tone. Nevertheless, the effects of E2 and estrogen receptors on many CSM components have not been examined. Additionally, many questions remain regarding mechanisms of the observed effects that estrogenic agonist and receptors produce on the CSM. For example, how do E2 and GPER inhibit ICa,L? What are the mechanisms that position GPER as an intrinsic component of β1AR signaling in the myocardium? What are the mechanisms whereby E2 inhibits the activities of SERCA and NCX? What are the mechanisms whereby E2 inhibits mitochondrial Ca2+ uptake? Further studies are needed to answer these questions. Through many examples, however, it is clear that GPER produces both E2-dependent and E2-independent effects on the CSM. While the search is ongoing for approaches to apply specific estrogen receptor agonists to the prevention of cardiovascular disease, the therapeutic potential of E2-independent effects of GPER and other estrogen receptors is as yet an unexplored territory.The moderating effects that estrogenic agonists and receptors exert on the CSM can explain many of their cardiovascular effects, such as preventing excessive cardiac contraction during sympathetic stress, limiting adverse outcomes related to CaQ-KT conceived the ideas, generated the figures, and wrote the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.