Early-phase drug discovery of ß-III-spectrin actin-binding modulators for treatment of spinocerebellar ataxia type 5.

ß-III-Spectrin is a key cytoskeletal protein that localizes to the soma and dendrites of cerebellar Purkinje cells and is required for dendritic arborization and signaling. A spinocerebellar ataxia type 5 L253P mutation in the cytoskeletal protein ß-III-spectrin causes high-affinity actin binding. Previously we reported a cell-based fluorescence assay for identification of small-molecule actin-binding modulators of the L253P mutant ß-III-spectrin. Here we describe a complementary, in vitro, fluorescence resonance energy transfer (FRET) assay that uses purified L253P ß-III-spectrin actin-binding domain (ABD) and F-actin. To validate the assay for high-throughput compatibility, we first confirmed that our 50% FRET signal was responsive to swinholide A, an actin-severing compound, and that this yielded excellent assay quality with a Z' value > 0.77. Second, we screened a 2684-compound library of US Food and Drug Administration-approved drugs. Importantly, the screening identified numerous compounds that decreased FRET between fluorescently labeled L253P ABD and F-actin. The activity and target of multiple Hit compounds were confirmed in orthologous cosedimentation actin-binding assays. Through future medicinal chemistry, the Hit compounds can potentially be developed into a spinocerebellar ataxia type 5-specific therapeutic. Furthermore, our validated FRET-based in vitro high-throughput screening platform is poised for screening large compound libraries for ß-III-spectrin ABD modulators.

Cardiac ryanodine receptor N-terminal region biosensors identify novel inhibitors via FRET-based high-throughput screening.

The N-terminal region (NTR) of ryanodine receptor (RyR) channels is critical for the regulation of Ca2+ release during excitation-contraction (EC) coupling in muscle. The NTR hosts numerous mutations linked to skeletal (RyR1) and cardiac (RyR2) myopathies, highlighting its potential as a therapeutic target. Here, we constructed two biosensors by labeling the mouse RyR2 NTR at domains A, B, and C with FRET pairs. Using fluorescence lifetime (FLT) detection of intramolecular FRET signal, we developed high-throughput screening (HTS) assays with these biosensors to identify small-molecule RyR modulators. We then screened a small validation library and identified several hits. Hits with saturable FRET dose-response profiles and previously unreported effects on RyR were further tested using [3H]ryanodine binding to isolated sarcoplasmic reticulum vesicles to determine effects on intact RyR opening in its natural membrane. We identified three novel inhibitors of both RyR1 and RyR2 and two RyR1-selective inhibitors effective at nanomolar Ca2+. Two of these hits activated RyR1 only at micromolar Ca2+, highlighting them as potential enhancers of excitation-contraction coupling. To determine whether such hits can inhibit RyR leak in muscle, we further focused on one, an FDA-approved natural antibiotic, fusidic acid (FA). In skinned skeletal myofibers and permeabilized cardiomyocytes, FA inhibited RyR leak with no detrimental effect on skeletal myofiber excitation-contraction coupling. However, in intact cardiomyocytes, FA induced arrhythmogenic Ca2+ transients, a cautionary observation for a compound with an otherwise solid safety record. These results indicate that HTS campaigns using the NTR biosensor can identify compounds with therapeutic potential.

Molecular Mechanism of a FRET Biosensor for the Cardiac Ryanodine Receptor Pathologically Leaky State.

Ca2+ leak from cardiomyocyte sarcoplasmic reticulum (SR) via hyperactive resting cardiac ryanodine receptor channels (RyR2) is pro-arrhythmic. An exogenous peptide (DPc10) binding promotes leaky RyR2 in cardiomyocytes and reports on that endogenous state. Conversely, calmodulin (CaM) binding inhibits RyR2 leak and low CaM affinity is diagnostic of leaky RyR2. These observations have led to designing a FRET biosensor for drug discovery targeting RyR2. We used FRET to clarify the molecular mechanism driving the DPc10-CaM interdependence when binding RyR2 in SR vesicles. We used donor-FKBP12.6 (D-FKBP) to resolve RyR2 binding of acceptor-CaM (A-CaM). In low nanomolar Ca2+, DPc10 decreased both FRETmax (under saturating [A-CaM]) and the CaM/RyR2 binding affinity. In micromolar Ca2+, DPc10 decreased FRETmax without affecting CaM/RyR2 binding affinity. This correlates with the analysis of fluorescence-lifetime-detected FRET, indicating that DPc10 lowers occupancy of the RyR2 CaM-binding sites in nanomolar (not micromolar) Ca2+ and lengthens D-FKBP/A-CaM distances independent of [Ca2+]. To observe DPc10/RyR2 binding, we used acceptor-DPc10 (A-DPc10). CaM weakens A-DPc10/RyR2 binding, with this effect being larger in micromolar versus nanomolar Ca2+. Moreover, A-DPc10/RyR2 binding is cooperative in a CaM- and FKBP-dependent manner, suggesting that both endogenous modulators promote concerted structural changes between RyR2 protomers for channel regulation. Aided by the analysis of cryo-EM structures, these insights inform further development of the DPc10-CaM paradigm for therapeutic discovery targeting RyR2.

