Mentoring programs are the most important factor in the achievement of students' human capital. However, in Iran's higher health education system,these initiatives have received less attention. The goal of this research is to reorganize the components of mentoring for medical university student achievement. This qualitative study was conducted using a Meta synthesis method. Keywords of mentoring medical students, mentoring academics students,human capital development, student development, and mentoring were searched in database: Science Direct, Springer, Wiley Online Library,ERIC, Sage, Emerald, Pub Med from 2000 to 2021. Out of total 91 studies, finally 51 studies were selected. The findings are divided into two parts. The first part deals with the characteristics of faculty members and students in thementoring programs of medical universities. These features include student-specific characteristics, faculty-specific characteristicsand common characteristics. In the second part of findings, the components of mentoring programs were extracted.These components include university, communication, soft capacities; before the implementation of the program, during program implementation,monitoring and evaluation of program implementation, and consequences of program implementation. The suggested components should be examined by managers of the higher health education system for student human capital development due to therelevance of mentoring programs in the development of medical students' human capital. Mentoring programs are used as a main tool in higher education to increase students' personal and professional growth  . In theIn general, regarding the importance and role of mentoring programs in student achievement, few studies were done in the field of healthsystem mentoring. Another noteworthy point is the lack of attention to this issue in the domestic research literature.For this reason, Meta synthesis and synthesis method of studies conducted in this field in the world were used.In our study, in addition to presenting a comprehensive and deeper view of this subject, an attempt was made to collect many of causesand factors of program failure according to a systematic and comprehensive method. The purpose of this study is therearrangement of mentoring components for the achievement of students in medical universities. Historically, the word "mentor" originated in the mid-eighteenth century, in the epic of Homer and Odyssey.The name is derived from the name of a friend who Odysseus entrusted his son to him as a trusted advisor  . NevertTo review and synthesize the research conducted on the topic, the seven-step Meta-synthesis method was used  . The fiThe fourth step is to extract information from the articles. As a consequence, the titles, purposes, and summaries of the outcomes of thearticles were recorded. The findings were then integrated, combined, and interpreted in the fifth stage. The qualitative content analysisapproach was employed in this stage. First, each article's essential themes were extracted independently. The major ideas were then divided intoprimary and secondary subcomponents based on their similarities and differences. The sixth step was to check the quality of the findings.To validate the findings, the method was reviewed by the research colleague. The seventh step was the provision of results.In all stages of the present study, the ethical principle of fidelity was observed in citing sources and using their results.To answer the first research question, i.e., what are the most important characteristics of medical students and faculty in theUniversity of Medical Sciences, the terms were categorized based on similarities and differences after extraction. 1. Characteristics of faculty; a) general characteristics: having no judgment and prejudice ( b) scientific characteristics: available  general characteristics: active listener ( b) academic characteristics: adaptability  common general characteristics: interest (positive attitude) ( b) common academic characteristics: knowledge sharing ( titude)  , 75; vtitude)  , 67; mtitude)  , 77; atitude)  , 33; rtitude)  ; mutualtitude)  ; mutualtitude)  , 59; ptitude)  , 73; stitude)  , 78; dtitude)  ; flexibtitude)  , 79; wtitude)  , 74; ctitude)  ; motivatitude)  , 65; atitude)  ; changetitude)  ; b) comsharing  , 77; csharing  , 72; asharing  , 59; csharing  ; consensharing  ; mutualsharing  , 78; msharing  , 73; ssharing  , 64; csharing  , 73; hsharing  , 78; msharing  ; settinsharing  , 74; msharing  , 58; ssharing  ; sharinsharing  ; adaptisharing  . In facIn order to answer the second research question, i.e. “what are the main components of medical universities’ mentoring programs”, a categorization was also made after extracting the key terms.1. Academic; a) university leadership: coordination among various shareholders in the university ( b) university management: development of internship programs  unirograms  , 81; arograms  ; develorograms  ; financrograms  ; financrograms  ; use ofrograms  , 72; crograms  , 49; srograms  , 73; programs  ; attentrograms  ; managerograms  ; develorograms  , 60; drograms  ; allocarograms  . This c2. Communication; a) development of communication: relationships based on mutual trust ( b) communication management: improving the quality of relationships  comonships  , 53; eonships  ; creationships  ; creationships  ; creationships  ; dynamionships  . The co3. Soft capacities; a) cultural norms: pro-mentoring academic culture ( b) moral norms: work ethics  mor ethics  , 53; a ethics  ; ethica ethics  ; develo ethics  ; creati ethics  . This c4. Prior to program implementation; a) pre-planning: knowledge and awareness of the benefits of the mentoring program ( b) preparation: preparation of student; preparation of professor  preofessor  , 73; fofessor  , 73; sofessor  , 76; sofessor  ; match ofessor  ; selectofessor  ; develoofessor  . This c5. During the program implementation; a) management of program implementation: developing leadership directives ( b) how to conduct sessions: recording and review of sessions by students  howtudents  ; holdintudents  ; time mtudents  ; extractudents  ; mutualtudents  ; standatudents  ; organitudents  , 39; itudents  , 70; gtudents  , 64; mtudents  ; activetudents  , 75; atudents  , 62; itudents  , 78; dtudents  , 62; dtudents  , 68; atudents  ; selecttudents  ; agreeitudents  ; designtudents  ; increatudents  ; teachitudents  ; simulatudents  ; precistudents  ; holdintudents  ; formintudents  ; studyitudents  ; prioritudents  ; regulatudents  ; contintudents  ; use oftudents  ; purpostudents  , 54; atudents  . This c6. Monitoring and evaluating the program implementation; a) performance monitoring: continuous monitoring ( b) program evaluation: continuous and regular feedback  outcomes for students: development of clinical skills ( b) academic outcomes: scholarly sociability  with 45%. It emphasizes theimportance and necessity of implementing mentoring programs for medical students. Of course, in order to have a successful program,all the steps must be performed accurately. In other words, all the identified components indicate what components make up a successful mentoring program,and they are all equally important in terms of performance.After analyzing and synthesizing the international studies, seven components were emerged in the field of student mentoring programs.The first component is academic. It includes leadership and management in the university. The important point is the participation of all stakeholdersin decision making in the university. Besides, the development of performance-based reward and punishment systems that are about theactions and performance of academics can be a motivating factor for better implementation of mentoring programs for medical students.Furthermore, formal mentoring programs should be systematically addressed in university documents to align with the university’s mission and goals.The results of this component are consistent with that of the previous studies  , 81. TThe third component is the soft capacities. It represents academic culture norms and moral norms. The foundation of any change is theacademic culture and the subcultures that define it. Moreover, a kind of collectivism must prevail in academic culture to form a common identityand language among academics in medical universities. Moral norms refer to ethical standards and attitudes such as academic ethics,moral obligation, ethical behavior, etc. The findings of this component are in line with earlier research  , 79. PThe fifth component is the implementation time of the program. It indicates how the mentoring program is implemented and managed.During the implementation of the program, a schedule should be designed in defining the objectives, tasks, and plans to achieve the goals.Moreover, regular sessions, commitment and mutual responsibility to fully implement the sessions, and diversification of communicationtools should be considered during the implementation of the program. Another significant consideration is the consistency of thesubstance and themes of the sessions, as well as the structuring of the program's execution with stated mentoring goals, which must becontinually reviewed. This component's findings are consistent with earlier study findings  , 80. TThis article attempted to provide a more comprehensive vision of mentoring programs via the synthesis and analysis of studies.Mentoring programs for medical students have a systematic method, and we need to pay attention to all phases to implement them effectively.A benefit of such research is that it combines the results of a single study on mentoring medical students, so allowing for a more thoroughgrasp of the issue because its components have yet to be identified in the worldwide mentoring literature in the medical sciences.As a result, the unique contribution of such research is the identification of components at both the individual and university levels.The disadvantage of this research is the lack of using quantitative methods that can be effective as a complement to the qualitative method.Therefore, further research is suggested to study this issue in the University of Medical Sciences with mixed methods research.Researchers can evaluate the situation of Iranian medical universities by converting the components of this research into a questionnaireusing advanced statistical methods such as multilevel analysis. Limitations of research: This research is qualitative and due to thenature of such research, it has little generalizability; another limitation of the Meta synthesis method is related to the gray literature.Other research in non-English languages on mentoring may not have been considered in this study. Finally, policy recommendations are offered for the health academic system:◦ Raising the awareness of mentoring programs and their importance in upstream academic health care documents and in the strategic programs of medical universities;◦ Sensitization of the medical university managers to the importance of these programs and the accurate design of all their phases, as well as a long-term capitalistic view of these programs;◦ Changing subcultures at the medical department level to create a university culture that supports mentoring programs;◦ Developing academic communication in medical universities to improve the social capital of the university and increase the effectiveness and efficiency of mentoring programs;◦ Preparing and training medical faculty and students to best implement the program;◦ Continuously monitoring program implementation along with reporting and receiving feedback on performance and using evaluation results to improve the program;◦ Providing more funding for medical student mentoring programs in terms of the scope and diversity of their benefits.A.K, E.K contributed to the conception and design of the work; the acquisition, analysis,or interpretation of data for the work. All Authors contributed in drafting and revising the manuscript critically for important intellectualcontent. All authors have read and approved the final manuscript and agree to be accountable for all aspects of the work in ensuring that questionsrelated to the accuracy or integrity of any part of the work are appropriately investigated and resolved.None declared.