Novel drug discovery platform for spinocerebellar ataxia, using fluorescence technology targeting ß-III-spectrin.

Numerous diseases are linked to mutations in the actin-binding domains (ABDs) of conserved cytoskeletal proteins, including ß-III-spectrin, α-actinin, filamin, and dystrophin. A ß-III-spectrin ABD mutation (L253P) linked to spinocerebellar ataxia type 5 (SCA5) causes a dramatic increase in actin binding. Reducing actin binding of L253P is thus a potential therapeutic approach for SCA5 pathogenesis. Here, we validate a high-throughput screening (HTS) assay to discover potential disrupters of the interaction between the mutant ß-III-spectrin ABD and actin in live cells. This assay monitors FRET between fluorescent proteins fused to the mutant ABD and the actin-binding peptide Lifeact, in HEK293-6E cells. Using a specific and high-affinity actin-binding tool compound, swinholide A, we demonstrate HTS compatibility with an excellent Z'-factor of 0.67 ± 0.03. Screening a library of 1280 pharmacologically active compounds in 1536-well plates to determine assay robustness, we demonstrate high reproducibility across plates and across days. We identified nine Hits that reduced FRET between Lifeact and ABD. Four of those Hits were found to reduce Lifeact cosedimentation with actin, thus establishing the potential of our assay for detection of actin-binding modulators. Concurrent to our primary FRET assay, we also developed a high-throughput compatible counter screen to remove undesirable FRET Hits. Using the FRET Hits, we show that our counter screen is sensitive to undesirable compounds that cause cell toxicity or ABD aggregation. Overall, our FRET-based HTS platform sets the stage to screen large compound libraries for modulators of ß-III-spectrin, or disease-linked spectrin-related proteins, for therapeutic development.

Structural and optical properties of individual Zn2GeO4 particles embedded in ZnO.

Functionalizing transparent conducting oxides (TCOs) is an intriguing approach to expand the tunability and operation of optoelectronic devices. For example, forming nanoparticles that act as quantum wells or barriers in zinc oxide (ZnO), one of the main TCOs today, may expand its optical and electronic tunability. In this work, 800 keV Ge ions have been implanted at a dose of 1 × 1016 cm-2 into crystalline ZnO. After annealing at 1000 °C embedded disk-shaped particles with diameters up to 100 nm are formed. Scanning transmission electron microscopy shows that these are particles of the trigonal Zn2GeO4 phase. The particles are terminated by atomically sharp facets of the type {11 [Formula: see text] 0}, and the interface between the matrix and particles is decorated with misfit dislocations in order to accommodate the lattice mismatch between the two crystals. Electron energy loss spectroscopy has been employed to measure the band gap of individual nanoparticles, showing an onset of band-to-band transitions at 5.03 ± 0.02 eV. This work illustrates the advantages of using STEM characterization methods, where information of structure, growth, and properties can be directly obtained.

Outcomes of clients in need of intensive team care in Flexible Assertive Community Treatment in Sweden.

BACKGROUND: Flexible Assertive Community Treatment (Flexible ACT) has been implemented in Sweden during recent years due to increasing interest in integrated services for people with severe mental illness. To date, few studies have been done on Flexible ACT effectiveness. AIMS: The overall aim of this study was to explore the extent to which clients assigned to the Flexible ACT board for ACT intensive care were stabilized with improved everyday functioning, social outcomes, and changes in healthcare use. METHODS: Ninety-three participants with psychosis, in need of ACT from six newly started Flexible ACT teams, were included. Data were collected using the Social Outcome Index scale (SIX), Practical and Social Functioning Scale, and a healthcare usage questionnaire. RESULTS: There was a significant positive change in everyday functioning and in the SIX-item 'friendship' at 18-months follow-up. A positive correlation was also found between everyday functioning and the SIX-item 'friendship' and a negative correlation between duration of ACT and everyday functioning. A significant increase in number of inpatient hospital days and psychiatric outpatient visits also occurred. CONCLUSION: Clients with psychosis who need ACT may benefit from Flexible ACT through improved social functioning. Being involved in meaningful activities and supported by others are key aspects of recovering from mental illness and are enhanced by Flexible ACT.

What influences a sustainable implementation of evidence-based interventions in community mental health services? Development and pilot testing of a tool for mapping core components.

BACKGROUND: An important aspect of research regarding the implementation of evidence-based practice is the sustainability and long-term stability of a programme. There is a need to measure these critical components for establishing successful programmes. AIM: The aim was to develop and pilot test the sustainable implementation scale (SIS) for measuring the critical components in the sustainable implementation of community mental health services. METHOD: The scale was based on implementation research and consisted of three subscales regarding (1) the organisational level, (2) the team level and (3) continuous support. Data from interviews and documents were collected from 14 programmes implementing the Individual Placement and Support model of supported employment. RESULTS: Internal consistency was acceptable for all subscales and for the scale as a whole. Regarding the scale, an analysis of the differences between fully established programmes and the programmes that were not established or were or only partially established after three years showed statistically significant differences, indicating that a greater number of implementation components were present in the fully established programmes. CONCLUSIONS: SIS showed both good reliability and acceptable internal consistency as well as the ability to predict programme survival.

Mental health first aid for the elderly: A pilot study of a training program adapted for helping elderly people.

OBJECTIVES: Epidemiological studies have shown a high prevalence of mental illness among the elderly. Clinical data however indicate both insufficient detection and treatment of illnesses. Suggested barriers to treatment include conceptions that mental health symptoms belong to normal aging and lack of competence among staff in elderly care in detecting mental illness. A Mental Health First Aid (MHFA) training program for the elderly was developed and provided to staff in elderly care. The aim of this study was to investigate changes in knowledge in mental illness, confidence in helping a person, readiness to give help and attitudes towards persons with mental illness. METHOD: Single group pre-test-post-test design. RESULTS: The study group included staff in elderly care from different places in Sweden (n = 139). Significant improvements in knowledge, confidence in helping an elderly person with mental illness and attitudes towards persons with mental illness are shown. Skills acquired during the course have been practiced during the follow-up. CONCLUSIONS: The adaption of MHFA training for staff working in elderly care gives promising results. Improvements in self-reported confidence in giving help, attitudes towards persons with mental illness and actual help given to persons with mental illness are shown. However, the study design allows no firm conclusions and a randomized controlled trail is needed to investigate the effectiveness of the program. Outcomes should include if the detection and treatment of mental illness among the elderly actually improved.

How mental health literacy and experience of mental illness relate to stigmatizing attitudes and social distance towards people with depression or psychosis: A cross-sectional study.

Background Evidence suggests that mental health literacy among the public is low, and stigmatizing attitudes are widespread. So far the effects of anti-stigma campaigns are small, and studies demonstrate that negative attitudes have been quite stable through recent decades. Aims To investigate the relationships between mental health literacy, experience of mental illness and stigmatizing attitudes/social distance towards people with depression or psychosis. Methods A cross-sectional study in which staff members from public services in Sweden (n = 1027) completed questionnaires covering demographic data, self-reported experience of mental illness, identification of a vignette for depression or psychosis, beliefs about helpful interventions for the illness presented in the vignette, and attitudes and social distance towards people with the illness. Results About 50% of participants could identify depression and less than 40% psychosis. A higher degree of mental health literacy was related to less stigma and social distance but mainly towards people with depression. A similar relationship was shown for having personal or family experience of mental illness and attitudes/social distance. Negative attitudes and social distance were significantly higher in all aspects measured towards a person with psychosis than a person with depression. Conclusions A higher degree of mental health literacy relates to more positive attitudes and less desire for social distance towards people with depression. The differences between depression and psychosis should be taken into account in anti-stigma interventions.

Mental health professional experiences of the flexible assertive community treatment model: a grounded theory study.