Furthermore, intra-islet paracrine activation of β-cell Gi/o-GPCRs and NKAs by δ-cell somatostatin secretion slowed Ca2+ oscillations, which decreased insulin secretion. β-cell membrane potential hyperpolarization resulting from Gi/o-GPCR activation was dependent on NKA phosphorylation by Src tyrosine kinases. Whereas, β-cell NKA function was inhibited by cAMP-dependent PKA activity. These data reveal that NKA-mediated β-cell membrane potential hyperpolarization is the primary and conserved mechanism for Gi/o-GPCR control of electrical excitability, Ca2+ handling, and insulin secretion.G i/o protein-coupled receptors (Gi/o-GPCRs) limit β-cell insulin secretion by decreasing Ca2+ entry; however, the underlying mechanism has not been identified. Here, the authors show that Gi/o-GPCRs hyperpolarize mouse and human β-cell membrane potential by activating Na+/K+ATPases.G It was discovered more than 50 years ago that Gi/o protein-coupled receptors (Gi/o-GPCRs) play a critical role in limiting insulin secretion in-part by decreasing β-cell electrical excitability and subsequent Ca2+ influx10. However, the exact mechanism(s) of Gi/o-GPCR control of β-cell electrical excitability, Ca2+ handling, and insulin secretion remain poorly understood.Pancreatic β-cell glucose-stimulated insulin secretion (GSIS) is essential for maintenance of euglycemiai/o-GPCRs such as somatostatin receptors (SSTRs), α2A-adrenergic receptors (ADRs), and D2-like dopamine receptors (DRDs)16. As a consequence, treatment of islets with Gi/o-GPCR ligands , adrenaline, or dopamine) activates hyperpolarizing currents and reduces intracellular cAMP ([cAMP]i) levels, which results in decreased intracellular Ca2+ ([Ca2+]i) and diminished insulin secretion21. Insulin secretion is inhibited by Gi/o-GPCRs, therefore, these signals are critical for preventing excessive insulin secretion under hypoglycemic as well as stimulatory conditions22. Indeed, loss of intact α2-ADR signaling leads to a drop in blood glucose levels under fasting and fed conditions due to elevated insulin secretion23, while α-ADR agonists attenuate GSIS25. Intra-islet communication is also mediated via Gi/o signaling 30, which tunes β-cell Ca2+ handling and insulin secretion. Thus, inhibition of islet Gi/o-GPCRs with pertussis toxin, also known as islet activating protein31, significantly stimulates hormone secretion, highlighting the importance of Gi/o-GPCRs in regulating islet function. As numerous Gi/o-GPCRs control physiological β-cell function, perturbations in these pathways impair β-cell GSIS and are in some instances associated with increased risk of developing diabetes34. For example, glucose-stimulated SST secretion is blunted during the pathogenesis of type 2 diabetes (T2D), which diminishes SSTR-mediated control of β-cell function33. Moreover, polymorphisms that increase α2A-ADR expression result in increased risk of developing T2D due to suppression of GSIS34. Taken together, these findings strongly suggest that Gi/o signaling plays a key role in regulating β-cell Ca2+ handling and insulin secretion; however, the underlying mechanism has not been conclusively identified for more than half a century.β-cells express numerous GVm hyperpolarization is predominantly mediated by K+ efflux; thus, it has been generally accepted that Gi/o-GPCR signaling activates an outward K+ conductance. ATP-sensitive K+ (KATP) channels can be ruled out as the source, because Gi/o-GPCR activation induces β-cell Vm hyperpolarization in the presence of sulfonylureas and in KATP channel-deficient islets35. As is the case in numerous other tissues, Gi/o signaling-induced β-cell Vm hyperpolarization has been widely ascribed to activation of G protein-gated inwardly-rectifying K+ (GIRK) channels36. RNA sequencing studies show that both mouse and human β-cells express low levels of GIRK channel transcripts 16, and immunofluorescent staining of mouse pancreatic sections confirms the expression of GIRK channel proteins in β-cells36. However, while Gi/o signaling activates robust inwardly-rectifying K+ currents in other cell types where GIRK channels are expressed37, currents with GIRK-like characteristics have not been reproducibly observed in primary β-cells. Moreover, there are conflicting reports detailing the effect of GIRK channel inhibitors on β-cell electrical activity and Ca2+ handling. For example, one study determined that pharmacological GIRK channel inhibition blocks adrenaline-induced Vm hyperpolarization in rat β-cells36, but another manuscript found that GIRK channel inhibition does not prevent adrenaline-induced Vm hyperpolarization in mouse β-cells19. Furthermore, treatment with a wide range of other K+ channel blockers failed to inhibit adrenaline-induced β-cell Vm hyperpolarization. These observations suggest that Gi/o signaling-induced β-cell Vm hyperpolarization is not mediated by GIRKs or other K+ channels.β-cell +/K+ ATPases (NKAs) can also be activated by Gi/o-GPCR signaling leading to Vm hyperpolarization39. NKA α1 pore-forming subunits (encoded by ATP1A1) are highly expressed in mouse and human β-cells13 where they preserve steep ionic gradients that are essential for setting and maintaining Vm. Active β-cell NKAs generate a net outward cationic flux by extruding three intracellular Na+ ions in exchange for two extracellular K+ ions, resulting in Vm hyperpolarization39. Inhibition of β-cell NKAs with ouabain leads to Vm depolarization and enhanced insulin secretion41, illustrating the key role that NKAs serve in preventing excessive insulin secretion. Importantly, NKA activity is regulated by several β-cell protein kinases that are influenced by Gi/o-GPCR signaling. For example, protein kinase A (PKA), which limits NKA activity, is inhibited by SSTR signaling in a cAMP-dependent manner; whereas, Src tyrosine kinases (STKs) augment NKA function and are activated by SSTR signaling46. Thus, Gi/o signaling is predicted to influence β-cell Ca2+ handling through changes in NKA α1 subunit phosphorylation.Electrogenic NaVm hyperpolarization is a principal mechanism for Gi/o-GPCR control of β-cell Ca2+ handling and insulin secretion. Activation of β-cell Gi/o signaling generated ouabain- and K+-sensitive outward currents as well as [Ca2+]i oscillations independently of KATP in both mouse and human β-cells. Inhibition of SST secretion from δ-cells increased islet [Ca2+]i, accelerated islet [Ca2+]i oscillations, and enhanced GSIS, demonstrating the critical role of δ-cell paracrine signaling in tuning β-cell Ca2+ handling and insulin secretion, likely through NKA activation. cAMP-dependent PKA activation decreased β-cell NKA function, whereas stimulation of tyrosine kinases  initiated islet [Ca2+]i oscillations. These results strongly suggest that phosphorylation by PKA and tyrosine kinases serve as crucial counter-regulatory mechanisms for modulation of β-cell NKA activity. Therefore, these findings illuminate a conserved mechanism for Gi/o-GPCR control of β-cell NKAs, which plays a key role in regulating β-cell electrical excitability, Ca2+ handling, and insulin secretion.Here we show that NKA-mediated β-cell i/o signaling activates hyperpolarizing GIRK channels36; however, measurement of β-cell GIRK currents has proven difficult utilizing traditional voltage-clamp recording techniques. Thus, we employed a modified recording paradigm to elicit quantifiable SST-induced β-cell currents. SST-mediated changes in β-cell Vm were monitored in intact islets and whole-cell currents were recorded in response to voltage ramps before and after each treatment49. β-cell currents displayed minimal trace-to-trace variability in response to repeated voltage ramps indicating stable voltage-clamping; however, to account for small current distortions arising from electrical activity in neighboring β-cells within an intact islet50, all currents were calculated from the median of at least 10 consecutive traces. In wild type (WT) mouse islets maximally stimulated with 20 mM glucose and 1 mM tolbutamide21, 200 nM SST induced outward β-cell currents with little rectification ; SST also hyperpolarized Vm .It is generally accepted that β-cell GKcnj6 is the most abundant islet GIRK channel transcript13, GIRK2 deficient (GIRK2 KOPanc) mouse islets were utilized to determine the contribution of these channels to SST-induced β-cell currents. As observed in WT β-cells, 200 nM SST elicited outward non-rectifying currents in β-cells without GIRK2 channels  and hyperpolarized Vm . Tolbutamide-stimulated mouse islets displayed transient glucose-mediated (20 mM) [Ca2+]i decreases, after which [Ca2+]i stabilized at an elevated level . GIRK channels were also pharmacologically inhibited in WT islets with 200 nM tertiapin-Q (TPQ) to confirm GIRK channel-independent SST-induced β-cell currents19. In the presence of TPQ, 200 nM SST elicited outward β-cell currents with minimal inward rectification  and hyperpolarized Vm . Although β-cell currents trended lower in the presence of TPQ, SST-induced β-cell currents were not significantly decreased when GIRK channels were inhibited. Furthermore, 76.8 ± 10.5% of islets displayed SST-induced [Ca2+]i oscillations following treatment with 200 nM TPQ, which was not significantly different than with SST alone . These results demonstrate that while GIRK channels likely play a role in Gi/o-GPCR regulation of β-cell electrical activity, SST-induced β-cell hyperpolarization is largely independent of GIRK channel activity.As transcriptome studies indicate that vel Fig. . Under tt GIRK2 KPanc mous+, which suggests that SST-induced β-cell currents are not mediated by K+ channels  or removal of extracellular K+ . Oua treatment subsequent to removal of extracellular K+ had no additional effect on islet [Ca2+]i, suggesting that islet NKAs are completely inhibited under these conditions  and without tolbutamide-mediated KATP inhibition . Importantly, KATP activation with 125 µM diazoxide (DZ) during NKA inhibition  decreased β-cell [Ca2+]i by 82.5 ± 15.7% , which demonstrates that NKA-mediated Vm depolarization does not prevent Vm hyperpolarization by K+ channels. Following sustained increases in islet [Ca2+]i that result from NKA inhibition (0 mM extracellular K+), SST-induced [Ca2+]i oscillations were rapidly restored by supplementation with 5 mM extracellular K+  [Ca2+]i oscillations  channels are also expressed in β-cells, these channels are largely inactive at voltages above −50 mV53, and are thus unlikely to account for sustained (5–10 min) SST-mediated increases in islet [Na+]i.NKA activity would be predicted to maintain low β-cell [Nai inhibit NKA activity21 and we observed a correlation between increasing [cAMP]i and termination of SST-induced islet [Ca2+]i oscillations ; after FSK treatment islet [Ca2+]i plateau fraction also increased by 40.5 ± 2.9% compared to with SST alone . These results suggest that increases in [cAMP]i can block SST-induced islet [Ca2+]i oscillations in-part by inhibiting β-cell NKA activity.It is established that elevations in [cAMP]ons Fig. . Thus, 5SST Fig. E–G and pion Fig. . Furtheri/o-GPCRs in addition to SSTRs including α2A ADRs15. Thus, we examined whether β-cell NKA activation is a conserved mechanism for Gi/o signaling-mediated control of islet [Ca2+]i. In the presence of 20 mM glucose and 1 mM tolbutamide islets did not oscillate until treated with 200 nM of the α-ADR activator clonidine (Clon)55, after which all exhibited [Ca2+]i oscillations  and a 64.0 ± 3.3% decrease in [Ca2+]i plateau fraction . Treatment with 150 µM Oua terminated Clon-induced [Ca2+]i oscillations in 94.9 ± 3.0% of islets  and increased [Ca2+]i plateau fraction by 61.1 ± 3.4% , which was indistinguishable from islets before Clon treatment. These findings indicate that stimulation of NKA activity is a conserved mechanism for Gi/o -GPCR control of β-cell [Ca2+]i.β-cells express a number of G57, which is predicted to impact β-cell Ca2+ handling. Therefore, Gi/o-coupled Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) driven by an optimized RIP were employed to selectively activate Gi/o signaling in β-cells  [Ca2+]i oscillations were observed in 88.5 ± 9.0% of islets . Interestingly, only a small subset of β-cells expressed Gi/o-DREADDs in each islet (based on mCherry fluorescence), suggesting that electrical coupling between β-cells amplifies Gi/o signaling-induced islet [Ca2+]i oscillations. After treatment with 150 µM Oua, [Ca2+]i oscillations ceased in 93.4 ± 4.3% of islets  and islet [Ca2+]i plateau fraction increased 35.7 ± 1.8% , which was indistinguishable from islets before CNO treatment. These data confirm that under depolarizing conditions direct stimulation of β-cell Gi/o signaling transiently hyperpolarizes Vm by activating NKAs.SSTRs and ADRs have also been shown to control α-cell functionets Fig. D, E and s-coupled glucagon-like peptide-1 receptors (GLP1Rs) and glucagon receptors (GCGRs) by glucagon secreted from α-cells helps to maintain β-cell [cAMP]i levels58. Because SST is a potent inhibitor of glucagon secretion, we next set out to determine if some SST-mediated changes in islet [Ca2+]i oscillations were due to altered paracrine signaling from α-cells. Following treatment with 500 nM of the GLP1R antagonist exendin-3 (9–39) (Ex9) [Ca2+]i oscillations were observed in only 18.7 ± 18.7% of islets and islet [Ca2+]i plateau fraction was unaffected . Even under conditions of GLP1R blockade glucagon may still elevate β-cell [cAMP]i through GCGR signaling. Therefore, we assessed whether selective α-cell SSTR signaling could influence islet [Ca2+]i oscillations. For this, we utilized the selective SSTR2 agonist L-054,26416 to activate SSTR2s, which are exclusively expressed in mouse α-cells and are required for SST-mediated suppression of glucagon secretion59. Treatment with 500nM L-054,264, did not stimulate islet [Ca2+]i oscillations and had no effect on islet [Ca2+]i plateau fraction  rather than by diminishing paracrine activation of β-cell Gs-GPCRs .Paracrine activation of β-cell Gceptors GP1Rs and 2+]i oscillations; this was accomplished utilizing islets from transgenic mice selectively expressing Gi/o-coupled DREADDs in δ-cells (δGi-DREADDs) to inhibit SST secretion during glucose-stimulated [Ca2+]i oscillations  and increased islet [Ca2+]i area under the curve (AUC) by 68.8 ± 18.3%  relative to the period prior to addition of CNO. However, δGi-DREADD activation had no effect on islet [Ca2+]i plateau fraction , [Ca2+]i AUC decreased by 54.0 ± 19.8% , and [Ca2+]i plateau fraction decreased by 15.8 ± 4.4% . Similarly, 200 nM SST decreased δGi-DREADD islet [Ca2+]i AUC by 21.2 ± 11.4% in the absence of CNO .We next sought to determine if endogenous islet SST controls i oscillation frequency, we examined the effect of δ-cell Gi/o-GPCR activation on SST and insulin secretion. δGi-DREADD activation had no effect on insulin secretion at 2 or 7 mM glucose; however, at 9 mM glucose δ-cell Gi/o signaling increased insulin secretion from 81.5 ± 7.7 to 352.2 ± 59.4 pg insulin h−1·islet−1 . Importantly, δGi-DREADD activation decreased SST secretion from 0.743 ± 0.082 to 0.267 ± 0.003 pg SST h−1 islet−1  under these conditions. Insulin secretion from WT islets was not affected by CNO at either 1 or 11 mM glucose  channel activity16, we went on to examine if NKAs contribute to this effect. Transcriptional analysis indicates that human β-cells express high levels of ATP1A1 transcript (gene encoding the NKA α1 subunit)13. Immunofluorescence staining of human pancreatic sections confirmed that insulin positive β-cells also stain positive for NKA α1, and revealed a predominantly cell membrane-restricted localization  was utilized60. Treatment with 400 nM SST at 7 mM glucose decreased β-cell [Ca2+]i AUC by 37.2 ± 0.8%  and [Ca2+]i plateau fraction by 36.4 ± 2.9% . Moreover, 400 nM SST prompted significant decreases in human islet [Ca2+]i AUC, which was reduced by 79.9 ± 16.4% in the absence of extracellular K+ . As human islets express a number of GIRK channel transcripts16, TPQ was utilized to assess the contribution of human β-cell GIRK channels to Gi/o-GPCR-mediated Vm hyperpolarization. When GIRK channels were inhibited with 200 nM TPQ, SST mediated human islet [Ca2+]i decreases that were indistinguishable from human islets not treated with TPQ  and activated outward currents that were inhibited by removal of extracellular K+ . SST-induced human β-cell currents were outward at voltages below the equilibrium potential of K+  and [Ca2+]i plateau fraction was reduced by 99.5 ± 0.3%  compared to SST alone. Furthermore, H89 abolished FSK-mediated increases in islet [Ca2+]i AUC  compared to before treatment, which was inhibited by Oua . SST-induced islet Na+ efflux  was also inhibited following GLP1R activation with 400 nM liraglutide , indicating that Gs protein-coupled receptor (Gs-GPCR)-mediated increases in β-cell [cAMP]i block NKA activity. Interestingly, in islets pretreated with 1 µM of myristoylated protein kinase inhibitory peptide 14–22 amide (PKI) for 1 h to inhibit PKA activity, SST increased Na+ efflux by 63.9 ± 2.6%  compared to before treatment, but Lira-mediated inhibition of SST-induced islet Na+ efflux was completely blocked . Treatment with 150 µM Oua was able to prevent SST-induced islet Na+ efflux even in the presence of PKI . These findings indicate that β-cell NKAs are likely inhibited by cAMP-dependent PKA activity.FSK-mediated elevations in [cAMP]els Fig. ; as a reAUC Fig.  as well 42, which interacts with and enhances NKA function46. Therefore, a STK inhibitor dasatinib (Dasa) was employed to investigate the role of STK signaling in Gi/o-GPCR control of β-cell NKA function . Dasa also increased islet [Ca2+]i plateau fraction by 40.7 ± 3.1% compared to SST alone . The average size of islets that oscillated in the presence of Dasa was 71.0 ± 8.6% larger than islets that stopped oscillating , which may indicate that bifurcated islet Ca2+ responses were due in-part to incomplete penetration of Dasa into larger islets.It has been shown that SSTR signaling stimulates STK activityion Fig. . All isl64, a Shp2 inhibitor NSC 87877 (NSC) was utilized to assess whether this pathway regulates β-cell NKAs . Furthermore, NSC increased islet [Ca2+]i plateau fraction by 39.9 ± 6.8% compared to SST alone . As with Dasa, the average size of islets that continued oscillating with NSC was 75.7 ± 16.7% larger than islets that stopped oscillating , which again suggests only partial Shp2 inhibition in larger islets.As SSTR signaling activates Src homology region 2 domain-containing phosphatase-2 (Shp2) that in turn stimulates STK functionKAs Fig. . All isl65. Therefore, we examined if insulin enhances β-cell NKA activity. Islet [Ca2+]i imaging experiments were carried out at 25 °C to suppress endogenous insulin secretion66. Treatment with 1 µM insulin-induced [Ca2+]i oscillations in 69.5 ± 10.4% of islets . Subsequent addition of 100 nM Dasa had no effect on the percentage of islets displaying insulin-induced [Ca2+]i oscillations ; islet cell lysates were immunoblotted for total NKA α1 protein as well as for NKA α1 phosphorylated at a putative tyrosine kinase phosphorylation site (tyrosine 10 (Y10))69. Phosphorylation of NKA α1 at residue Y10 (p-ATP1A1Y10) increased by 65.3 ± 12.3% in islets treated with SST (P < 0.05) and by 64.0 ± 23.4% in islets treated with insulin  relative to vehicle controls. As previous studies have shown that phosphorylation of NKA α1 at residue Y10 increases NKA activity69, this result suggests that Gi/o-GPCR (and insulin receptor) signaling augments β-cell NKA function in-part through phosphorylation of the NKA α1 subunit.Phosphorylation of the NKA α1 subunit by tyrosine kinases has been shown to regulate pump activityi/o-GPCR signaling modulates β-cell [Ca2+]i oscillations, which in turn regulate pulsatile insulin secretion21. Here, we elucidate the mechanistic underpinnings of β-cell Gi/o signaling-induced Vm hyperpolarization. We found that Gi/o-GPCR activation stimulates β-cell NKAs, which hyperpolarizes Vm independently of KATP channel activity and leads to decreased islet [Ca2+]i oscillation frequency. Furthermore, STK activation was required for Gi/o signaling-induced activation of β-cell NKAs, whereas NKA-mediated Vm hyperpolarization was inhibited by cAMP-dependent PKA activation. This indicates that multiple signaling modalities converge to control NKA function and thus islet [Ca2+]i handling. These results demonstrate the importance of δ-cell SST secretion in controlling islet [Ca2+]i oscillations through SSTR-mediated regulation of β-cell NKA activity. Moreover, this work reveals the conserved  mechanism for Gi/o-GPCR control of β-cell NKA function. Therefore, the data establish that regulation of β-cell NKA activity by Gi/o signaling plays a critical role in tuning the frequency of islet [Ca2+]i oscillations, and thus the kinetics of insulin secretion.Islet Gi/o signaling hyperpolarizes β-cell Vm55, which has been attributed to Gi/o-GPCR-mediated activation of a K+ conductance. Stimulation of GIRK channel activity accounts for Gi/o signaling-induced Vm hyperpolarization in many tissues37 and this has also been proposed for β-cells36. While a small SST-induced GIRK-like conductance has been recorded in human β-cells16, elegant studies by Sieg et al. demonstrated that GIRK channels are not activated in mouse β-cells by ADR stimulation19. We also observed minimal activation of a SSTR-mediated GIRK conductance in mouse or human β-cells. However, as GIRK channel inhibition increased islet [Ca2+]i plateau fraction in islets undergoing SST-induced [Ca2+]i oscillations, GIRK channels are predicted to play a conserved role in Gi/o-GPCR-mediated β-cell Vm hyperpolarization. To further enhance β-cell GIRK channels responsible for this change in islet [Ca2+]i, K+ was removed from the extracellular solution. Surprisingly, SST did not stimulate β-cell currents or hyperpolarize Vm in the absence of extracellular K+, suggesting that SST-induced Vm hyperpolarization may not be due to a K+ conductance. Furthermore, SST-activated currents were present in β-cells lacking GIRK2 channels, were not inhibited by TPQ, and interestingly were outward at voltages below the equilibrium potential of K+, further establishing that a non-K+ conductance is activated by β-cell SSTR signaling. As Gi/o-GPCR signaling activates NKAs in other tissues and removal of extracellular K+ inhibits NKA activity, we considered NKAs a strong candidate for SST-induced β-cell Vm hyperpolarization40. This was confirmed using the NKA inhibitor Oua, which blocked SST-induced islet [Ca2+]i decreases. Moreover, epinephrine has been shown to hyperpolarize β-cell Vm and stimulate NKAs in certain tissues38, and indeed, our data confirmed that α2A-ADR signaling decreases β-cell [Ca2+]i by activating NKAs. Taken together, these results suggest that while activation of both NKAs and GIRK channels control islet [Ca2+]i plateau fraction, NKA activation serves a predominant and conserved role in Gi/o signaling-mediated β-cell Vm hyperpolarization.Numerous studies have shown that Gi/o-GPCR control of NKA activity are highly tissue-specific, but are strongly linked to the phosphorylation status of the NKA α subunit45. Our data show that β-cell NKA activity is inhibited by