BACKGROUND: Despite the lack of evidence for effectiveness of the Flexible Assertive Community Treatment (Flexible ACT), the model is considered feasible and is well received by mental health professionals. No current studies have adequately examined mental health professional experiences of working with Flexible ACT. AIMS: The aim of this study was to explore mental health professional experiences of working with the Flexible ACT model compared with standard care. METHOD: The study was guided by grounded theory and based on the interviews with 19 theoretically chosen mental health professionals in Swedish urban areas primarily working with consumers with psychosis, who had worked with the Flexible ACT model for at least 6 months. RESULTS: The analysis resulted in the core category: "Flexible ACT and the shared caseload create a common action space" and three main categories: (1) "Flexible ACT fills the need for a systematic approach to crisis intervention"; (2) "Flexible ACT has advantages in the psychosocial working environment"; and (3) "Flexible ACT increases the quality of care". CONCLUSIONS: Mental health professionals may benefit from working with the Flexible ACT model through decreased job-strain and stress, increased feeling of being in control over their work situation, and experiences of providing higher quality of care.

In cardiomyocytes, binding of unzipping peptide activates ryanodine receptor 2 and reciprocally inhibits calmodulin binding.

RATIONALE: One hypothesis for elevated Ca(2+) leak through cardiac ryanodine receptors (ryanodine receptor 2 [RyR2]) in heart failure is interdomain unzipping that can enhance aberrant channel activation. A peptide (domain peptide corresponding to RyR2 residues 2460-2495 [DPc10]) corresponding to RyR2 central domain residues 2460-2495 recapitulates this arrhythmogenic RyR2 leakiness by unzipping N-terminal and central domains. Calmodulin (CaM) and FK506-binding protein (FKBP12.6) bind to RyR2 and stabilize the closed channel. Little is known about DPc10 binding to the RyR2 and how that may interact with binding (and effects) of CaM and FKBP12.6 to RyR2. OBJECTIVE: To measure, directly in cardiac myocytes, the kinetics and binding affinity of DPc10 to RyR2 and how that affects RyR2 interaction with FKBP12.6 and CaM. METHODS AND RESULTS: We used permeabilized rat ventricular myocytes and fluorescently labeled DPc10, FKBP12.6, and CaM. DPc10 access to its binding site is extremely slow in resting RyR2 but is accelerated by promoting RyR opening or unzipping (by unlabeled DPc10). RyR2-bound CaM (but not FKBP12.6) drastically slowed DPc10 binding. Conversely, DPc10 binding significantly reduced CaM (but not FKBP12.6) binding to the RyR2. Fluorescence resonance energy transfer measurements indicate that DPc10-binding and CaM-binding sites are separate and allow triangulation of the structural DPc10 binding locus on RyR2 vs FKBP12.6-binding and CaM-binding sites. CONCLUSIONS: DPc10-RyR2 binding is sterically limited by the resting zipped RyR2 state. CaM binding to RyR2 stabilizes this zipped state, whereas RyR2 activation or prebound DPc10 enhances DPc10 access. DPc10-binding and CaM-binding sites are distinct but are allosterically interacting RyR2 sites. Neither DPc10 nor FKBP12.6 influences RyR2 binding of the other.

Experiences of a Mental Health First Aid training program in Sweden: a descriptive qualitative study.

Restricted mental health literacy and stigma are barriers to treatment of mental disorders. A Mental Health First Aid training program was tested for implementation in Sweden among employees in the public sector. The aim of the present qualitative study was to explore participants' experiences of the program in more depth, in conjunction with a randomized controlled study. Twenty four persons participated in a total of six focus groups 6-8 months after program participation. Data were analyzed using content analysis. The analysis resulted in five categories illustrating the participants' experiences of the course: increased awareness, knowledge and understanding; influence on attitude and approach; tool box and confidence; feedback on content and layout; and tangible examples of applied knowledge. The most central finding is the fruitfulness of the program's practical focus and use, the increased confidence and inclination to act following program participation, and the importance of experienced instructors.

FRET-based trilateration of probes bound within functional ryanodine receptors.