FSK- and GLP1R-mediated increases in [cAMP]i and that this effect depends on PKA activity. These data support previous findings indicating that Gi/o signaling-induced islet [Ca2+]i oscillations can be terminated by stimulating adenylyl cyclase (AC) activity, inhibiting phosphodiesterase (PDE) activity, or directly raising [cAMP]i21. Furthermore, as cellular metabolism increases β-cell [cAMP]i levels71, PKA-mediated NKA-inhibition would be predicted to be glucose-sensitive. These results strongly suggest that phosphorylation by PKA inhibits β-cell NKA activity.The mechanisms underlying Gi decreased only modestly immediately after SST treatment, which was likely a consequence of islet [Ca2+]i being clamped at an elevated level. Under these conditions, β-cell [cAMP]i was greatly reduced even prior to SST treatment, presumably due in-part to cAMP degradation by Ca2+-activated PDEs  as well as by diminished cAMP generation by Ca2+-inhibited ACs 73. Thus, it is probable that because [cAMP]i was already so low that Gi/o-GPCR activation could not reduce it further. Several minutes after SST treatment we observed β-cell [cAMP]i oscillations that were out-of-phase with [Ca2+]i oscillations, which is consistent with Ca2+-dependent inhibition of [cAMP]i accumulation . Taken together, these findings suggest that Gi/o-GPCR signaling (a) activates β-cell NKAs leading to inhibition of Ca2+ entry; (b) islet [Ca2+]i decreases drive cAMP generation leading to PKA-mediated NKA inhibition, which results in Ca2+ influx; (c) this in turn, reduces [cAMP]i and alleviates PKA inhibition of NKA, thus restoring tyrosine kinase-supported β-cell NKA Vm hyperpolarization. While SST-induced β-cell [Ca2+]i oscillations are regulated by [cAMP]i levels, there are other factors that could contribute to the oscillatory nature of Gi/o-GPCR-mediated β-cell NKA activity. For example, NKAs require sufficient [Na+]i levels to function74, thus NKA-mediated depletion of β-cell [Na+]i during periods of sustained activity may lead to NKA inactivation allowing for Vm depolarization and [Ca2+]i influx. Furthermore, as SSTRs and other Gi/o-GPCRs undergo desensitization and internalization when continually exposed to ligands75, receptor recycling would be predicted to play a role in β-cell NKA oscillations. However, when islet [Ca2+]i was clamped at a low level, SST-induced prolonged non-oscillatory decreases in islet [Na+]i that were inhibited with conditions that increase islet cAMP, suggesting that β-cell NKAs are primarily inhibited by cAMP-dependent PKA activity. Although, these findings indicate that Gi/o-GPCR-mediated β-cell NKA activity is tuned by fluctuations in PKA signaling, additional kinases that modulate NKA function  could also be impacted by changes in [cAMP]i80. Thus, future studies are required to more completely determine the mechanisms that control the oscillatory nature of Gi/o-GPCR-mediated β-cell NKA activity.β-cell [cAMP]i oscillations emerged over time, induction of SST-induced islet [Ca2+]i oscillations despite minimal initial [cAMP]i decreases suggested that cAMP was not the only signal controlling Gi/o-GPCR-mediated β-cell NKA oscillations. While cAMP-dependent changes in PKA activity modulate β-cell NKA function, Gi/o-GPCRs also stimulate cAMP-independent protein kinases that have been shown to influence NKA activity. For example, STK and Shp2, which activates STK, interact with SSTRs and are activated by SSTR signaling64. Moreover, STKs interact with and augment NKA activity46. As STK and Shp2 inhibition greatly diminished NKA-mediated decreases of islet [Ca2+]i, SST-induced STK signaling is predicted to facilitate activation of β-cell NKAs. Interestingly, the STK inhibitor used in these studies, Dasa, which is FDA approved for the treatment of Philadelphia chromosome-positive chronic myeloid leukemia, has been shown to decrease blood glucose levels in numerous clinical studies, most prominently in diabetic patients83. This suggests the possibility that Dasa increases human islet insulin secretion by inhibiting NKAs, and thus enhancing glucose-mediated Ca2+ entry.Although islet [cAMP]84. Our findings confirmed that insulin enhances β-cell NKA function; however, insulin-induced islet [Ca2+]i oscillations were only modestly affected by Dasa treatment, indicating that insulin stimulates β-cell NKAs independently of STK signaling, possibly through direct phosphorylation of NKA α1 subunits. Phosphorylation of NKA α1 by tyrosine kinases has been shown to augment NKA function85 and indeed, phosphorylation of islet NKA α1 residue Y10 increased following SST and insulin treatment. This establishes that Gi/o-GPCR-mediated β-cell NKA activation is due in-part to phosphorylation by tyrosine kinases. Therefore, it will be important to further examine the mechanisms underlying tyrosine kinase regulation of β-cell NKA function and its role in tuning islet [Ca2+]i oscillations as well as GSIS.Insulin receptors that interact with and control STK signaling are also tyrosine kinases and have been shown to stimulate NKAs in a variety of tissues86; thus, control of β-cell NKA function by SSTR signaling would be expected to be perturbed during T2D. Other islet cell types, including α- and δ-cells, also express high levels of NKA α1 subunit transcript13, which is supported by our immunofluorescence staining of human pancreatic sections that showed NKA α1 subunit expression in insulin-negative islet cells  and δ-cells 87. Moreover, NKAs have been shown to regulate α-cell Vm in response to fatty acid metabolism88. Thus, NKA control of plasma membrane Vm would be expected to influence Ca2+ handling in these other islet cell types and may help explain why GIRK channel inhibition fails to completely inhibit SST-induced α-cell Vm hyperpolarization57. Lastly, expression of specific NKA subunits, such as FXYD2, are altered in T2D human islets89 and in leptin receptor deficient diabetic mouse islets90. Thus, understanding the role(s) that NKA serves in all islet cell types under physiological and diabetic conditions will illuminate critical features of islet function and disfunction.SST secretion becomes defective during the pathogenesis of diabeteslls Fig. . Furtheri/o-coupled mechanism for controlling β-cell Ca2+ entry, and thus insulin secretion in response to numerous Gi/o-GPCR ligands that have been shown to limit insulin secretion . Moreover, we demonstrated that endogenous SSTR signaling tunes islet [Ca2+]i oscillations and presumably pulsatile insulin secretion by activating β-cell NKAs. Finally, we determined that Gi/o signaling stimulates β-cell NKA function by activating tyrosine kinases . Taken together, these findings reveal an essential and conserved NKA-mediated mechanism governing Gi/o-coupled signals known to regulate insulin secretion that remained elusive for over half a century  were generated by crossing mice expressing a mutant Gi/o-GPCR-P2A-mCitrine (B6.129-Gt(ROSA)26Sortm1Ute/J; Stock #: 026219; JAX) construct preceded by a loxP-flanked STOP cassette with mice expressing a SST-IRES-Cre 92. All δGi DREADD islets were prepared from mice heterozygous for SST-IRES-Cre as this transgene decreases endogenous SST expression in an allele dosage-dependent manner. Transgenic mice with pancreatic-specific knockout (KO) of Kcnj6 (gene encoding GIRK2) were generated by crossing animals with a loxP-flanked Kcnj6 exon 4 with mice expressing a Pdx1-Cre 94. All animals were housed in a Vanderbilt University IACUC (protocol # M1600063-01) approved facility on a 12-h light/dark cycle with access to standard chow  ad libitum. Mice were humanely euthanized by cervical dislocation followed by exsanguination. To preserve islet ion channel function mice were not treated with anesthesia.All mice were 10- to 16-week old, age-matched males on a C57Bl6/J background , Bar Harbor, ME). Transgenic mice expressing Ghttps:// www.chtn.org/). Written consent was obtained for deceased donors by the CHTN prior to reception of human pancreatic tissue.All studies detailed here were approved by the Vanderbilt University Health Sciences Committee Institutional Review Board IRB# 110164). Healthy human islets were provided from multiple isolation centers by the Integrated Islet Distribution Program (IIDP). Deidentified human donor information is provided in Table 10164. He60 and the coding sequence of hM4D(Gi)-mCherry 95 were cloned into a pLenti6 lentiviral transfer plasmid and utilized to produce 3rd-generation lentiviruses (LVs) that express Gi/o-DREADDs selectively in β-cells (βGi/o-DREADDs) as previously described96.Unless otherwise noted all chemicals and reagents were purchased from Sigma-Aldrich  or Thermo Fisher . Clozapine N-oxide (CNO) was purchased from Hello Bio . Clonidine hydrochloride (Clon), exendin-3 (9–39) amide (Ex9), forskolin (FSK), L-054,264, ouabain (Oua), and myristoylated PKI 14–22 amide (PKI) were purchased from R&D Systems . Tertiapin-Q (TPQ) was purchased from Alomone Labs . Dasatinib (Dasa), H-89, and NSC 87877 were purchased from Cayman Chemical . Liraglutide (Lira) was purchased from Novo Nordisk . An optimized rat insulin promoter (RIP)98; mouse islets were cultured in RPMI-1640 (Corning) media with 5.6 mM glucose supplemented with 15% FBS, 100 IU ml−1 penicillin, and 100 mg ml−1 streptomycin (RPMI) at 37 °C, 5% CO2. Upon arrival, human islets were allowed to recover for at least 2 h in CMRL-1066  media containing 5.6 mM glucose and supplemented with 20% fetal bovine serum (FBS), 100 IU ml−1 penicillin, 100 mg ml−1 streptomycin, 2 mM GlutaMAX, 2 mM HEPES, and 1 mM sodium pyruvate (CMRL) at 37 °C, 5% CO2. Mouse and human islets were cultured in poly-d-lysine-coated 35 mm glass-bottomed dishes  and all experiments were conducted within 48 h.Mouse pancreata were digested with collagenase P  and islets were isolated using density gradient centrifugation2, 10.0 EGTA, 10.0 HEPES, and 0.005 amphotericin B (adjusted to pH 7.2 with KOH). Mouse and human islets were patched in Krebs-Ringer HEPES buffer  containing (mM) 119.0 NaCl, 4.7 KCl, 2.0 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, and 10.0 HEPES (pH 7.35 adjusted by NaOH) supplemented with indicated glucose concentrations; a perforated whole-cell patch-clamp technique was utilized to record β-cell membrane potential (Vm) in current-clamp mode using an Axopatch 200B amplifier with pCLAMP10 software (Molecular Devices)100. Islet cells that did not display electrical activity at 2 mM glucose were identified as β-cells. After a perforated patch configuration was established  Vm depolarization and action potential (AP) firing were induced by exchanging the bath solution with KRHB supplemented with 20 mM glucose and 1 mM tolbutamide. After AP firing was observed, the amplifier was switched to voltage-clamp mode; Vm was held at −60 mV and the membrane voltage was ramped from −100 mV to −50 mV every 15 s for at least 3 min and the resulting β-cell currents recorded. The amplifier was then returned to current-clamp mode and Vm recorded. Mouse and human islets were perifused with further treatments and changes in β-cell Vm and currents measured as indicated in figure legends.Patch electrodes (3–4 MΩ) were backfilled with intracellular solution containing (mM) 90.0 KCl, 50.0 NaCl, 1.0 MgCl2+]i and [cAMP]i imaging, mouse islets were transduced for 4 h with an adenovirus (AV) expressing a CMV-jRGECO1a-P2A-cAMPr construct  and cultured 24 h at 37 °C, 5% CO2 prior to imaging. Alternatively, mouse islets were loaded with a Fura-2 AM Ca2+ indicator (2 µM) for 30 min before the start of an experiment. Human islets were either transduced for 4 h with an AV expressing RIP-GCaMP6s (VectorBuilder) and cultured 48 h before imaging or loaded with a Cal-590 Ca2+ indicator  for 1 h prior to the start of a study. Before each experiment, islet culture media was replaced with 2 mL KRHB supplemented with indicated glucose concentrations; after 10 min, the islets were treated as detailed in figure legends. Mouse islet jRGECO1a (excitation (Ex): 561 nm; emission (Em): 620 ± 50 nm) and cAMPr fluorescence  were simultaneously measured every 5 s utilizing an LSM 780 multi-photon confocal microscope equipped with Zeiss Zen software  as indicators of [Ca2+]i and [cAMP]i respectively. Mouse islet Fura-2 AM fluorescence  was measured every 5 s with a Nikon Ti2 epifluorescence microscope equipped with a Prime 95B camera with 25 mm CMOS sensors and Nikon Elements software ; the ratio of Fura-2 AM fluorescence excited at 340 nm and 380 nm was utilized as an indicator of [Ca2+]i. Human islet Cal-590  or β-cell GCaMP6s fluorescence  was measured every 5 s as an indicator of [Ca2+]i utilizing the LSM 780 microscope (×20 magnification).For simultaneous islet [Ca2+]i and [Na]i imaging, mouse islets were loaded with a Fura Red AM Ca2+ indicator  and an ION Natrium Green-2 (ING-2) Na+ indicator  for 1 h before the start of an experiment. Some experiments as indicated in figure legends were carried out with only ING-2. Before each experiment, islet culture media was replaced with KRHB supplemented with indicated glucose concentrations; after 10 min, the islets were treated as detailed in figure legends. Mouse islet Fura Red  fluorescence was measured every 5 s with the Nikon Ti2 (×10 magnification); the ratio of Fura Red fluorescence excited at 500 ± 10 nm and 430 ± 12 nm was utilized as an indicator of [Ca2+]i. Mouse islet ING-2 fluorescence  was measured every 5 s as an indicator of [Na]i.For simultaneous islet i, [cAMP]i, [Na+]i, and NKA immunofluorescence were analyzed using Zeiss Zen software, Nikon Elements software, and the ImageJ Fiji image processing pack. Axon Clampfit software was utilized to quantify β-cell SST-induced currents and Vm as well as perform cross-correlation analyses. All β-cell SST-induced currents are median values of 10 or more consecutive traces. Islet [Ca2+]i plateau fraction was defined as the fraction of time during which islet [Ca2+]i was ≥50% of islet [Ca2+]i oscillation amplitude. Heatmaps were generated using the MATLAB imagesc function. Period analysis of δGi islet [Ca2+]i oscillations was carried out utilizing the MATLAB detrend and findpeaks functions. Immunoblots were analyzed using Bio-Rad Image Lab 5.0. Figures were prepared utilizing Adobe Illustrator. Statistical analyses were carried out utilizing Microsoft Excel and GraphPad Prism 9.2.0 as indicated in figure legends; data were compared utilizing paired or unpaired two-sample t-tests, one-sample t-tests, or one-way analysis of variance (ANOVA) with Šidák’s post-hoc multiple comparisons tests. Data were normalized when appropriate as indicated in figure legends. Unless stated otherwise, data are presented as mean values ± standard error (SEM) for the specified number of samples (n). Differences were considered significant for P ≤ 0.05.Islet [CaFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


The term ‘Ca2+ homeostasis’ suggests that balancing resting Ca2+ levels is a rather static process. However, direct ER Ca2+ imaging shows that resting Ca2+ levels are maintained by surprisingly dynamic Ca2+ fluxes between the ER Ca2+ store, the cytosol, and the extracellular space. The data show that the ER Ca2+ leak, continuously fed by the high-energy consuming SERCA, is a fundamental driver of resting Ca2+ dynamics. Based on simplistic Ca2+ toolkit models, we discuss how the ER Ca2+ leak could contribute to evolutionarily conserved Ca2+ phenomena such as Ca2+ entry, ER Ca2+ release, and Ca2+ oscillations.At any moment in time, cells coordinate and balance their calcium ion (Ca Despitelum (ER) , our undsolution . These e signals .2+-concentration is about 100 nM. Upon stimulation, cytosolic Ca2+ concentrations rise to 0.5–1 µM and can reach tens of micromolar close to active Ca2+-channels  ∼100–200 µM] while maintaining high responsiveness to Ca2+.In the cytosol, the resting Cachannels . In the 2+ imaging, three fundamentally different principles were developed. One is based on the direct loading of cells with synthetic acetoxymethyl (AM)-estered Ca2+ indicators, such as Mag-Fura2-AM  or disry cells’ ] conditi2+ concentration in the ER lumen is about thousand-fold higher than in the cytosol and about 10–20x times lower than in the extracellular space  is needed.In a simplified view, a living cell generates the resting membrane potential by potassium ions that leak from inside the cell to the outside, via Ktrogenic . The molly clear , but the (2+ loss. The data confirmed that acute application of SOCE-blockers (SKF-96365/BTP-2) induces an immediate drop in ER Ca2+ levels  . We test+ levels   (2+ influx (The resting Capression  and seemctivity’ . The Ca2ll known  but in hd VGCCs) . We thin+ influx .2+ fluxes via the extracellular space? Perhaps, resting Ca2+ fluxes are needed to signal neuronal health. More SOCE-like Ca2+ entry or less active removal of cytosolic Ca2+ would lead to cellular Ca2+ overload. This has clinical implications. For instance, when SOCE blockers are used to prevent acute or neurodegenerative Ca2+ overload, resting homeostatic Ca2+ influx would be reduced. This would also reduce ER Ca2+ levels and thereby induce ER stress signaling  (see later).Theoretically, fast passive Casolution . When weobserved . We cannny years . It can + sparks  depend o2+ imaging (ER/cytosol) to find out how ER Ca2+ dynamics shape homeostatic Ca2+ fluxes. Astrocytes are well suited as a prototypical Ca2+ model , but no ryanodine receptors. Notably, there is just one SERCA  and a high amount of plasma membrane Ca2+-ATPases . Sec61a and other ER Ca2+ leak channel candidates  are highly expressed  and dual color Ca2+ model . The cel RNA-seq . The celxpressed . Based o2+ release .Cultured cortical astrocytes show depolarization-dependent ER Ca release , but do  release . Surprisels high   slope relationship with a spatiotemporal time-lag in the range of seconds  oscillations and are involved in the oscillation cycle, and might account for the temporal shift between ER and cytosolic calcium signals (2+ uniporter complex (MCU complex)  . Howeverchondria . For insbolished . However elegans . How mit2+ signal is IP3-induced Ca2+ release (IP3-iCR). In astrocytes, IP3-iCR can be activated by adenosine via the highly expressed metabotropic receptor Adora1a  that continuously promotes resting homeostatic Ca2+ influx.For clinical research, it will be important to know how the ‘Cal damage . It can


Developments in investigational agents and novel regimens in acute myeloid leukemia (AML) were reported in the 2022 American Society of Hematology (ASH) annual meeting. Encouraging efficacy data were presented from first-in-human studies of two investigational menin inhibitors, SNDX-5613 and KO-539, in relapsed and refractory (R/R) acute myeloid leukemia (AML) with KMT2A rearrangement or mutant NPM1, with overall response rates (ORR) of 53% (32/60) and 40% (8/20), respectively. The addition of the novel drug pivekimab sunirine, a first-in-class antibody–drug conjugate targeting CD123, to azacitidine and venetoclax in R/R AML resulted in an ORR of 45% (41/91), which rose to 53% in those who were venetoclax naïve. Additional novel triplet treatment combinations included the addition of magrolimab, an anti-CD47 antibody, to azacitidine and venetoclax, with an ORR of 81% (35/43) in newly diagnosed AML, including an ORR of 74% (20/27) in TP53 mutated AML. The addition of the FLT3 inhibitor gilteritinib to azacitidine/venetoclax was also featured, with an ORR of 100% (27/27) in newly diagnosed AML and an ORR of 70% (14/20) in R/R AML. To the editorEach year, exciting new developments and innovations in hematology are presented at the American Society of Hematology (ASH) meeting. In this article, we review some of the most impressive developments in investigational agents and novel regimens in acute myeloid leukemia (AML) from ASH 2022.Several presentations featured exciting activity from the novel class of menin inhibitors, which target the interaction of menin with the epigenetic regulators of KMT2A in AML. This inhibits leukemogenesis in AML cases featuring KMT2A rearrangement (KMT2Ar) or mutant NPM1 (mNPM1), two previously untargeted genetic alterations that represent 3–11% and 30% of newly diagnosed AML patients, respectively [An alternative menin inhibitor KO-539 (ziftomenib) was featured in Dr. Erba’s presentation of preliminary results from the KOMET-001 first-in-human Phase 1/2 trial of ziftomenib in adults with R/R AML. Preliminary efficacy data showed that in a similarly heavily pretreated population, the 600 mg dose had meaningful efficacy, with an ORR of 40% (8/20) and CRc in 35% (7/20) in mNPM1 AML, although outcomes were worse with KMT2Ar AML . Differentiation syndrome (DS) was a notable adverse event, with at least grade 3 severity occurring in 27% (8/29) of KMT2Ar patients including 1 death, although no grade 3 DS occurred in mNPM1 patients. However, DS was associated with improved response (75% of mNPM1 patients), and DS severity decreased with improved protocol guidance [Pivekimab sunirine  is a first-in-class antibody–drug conjugate (ADC) targeting CD123, which is expressed on most AML blasts, with a novel indolinobenzodiazepine pseudodimer (IGN) payload that alkylates DNA. In this phase 1b/2 study of the triplet regimen of PVEK, azacitidine (AZA), and venetoclax (VEN) presented by Dr. Daver, there was an ORR of 45% (41/91) with CRc of 25% (23/91), with higher responses (ORR 53% and CRc 38%) in the 47 patients who were VEN-naïve. The drug was well tolerated, with infusion reactions occurring in 22% (20/91) of patients, and only 5% discontinued treatment due to PVEK-related adverse events [Dr. Daver also presented data from the phase 1/2 study of the novel triplet regimen of magrolimab, an anti-CD47 antibody that blocks the “don’t eat me” signal on leukemia cells, with AZA and VEN in older/unfit or high risk AML. The ORR in newly diagnosed AML was similar to prior reported responses with AZA/VEN, with ORR 81% (35/43) including an ORR of 74% (20/27) with TP53 mutations. Median OS (mOS) was not reached in newly diagnosed non-secondary AML patients, with mOS 7.6 months in untreated secondary AML with TP53 mutation. Responses in R/R AML were more modest, with prior VEN exposed patients faring poorly (ORR 11%), resulting in closure of this study arm. Grade 3 anemia occurred in 23% (18/79), and median hemoglobin drop was 1.2 g/dL after magrolimab infusion [Another exciting novel triplet regimen was the addition of gilteritinib, an oral FLT3 inhibitor, to AZA/VEN in FLT3-mutated AML. The ORR in this phase I/II study presented by Dr. Short was 100% 27/27) with 92% CR in newly diagnosed patients, with median OS not yet reached and 1-year OS 85%. The ORR was 70% (14/20) in R/R patients, with 20% (4/20) achieving CR, and a median OS of 5.8 months. Outcomes were better in those who had not received prior VEN or gilteritinib, with mOS 10.5 months [/27 with Overall, the ASH 2022 annual meeting showcased several notable advances in the field of investigational therapies in AML, as summarized in Tables