To locate the biosensor peptide DPc10 bound to ryanodine receptor (RyR) Ca(2+) channels, we developed an approach that combines fluorescence resonance energy transfer (FRET), simulated-annealing, cryo-electron microscopy, and crystallographic data. DPc10 is identical to the 2460-2495 segment within the cardiac muscle RyR isoform (RyR2) central domain. DPc10 binding to RyR2 results in a pathologically elevated Ca(2+) leak by destabilizing key interactions between the RyR2 N-terminal and central domains (unzipping). To localize the DPc10 binding site within RyR2, we measured FRET between five single-cysteine variants of the FK506-binding protein (FKBP) labeled with a donor probe, and DPc10 labeled with an acceptor probe (A-DPc10). Effective donor positions were calculated from simulated-annealing constrained by both the RyR cryo-EM map and the FKBP atomic structure docked to the RyR. FRET to A-DPc10 was measured in permeabilized cardiomyocytes via confocal microscopy, converted to distances, and used to trilaterate the acceptor locus within RyR. Additional FRET measurements between donor-labeled calmodulin and A-DPc10 were used to constrain the trilaterations. Results locate the DPc10 probe within RyR domain 3, ?35 Å from the previously docked N-terminal domain crystal structure. This multiscale approach may be useful in mapping other RyR sites of mechanistic interest within FRET range of FKBP.

N-terminal and central segments of the type 1 ryanodine receptor mediate its interaction with FK506-binding proteins.

We used site-directed labeling of the type 1 ryanodine receptor (RyR1) and fluorescence resonance energy transfer (FRET) measurements to map RyR1 sequence elements forming the binding site of the 12-kDa binding protein for the immunosuppressant drug, FK506. This protein, FKBP12, promotes the RyR1 closed state, thereby inhibiting Ca(2+) leakage in resting muscle. Although FKBP12 function is well established, its binding determinants within the RyR1 protein sequence remain unresolved. To identify these sequence determinants using FRET, we created five single-Cys FKBP variants labeled with Alexa Fluor 488 (denoted D-FKBP) and then targeted these D-FKBPs to full-length RyR1 constructs containing decahistidine (His10) "tags" placed within N-terminal (amino acid residues 76-619) or central (residues 2157-2777) regions of RyR1. The FRET acceptor Cy3NTA bound specifically and saturably to these His tags, allowing distance analysis of FRET measured from each D-FKBP variant to Cy3NTA bound to each His tag. Results indicate that D-FKBP binds proximal to both N-terminal and central domains of RyR1, thus suggesting that the FKBP binding site is composed of determinants from both regions. These findings further imply that the RyR1 N-terminal and central domains are proximal to one another, a core premise of the domain-switch hypothesis of RyR function. We observed FRET from GFP fused at position 620 within the N-terminal domain to central domain His-tagged sites, thus further supporting this hypothesis. Taken together, these results support the conclusion that N-terminal and central domain elements are closely apposed near the FKBP binding site within the RyR1 three-dimensional structure.

Large-scale opening of utrophin's tandem calponin homology (CH) domains upon actin binding by an induced-fit mechanism.

We have used site-directed spin labeling and pulsed electron paramagnetic resonance to resolve a controversy concerning the structure of the utrophin-actin complex, with implications for the pathophysiology of muscular dystrophy. Utrophin is a homolog of dystrophin, the defective protein in Duchenne and Becker muscular dystrophies, and therapeutic utrophin derivatives are currently being developed. Both proteins have a pair of N-terminal calponin homology (CH) domains that are important for actin binding. Although there is a crystal structure of the utrophin actin-binding domain, electron microscopy of the actin-bound complexes has produced two very different structural models, in which the CH domains are in open or closed conformations. We engineered a pair of labeling sites in the CH domains of utrophin and used dipolar electron-electron resonance to determine the distribution of interdomain distances with high resolution. We found that the two domains are flexibly connected in solution, indicating a dynamic equilibrium between two distinct open structures. Upon actin binding, the two domains become dramatically separated and ordered, indicating a transition to a single open and extended conformation. There is no trace of this open conformation of utrophin in the absence of actin, providing strong support for an induced-fit model of actin binding.

Perceived and anticipated discrimination in people with mental illness--an interview study.

BACKGROUND: Studies on perceived discrimination of people with mental illness are largely lacking. The purpose of the study was to investigate perceived discrimination in a sample of users in contact with mental health services in Sweden. METHODS: Interviews were conducted with 156 users, asking for perceived and anticipated discrimination during the last 2 years. Background characteristics were also collected. RESULTS: Perceived discrimination was common. Highest frequencies were reported regarding family (54%), avoidance by people who knew about the mental illness (53%) and in making or keeping friends (50%). A majority of those anticipating discrimination regarding job or education seeking, or starting a close relationship did not report having been discriminated in these areas. Previous hospitalizations were associated with discrimination, and age with anticipated discrimination. CONCLUSIONS: Public stigma and self-stigma have been reported to have a number of negative consequences for people with mental illness. Discrimination is part of this complex situation and this study showed that this largely affects a number of individual life areas posing an obstacle for social integration. Anticipated discrimination or self-stigma was also prevalent and it is pointed out that this to a great extent is an obstacle on its own without being promoted by actual experiences of discrimination.

A 2-year follow-up study of people with severe mental illness involved in psychosocial rehabilitation.

UNLABELLED: BACKGROUNDS. A focus on psychiatric rehabilitation in order to support recovery among persons with severe mental illness (SMI) has been given great attention in research and mental health policy, but less impact on clinical practice. Despite the potential impact of psychiatric rehabilitation on health and wellbeing, there is a lack of research regarding the model called the Psychiatric Rehabilitation Approach from Boston University (BPR). AIM: The aim was to investigate the outcome of the BPR intervention regarding changes in life situation, use of healthcare services, quality of life, health, psychosocial functioning and empowerment. METHODS: The study has a prospective longitudinal design and the setting was seven mental health services who worked with the BPR in the county of Halland in Sweden. In total, 71 clients completed the assessment at baseline and of these 49 completed the 2-year follow-up assessments. RESULTS: The most significant finding was an improved psychosocial functioning at the follow-up assessment. Furthermore, 65% of the clients reported that they had mainly or almost completely achieved their self-formulated rehabilitation goals at the 2-year follow-up. There were significant differences with regard to health, empowerment, quality of life and psychosocial functioning for those who reported that they had mainly/completely had achieved their self-formulated rehabilitation goals compared to those who reported that they only had to a small extent or not at all reached their goals. CONCLUSIONS: Our results indicate that the BPR approach has impact on clients' health, empowerment, quality of life and in particular concerning psychosocial functioning.

Mapping attitudes and awareness with regard to national guidelines: an e-mail survey among decision makers.

INTRODUCTION: The adoption of evidence-based guidelines within the mental health field has been slow. Changing inadequate practice is therefore a formidable challenge for mental health-care managers. AIM: To explore decision-makers' attitudes and awareness regarding the national guidelines for psychosocial interventions targeting people with schizophrenia. METHOD: A questionnaire distributed by e-mail to 592 Swedish decision-makers was analysed using descriptive and comparative techniques. RESULTS: Significantly more of the top-level mental health-care managers than politicians stated that they knew about the national guidelines (i.e. their release and content) and they considered the guidelines to be a good source of support for planning and allocating resources. CONCLUSION: If those responsible for allocating resources (i.e. politicians) are unaware of the dissemination of national guidelines or their content, and they do not perceive the national guidelines to be a good source of support for planning and allocating resources, this is likely to have a negative influence on the remit of nurse managers as well as nursing practice. IMPLICATIONS FOR NURSING MANAGEMENT: Top-level mental health-care managers have a vital role to play in the implementation of national guidelines. However, our findings indicate that implementing national guidelines in practice could be virtually impossible without strategic government support.

Kinetics and mapping of Ca-driven calmodulin conformations on skeletal and cardiac muscle ryanodine receptors.

Calmodulin transduces [Ca2+] information regulating the rhythmic Ca2+ cycling between the sarcoplasmic reticulum and cytoplasm during contraction and relaxation in cardiac and skeletal muscle. However, the structural dynamics by which calmodulin modulates the sarcoplasmic reticulum Ca2+ release channel, the ryanodine receptor, at physiologically relevant [Ca2+] is unknown. Using fluorescence lifetime FRET, we resolve different structural states of calmodulin and Ca2+-driven shifts in the conformation of calmodulin bound to ryanodine receptor. Skeletal and cardiac ryanodine receptor isoforms show different calmodulin-ryanodine receptor conformations, as well as binding and structural kinetics with 0.2-ms resolution, which reflect different functional roles of calmodulin. These FRET methods provide insight into the physiological calmodulin-ryanodine receptor structural states, revealing additional distinct structural states that complement cryo-EM models that are based on less physiological conditions. This technology will drive future studies on pathological calmodulin-ryanodine receptor interactions and dynamics with other important ryanodine receptor bound modulators.