Mentoring’s pivotal role in nurturing professional identity formation (PIF) owes much to its combined use with supervision, coaching, tutoring, instruction, and teaching. However the effects of this combination called the ‘mentoring umbrella’ remains poorly understood. This systematic scoping review thus aims to map current understanding.A Systematic Evidence-Based Approach guided systematic scoping review seeks to map current understanding of the ‘mentoring umbrella’ and its effects on PIF on medical students and physicians in training. It is hoped that insights provided will guide structuring, support and oversight of the ‘mentoring umbrella’ in nurturing PIF. Articles published between 2000 and 2021 in PubMed, Scopus, ERIC and the Cochrane databases were scrutinised. The included articles were concurrently summarised and tabulated and concurrently analysed using content and thematic analysis and tabulated. The themes and categories identified were compared with the summaries of the included articles to create accountable and reproducible domains that guide the discussion.A total of 12201 abstracts were reviewed, 657 full text articles evaluated, and 207 articles included. The three domains identified were definitions; impact on PIF; and enablers and barriers. The mentoring umbrella shapes PIF in 3 stages and builds a cognitive base of essential knowledge, skills and professional attitudes. The cognitive base informs thinking, conduct and opinions in early supervised clinical exposure in Communities of practice (COP). The COPs’ individualised approach to the inculcation of desired professional characteristics, goals, values, principles and beliefs reshapes the individual’s identity whilst the socialisation process sees to their integration into current identities.The mentoring umbrella’s provides personalised longitudinal support in the COP and socialisation process. Understanding it is key to addressing difficulties faced and ensuring holistic and timely support.The online version contains supplementary material available at 10.1186/s12909-022-03589-z. Mentoring plays a critical role in nurturing professional identity formation (henceforth PIF) or helping medical students and physicians (henceforth physicians in training) “think, act and feel like physicians” . This roThe first is evidence that role modelling, supervision, coaching, tutoring, teaching and instruction take on characteristics that liken them to mentoring when applied in a longitudinal manner to enduring and personalised educational relationships . KrishnaThe second is the notion that professional identity is part of a larger concept of identity and that self-concepts of identity are intimately related and informed by self-concepts of personhood or “what makes you, you” . As suchRadha Krishna and Alsuwaigh ’s Ring TIt is suggested that better understanding of these values, beliefs, and principles will reveal how a physician in training’s views their roles, responsibilities, and place within a team, family unit, professional community, and society and provide insights into the physician in training’s thinking, conduct and coping in the face of different situational, environmental, and/or relational influences –22.At the core of the Ring Theory is the Innate Ring that houses the individual’s spiritual, religious and/or theist beliefs, values, moral ideals, and ethical principles. These are shaped by the individual’s demographical and historical features such as the ethnicity, culture, religion, family unit, gender, society, country, and social group they were born into. These considerations influence the individual’s Innate Identity and their thinking, goals, motivations, and actions.The Individual Ring represents conscious function which includes the ability to think, feel, communicate, carry out actions, and interact with the environment. The Individual Ring houses the individual’s values, beliefs, principles, biases, preferences, thoughts, emotions, experiences, decision making and personality which shape Individual Identity.The Individual Ring also acts to balance the thinking, goals, motivations, and actions drawn from the Innate, Relational and Societal Identities.The Relational Ring consists of personal relationships deemed to be important to the individual, and the values and beliefs that stem from and inform these relationships. The Societal Ring contains societal, religious, professional, and legal expectations set out in the individual’s society to guide and police conduct. One’s professional identity resides here.These identities may come into conflict when professional involvement in cases such as those involving palliative sedation, withdrawal or withholding of treatment, termination of pregnancy or familial determination arise.A structured ‘mentoring umbrella’ approach replete with a combination of mentoring, supervision, coaching, tutoring, teaching and instruction may be key to structuring and guiding this professional identity formation process. Indeed, Krishna et al. (2018) suggest that the most significant role of this holistic approach is its ability to support students, residents and junior doctors during periods of negotiation where new experiences and obstacles are either accepted, adapted to fit their particular circumstances or needs (compromised) or rejected . Kuek, NWith Sarraf-Yazdi et al. (2021) suggesting that mentoring helps each of the four identities adapt to the inculcation of these new professional values and responsibilities, evaluating elements of the ‘mentoring umbrella’ more closely may clarify its role within any proposed PIF focused training program.A Systematic Evidence-Based Approach guided systematic scoping review (henceforth SSR in SEBA) is used to map what is known about the effects of mentoring, supervision, coaching, tutoring, teaching and instruction upon PIF –36. GiveThis SSR in SEBA is overseen by an expert team comprised of medical librarians from the Yong Loo Lin School of Medicine (YLLSoM) and the National Cancer Centre Singapore (NCCS), and local educational experts and clinicians at NCCS, the Palliative Care Institute Liverpool, YLLSoM and Duke-NUS Medical School who guide, oversee and support all stages of SEBA to enhance the reproducibility and accountability of the process –49 Fig..Fig. 2ThThe research and expert teams set out the overarching goals, study population, context and remediation programs to be evaluated.The PICOS format was used to guide the inclusion criteria Table What is known about the effect of mentoring, supervision, coaching, tutoring, teaching and instruction on professional identity formation amongst medical students, residents and junior doctors?”To identify the research question, the expert and research teams were guided by the Population, Intervention, Comparison, Outcome and Study Design (PICOS) elements of the inclusion criteria , 51. Thest January 2000 to 31st December 2020 to account for prevailing manpower and time constraints. Additional ‘snowballing’ of references of the included articles ensured a more comprehensive review of the articles [In keeping with Pham, Rajić ’s recommarticles .Using an abstract screening tool, the research team independently reviewed abstracts to be included and employed ‘negotiated consensual validation’ to achieve consensus on the final list of articles to be included .Guidance on the conduct of narrative synthesis in systematic reviews” by Popay, Roberts [identifying and operationalizing a priori coding categories” from “The Development of Professional Identity” by Cruess and Cruess [. ‘Negotiated consensual validation’ was used as a means of peer debrief in all three teams to further enhance the validity of the findings [The split approach  sees con Roberts . The sec Roberts  to find  Roberts  to “idend Cruess . ‘Negotifindings .The Jigsaw Perspective employs Phases 4 to 6 of France et al. ’s adaptaThemes/categories were compared with the tabulated summaries   and hinder (barriers) the impact of mentoring, supervision, coaching, teaching and instruction upon PIF. These may be divided into mentee, mentor and institutional factors.Mentee related factors influencing the efficacy of the mentoring umbrella include being motivated, proactive, invested in the mentoring process and relationships, reflective, willingness to take feedback and make necessary adaptations and assign sufficient time to training , 169–176Mentor related factors consider all the roles played under the aegis of the mentoring umbrella. These include being motivated and invested in mentoring, having the abilities, availabilities and experience required, possessing good listening and communication skills, a commitment to self-improvement and learning, being open to feedback and learning from the mentee, being able to provide holistic and longitudinal support and understanding and abiding by the expectations and standards of practice expected of a mentor , 178.The host organization plays a critical role in matching, training, supporting and structuring the training process. The implementation of protected time and formal recognition of participation in mentoring help maintain motivation.The host organization also plays a part in establishing clear codes of conduct, roles and responsibilities and expectations of all stakeholders, structuring the mentoring process, providing it a formal place in the curriculum, assessing and overseeing the program , 174. ThThe themes drawn from evidenced-based publications were compared with those from non-data based articles  found that the themes from both groups to be similar and non-data based articles did not bias the analysis untowardly.Most of the included articles were data-driven (175 out of 207) whilst the remaining articles were non-data-based articles . Despite non-data-based articles forming a small minority of articles, we examined themes drawn from the non-data-driven publications and compared them with those from data-based articles . This process revealed similarities between the two groups suggesting that non-data-based articles did not bias the analysis untowardly.A majority of articles only stated the outcomes of the mentoring umbrella without addressing mechanisms via which they exert their influence , 182. GiIn answering its primary question, this SSR in SEBA of the mentoring umbrella’s effects on PIF provides a number of insights into the mentoring umbrella’s influence on the stages of PIF development and the role of the host organization.When applied longitudinally to an individualised learning relationship, across different settings involving one learner or a small group of learners with common goals, abilities and experiences, the mentoring umbrella provides an individualised perspective of development. This approach accounts for the physician-in-training’s and the instructor’s, teacher’s, coach’s, supervisor’s and tutor’s abilities, availabilities, attitudes, context, competencies, demographics, experiences, goals, motivations, and needs, in addition to building upon the physician in training’s successes, failures and reflections to enhance their longer term development. The overlapping elements within the mentoring umbrella provide synergistic support in addressing the influences of the physician-in-training’s societal, professional, clinical, academic, research, and personal considerations, the regnant sociocultural considerations, the influence of prevailing healthcare and educational system and the impact of the local hidden, informal and formal curriculum, upon PIF. This affirms the notion that the mentoring umbrella may be applied widely and in the stage based manner that allows them mentoring umbrella to shape PIF.The first stage of mentoring umbrella’s influence on PIF begins with the building of a ‘cognitive base’ of knowledge, skills, relevant expectations, roles, responsibilities around the physician-in-training’s goals, abilities, milestones, experience and setting. The cognitive base also inculcates regnant standards of professionalism and sociocultural considerations. Much of this personalisation in this stage falls upon tutoring, teaching and instruction.Applied longitudinally, the mentoring umbrella also advance mutual understanding, trust and open communication, networking, interprofessional collaborations, research output, and enhances clinical and research competencies.a persistent, sustaining social network of individuals who share and develop an overlapping knowledge base, set of beliefs, values, history and experiences focused on a common practice and/or enterprise” [Early exposure to clinical practice builds upon a personalised cognitive base and occurs in communities of practice. Barab et al. (2004) define CoPs as “erprise” . Here tha representation of self, achieved in stages over time during which the characteristics, values, and norms of the medical profession are internalised, resulting in an individual thinking, acting and feeling like a physician”.Cruess et al. (2015)  describeWhilst technically part of the COP, the precise mechanism in which the socialisation process helps the integration of new values, beliefs and principles are integrated into current identities remains unclear. However it does appear that within the socialisation process the mentoring umbrella provides physicians in training with personalised, responsive, appropriate and timely support as they confront ethical, cultural, philosophical, religious and social issues that conflict with their Innate, Individual, Relational and Societal values, beliefs and principles. Here, coaching’s ability to observe , 67, lisThis review also underscores the role of the host organisation , 171 in Here, the various aspects of the mentoring umbrella encapsulate many of the primary influences upon PIF set out by Cruess and colleagues  , 184. PeOne of the main limitations of this study was its inability to differentiate residents and junior doctors in training from more senior doctors such as consultants, attendings, specialists and senior consultants who have completed their training and physicians who are not in training programs. This limited the number of articles included. In addition, difficulties separating these groups also made analysis of the data difficult given the different levels of experience, roles, responsibilities and needs amongst the included groups of physicians given the diversity of the training programs and different settings and educational and healthcare programs adopted.Moreover, whilst this study was intended to analyse the wide range of current literature on mentoring and PIF programs, our review was limited by a lack of consistent reporting of current programs. Furthermore, most of the included papers were largely drawn from North American and European practices potentially limiting the applicability of these findings in other healthcare settings. This was compounded by our focus upon articles that were published in English.Whilst taking into account the limited resources and availability of the research and experts teams and limiting the review to the specified dates to increase the chances of completing the review, this too could have seen important articles excluded.This SSR in SEBA highlights the role of mentoring umbrella in nurturing PIF. Whilst the three stages built on posits by Cruess and colleagues remain to be evidenced, it does underline the need for longitudinal and holistic evaluation of the mentoring umbrella’s impact on PIF. Further understanding of the mentoring umbrella and its role in PIF also demands better appreciation of the need for personalised, holistic and longitudinal assessments and individualised and timely support. These gaps represent some of the key areas for future studies seemingly as the role of portfolios and longitudinal assessment measures to enhance support of evolving concepts of PIF develop.Additional file 1: Appendix A. Tabulated Summaries


An increase in intracellular calcium concentrations ([Ca2+]i) is a common event in most stress-induced signal transduction pathways. In recent years, significant progress has been made in research related to the early events of stress signaling in plants, particularly in the identification of primary stress sensors. This review highlights current advances that are beginning to elucidate the mechanisms by which abiotic environmental cues are sensed via Ca2+ signals. Additionally, this review discusses important questions about the integration of the sensing of multiple stress conditions and subsequent signaling responses that need to be addressed in the future.Plants are exposed to various environmental stresses. The sensing of environmental cues and the transduction of stress signals into intracellular signaling are initial events in the cellular signaling network. As a second messenger, Ca Plants encounter various abiotic stresses in their natural environment, for example, mechanical stimuli, drought, flood, cold, and salinity stress. These adverse conditions often limit plant growth and crop productivity worldwide . To survTypically, a primary abiotic stress sensor is required to perceive external environmental changes; in response to such changes, the sensor remodels signal transduction pathways and initiates appropriate responses that allow the plant to adapt to the stress condition. Thus, perception of the external environment by a sensor is the earliest step in the conversion of external stimuli into cellular signals by plants.2+), an indispensable second messenger, is considered to be a critical element by which plants modulate a complicated network of signaling pathways to respond to various abiotic stresses (2+]i) is one of the earliest signaling events when plants are challenged with abiotic stimuli, and the resulting Ca2+ signaling regulates many processes in plants, including transcriptional regulation and subsequent physiological as well as developmental responses , the link between abiotic stress sensors and the Ca2+ influx channels that are regulated by these sensors is still largely unknown. Here, we mainly discuss recent findings related to Ca2+ signaling to reveal the mechanisms by which plants sense stressful environment; these findings have advanced our understanding of plant sensory mechanisms.In the resting state, i increase by activating certain Ca2+-permeable channels, allowing plant cells to perceive changes in the physical properties of the membrane -like proteins (MSLs), Mid1-complement activity protein channels (MCAs), two-pore potassium channels (TPKs), Piezo channels (PZO), and reduced hyperosmolality induced i and is hypersensitive to growth inhibition by Na+  to form glycosylinositol phosphorylceramide (GIPC) sphingolipids in the external layer of the plasma membrane, and it is essential for NaCl-triggered depolarization of the cell-surface potential  mutant, indicating that TPC1 mediates Ca2+ release from the vacuole and facilitates the propagation of Ca2+ waves  is required for NaCl-activated Ca2+ influx through the plasma membrane in root epidermal cells. The loss-of-function mutation of AtANN4 also disrupts the salt-induced [Ca2+]i increases in Arabidopsis , which was the first potential cold sensor to be identified in rice. Overexpression of COLD1 significantly improves survival rates after chilling treatment, whereas mutants with deficiency or decreased expression of COLD1 are sensitive to chilling stress , which is produced extracellularly in response to external stresses and internal cues. Extracellular hydrogen peroxide (eH2O2) plays a vital role in many physiological processes during the lifecycle of plants, such as root development, pollen tube growth, organ wilting, and responses to biotic and abiotic stress (Reactive oxygen species (ROS) play a key role in plant cell signaling. Plant cells generate various ROS, including hydrogen peroxide , which regulates ABA signaling and stomatal closure in guard cells  and stimulating stomatal closure  EF-hands and promotes H2O2 which then subsequently activates Ca2+ channels in response to a range of abiotic stresses  mutants in Arabidopsis and showed that HPCA1 encodes a leucine-rich repeat receptor kinase that localizes to the cell surface. It was the first molecular component found to be responsible for sensing eH2O2  and Shenzhen Peacock Plan (KQTD2017032715165926).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A common hypothesis is that Ca2+ entering through ciliary ion channels may reduce cystogenesis. The cilia have at least two Ca2+-conducting channels: polycystin-2 (PC2) and TRPV4  cation channel, subfamily V, member 4), but how substantially they can increase intraciliary Ca2+ is unknown. By recording channel activities in isolated cilia, conditions are identified under which the channels can increase free Ca2+ within the cilium by at least 500-fold through regenerative (positive-feedback) signaling. Ca2+ that has entered through a channel can activate the channel internally, which increases the Ca2+ influx, and so on. Regenerative signaling is favored when the concentration of the Ca2+ buffer is reduced or when a slower buffer is used. Under such conditions, the Ca2+ that enters the cilium through a single PC2 channel is sufficient to almost fully activate that same channel. Regenerative signaling is not detectable with reduced external Ca2+. Reduced buffering also allows regenerative signaling through TRPV4 channels, but not through TRPM4  channels, which are activated by Ca2+ but do not conduct it. On a larger scale, Ca2+ that enters through TRPV4 channels can cause secondary activation of PC2 channels. I discuss the likelihood of regenerative ciliary Ca2+ signaling in vivo, a possible mechanism for its activation, and how it might relate to cystogenesis.Polycystic kidney disease (PKD) is a leading cause of end-stage renal disease. PKD arises from mutations in proteins, one a Ca In PKD, renal epithelial cells often have reduced cytoplasmic Ca2+ , a hereditary disorder that often causes end-stage renal disease, is a ciliopathy. It results from mutations in proteins that are expressed in part in the cilia of renal epithelial cells . Cystic mic Ca2+ , and reshenotype . Thus itnal Ca2+ .2+ into the renal primary cilium. The first of these is polycystin-2 . PC2 is a member of the TRPP subfamily of transient receptor potential (TRP) cation-conducting channels  mechanism. A small Ca2+ influx would increase the Ca2+ concentration at the internal face of the channel. That would further activate the channel, increasing the Ca2+ influx, and so on. In this report, I identify laboratory conditions that allow powerful amplification of ciliary Ca2+ signals through positive feedback. With reduced Ca2+ buffering, Ca2+ influx through a single PC2 channel is sufficient to drive that same channel to an almost fully open state near a typical resting membrane potential. On a larger scale, activation of TRPV4 channels can create sufficient intraciliary Ca2+ to secondarily activate PC2 channels. I discuss the likelihood of regenerative Ca2+ signaling in vivo. I also propose a means for its activation and relate that to a possible mechanism of cystogenesis in PKD.Both PC2  and TRPVnal Ca2+ . One can2+ external solution), the cilium was excised from the cell. This left the cilium inside the recording pipette in the inside-out configuration. The pipette containing the cilium was then transferred among different solutions that bathed the cytoplasmic face of the membrane. A schematic diagram appears elsewhere , and GSK1016790A, a TRPV4 agonist (50 nM) were added to some internal solutions as noted.The beads coated with cells were stored in a standard external (extracellular) solution containing (in mM) NaCl 140, KCl 5, CaCl current . All int2+ buffering. Voltage was maintained at +60 mV. If no large-conductance channels were seen to open within 2 min, active TRPP2 channels were judged to be absent.For some studies, I selected cilia with no detectable PC2 channels. To check for the presence of TRPP2 channels, the cilium was exposed to one of the solutions with reduced CaK'Ca between Ca2+ and each of the three Ca2+ buffers used were determined by Scatchard analysis (2+-selective electrode (Orion 932000). The K'Ca values were 6.7 × 106 M−1 for BAPTA, 2.0 × 106 M−1 for EGTA, and 0.78 × 106 M−1 for dibromoBAPTA. The x-intercept of the Scatchard plot also yielded the purity of the chelator, which was 84–88%. The impurity is reportedly water (2+ solutions prepared. Our double-distilled water contained 9 ± 1 µM Ca2+ (n = 6 determinations), and this is included in the values shown for [Ca2+]total. The low-Ca2+ solutions buffered with BAPTA or EGTA were not sensitive to small errors in the concentrations of total Ca2+ or buffer. In all cases, a 2% error (high or low) in either quantity gave a concentration of free Ca2+ between 13 and 21 nM.Apparent association constants analysis  with a Cly water . CalciumBAPTA, EGTA, GSK1016790A, and U-73122 were purchased from MilliporeSigma and dibromoBAPTA from Santa Cruz Biotechnology. GSK1016790A was diluted from a 100 µM stock solution and U-73122 from a 5 mM stock solution, both in DMSO.For voltage jumps, the cilium was clamped at a prepulse potential for 10 s, which was then stepped to a more positive (postpulse) potential for 10 s. Prepulse potentials tested ranged from −140 mV to +20 mV and postpulse potentials from +20 mV to +80 mV. For analysis, the mean current in the absence of detectable channel openings (leak current) was subtracted in QuB . Transient currents are defined as the mean current during the first 3 s of the postpulse minus the mean current during the final 3 s of the postpulse. For steady-state currents, mean currents over a 20 s recording, minus the leak current, are reported.For macroscopic TRPV4 current-voltage relations, current was measured as voltage was continuously varied from −100 or −140 mV to +100 mV. Current at −90 mV was figured as the mean of the values from −100 mV to −80 mV, and current at +90 mV as the mean of the values from +80 mV to +100 mV. For the single-channel study of TRPV4, 20 s recordings were made at each voltage. Single-channel currents were determined from amplitude histograms.p < 0.05.Results are expressed as mean ± standard error. Data sets were compared with a nonparametric test, either the Wilcoxon signed rank test (for paired measures) or the Mann-Whitney rank sum test (for unpaired measures). Our criterion for significance was 2+ entering the cilium through PC2 channels might accumulate inside the cilium to a concentration sufficient to further activate those same PC2 channels. I have been able to verify the existence of such conditions in the laboratory.One can predict that there are conditions under which Ca2+ , while maintaining the low free Ca2+ concentration, prevented the transient current . Prepulse voltages sufficient to cause transient activation varied from one cilium to the next. In the primary cilium of mIMCD-3 cells, in the absence of agonists, PC2 channels are the only large-conductance channel seen, and the only channel activated by micromolar concentrations of intraciliary Ca2+ . Under cuffering . The tra was low , left. Auffering , right. versible . In eachmM BAPTA . The dif2+ to enter the cilium. Even at a steady potential, though, PC2 channels might conduct enough Ca2+ into the cilium to increase their own activities. This was also confirmed experimentally. In 12 of 13 cilia tested, channel activity over some range of holding potentials was greater when Ca2+ buffering was reduced .The voltage prepulses I tested temporarily increased the driving force for Ca reduced , left thuffering , right. uffering  and was uffering . In the uffering . The dif2+ buffering as the potential was changed from −20 to +20 mV . EGTA should be less effective than BAPTA at restricting the accumulation of intraciliary Cafor EGTA . Thus a otential . The tra channel , and the channel . In 11 c channel . The eff2+ buffer instead of BAPTA (each at 0.25 mM). This was seen in cilia with several PC2 channels .In most cilia tested, steady-state channel activity was greater at some holding potentials with EGTA as the Cachannels  and in 3channels . The effchannels . In the channels . The dif2+, in all cases well below the ∼1 µM or more needed to activate PC2 in previous reports ; with 0.1 µM external Ca2+ the value was 0.8 ± 0.4 pA (n = 9 cilia) . This was true (p = 0.0024) even if the three very high values from the population tested in 2 mM Ca2+ were excluded. With reduced external Ca2+, steady-state PC2 current at +40 or +60 mV did not depend on the concentration of the Ca2+ buffer . With reduced cytoplasmic Ca2+ buffering, mean steady-state current was greater with 2 mM external Ca2+  than with 0.1 µM external Ca2+  .Each experiment shown so far compares two internal solutions that were prepared to have identical concentrations of free Ca reports . Voltagen of PC2 . With 2 9 cilia) . The val+ buffer . The mea3 cilia) . The dif2+ that activates PC2 channels when Ca2+ buffering is reduced may enter through the PC2 channels themselves, but the experiments thus far do not allow that conclusion. An ideal control would be a PC2 channel that has the usual sensitivity to intraciliary Ca2+ but does not conduct Ca2+. If transient activation were to be seen with such a channel, it would indicate that there is a Ca2+ source other than flow through PC2 itself. No such PC2 variant has been described, but the renal primary cilium has another channel that can serve this purpose: TRPM4. These channels are present in the primary cilia of most mIMCD-3 cells and, like other TRPM4 channels reported, are activated by internal Ca2+ but do not conduct Ca2+ . When a cilium was moved to an internal solution with just 15 nM free Ca2+ but also reduced Ca2+ buffering (0.1 mM BAPTA), almost no TRPM4 current was seen (n = 18 cilia). The reduction of TRPM4 current is significant . On return to the internal solution with 10 µM free Ca2+, the TRPM4 current was restored (2+ and reduced buffering to that activated in 10 µM free Ca2+ was 0.03 ± 0.01 (n = 18 cilia).The top trace of 2+-activated current was conducted through TRPM4 channels, the experiment was repeated in cells in which TRPM4 was knocked down with shRNA (2+ was 0.21 ± 0.05 pA (n = 8 cilia), which is significantly less that the current in wild-type cells . With reduced Ca2+ buffering, a prepulse to a negative voltage did not give rise to a detectable transient TRPM4 current in cilia from wild-type cells when the voltage was jumped from −120 or −140 mV to +60 mV (n = 10 cilia). In the absence of PC2 channels, I found no evidence of a Ca2+ influx that could activate TRPM4 even with reduced Ca2+ buffering. This supports the hypothesis that the activation of PC2 described above occurs following an influx of Ca2+ through the PC2 channels themselves.To verify that the Cath shRNA . In cilio +60 mV . Followi2+ buffered with 0.25 mM BAPTA, the ciliary macroscopic current-voltage relation showed a small leak conductance (n = 18 cilia). The mean reversal potential of the additional current (intersection of the red and black recordings) was −9.0 ± 0.6 mV (n = 14 cilia).The primary cilia of mIMCD-3 cells also express TRPV4 channels. To study TRPV4 currents in isolation, cilia lacking active PC2 channels were again selected. With internal Caductance , black. ductance  activateductance , red. Thductance , orange.2+ , 50 nM internal GSK1016790A activated an outwardly rectifying current (n = 7 cilia). With 2 mM BAPTA, the additional current averaged 3.8 ± 1.3 pA at −90 mV and 3.7 ± 3.6 pA at +90 mV (n = 7 cilia). The effect of buffer concentration is significant at both potentials .TRPV4 channels can be activated by internal Cauct Ca2+ . In otheuct Ca2+ . I was a current , orange,2+ and can be activated by intraciliary Ca2+. Thus a Ca2+ influx through one of these channel types might activate the other. I tested whether ciliary PC2 channels could show a secondary activation following direct activation of TRPV4 in the presence of 0.25 mM BAPTA. 2+ and no agonist, the PC2 channel opened only at strongly depolarizing potentials (2+. The influx of Ca2+ and Na+ is cancelled by an efflux of K+.) On addition of 50 nM GSK1016790A, the open probability of the single PC2 channel at −5 mV increased from almost 0 to a value approaching 1 . In the presence of GSK1016790A, the mean current in the same cilia at −5 mV averaged 37 ± 21 pA (n = 8 cilia). The effect of GSK1016790A on PC2 activity is significant .In a second cilium, the agonist activated a mean PC2 current at −5 mV of 177 pA , right, 2+ signaling in the primary cilia of renal epithelial cells. 2+ from the external compartment and also are activated by intraciliary Ca2+. This can result in positive feedback: As Ca2+ enters the cilium, it increases the activity of PC2 and TRPV4 channels, causing further Ca2+ entry, which causes further activation, and so on. Intraciliary Ca2+ also increases the open probability of TRPM4 channels, but those do not conduct Ca2+. Opening any of these channels should depolarize the cilium. That may further contribute to a regenerative response, since the PC2 channels are also activated by depolarization . The interaction potentiates Ca2+-induced Ca2+ release unless the channel function of PC2 is eliminated by mutation. The potentiation is sensitive to the speed and concentrations of the Ca2+ buffers chosen. A similar system was described in oocytes overexpressing PC2 and IP3R . The additional intraciliary Ca2+ arose from Ca2+ flowing through PC2 channels into the cilium, where it was able to overcome the dilute Ca2+ buffer and accumulate. Consistent with this explanation, positive feedback could not be achieved with low external Ca2+, nor with a channel (TRPM4) that is activated by Ca2+ but does not conduct it. When BAPTA was replaced with EGTA, a buffer that is slower to bind Ca2+, positive feedback was enhanced  The pr to 1 mM , and the to 1 mM . One can of cAMP . Consistactivity . Althougactivity .2+, perhaps regenerative, in vivo. Such an increase has been reported on deflection of the cilium (2+ increase is mediated by ciliary channels or by backflow of Ca2+ from the cell body into the cilium (N-terminal fragment of PC1 activates PC2 channels and initiates an influx of Ca2+ into the primary cilium (in vivo (It also remains to learn what stimuli might initiate an increase in intraciliary Cae cilium . Howevere cilium . In mIMCy cilium . Also in(in vivo .2+ influx in response to hypotonicity (2+, which could then inhibit the ciliary cyclases, reduce ciliary cAMP, and slow the progression of PKD. The increased ciliary Ca2+ could secondarily activate PC2, but such a model does not explain why the channel function of PC2 in particular is needed to prevent cystogenesis. In a model of autosomal recessive PKD, increased water intake, which can reduce the osmolality of the urine, did slow the progression of the cystic disease (An unexplored possibility is that TRPM3 and TRPV4 may be osmotically activated in the cilium. TRPM3 and TRPV4 are typically activated by hypoosmolality , althougtonicity . Reducin disease . Increas disease  and has  disease , and exi disease  have not