Screening for Novel Type 2 Ryanodine Receptor Inhibitors by Endoplasmic Reticulum Ca2+ Monitoring.

Type 2 ryanodine receptor (RyR2) is a Ca2+ release channel on the endoplasmic (ER)/sarcoplasmic reticulum that plays a central role in the excitation-contraction coupling in the heart. Hyperactivity of RyR2 has been linked to ventricular arrhythmias in patients with catecholaminergic polymorphic ventricular tachycardia and heart failure, where spontaneous Ca2+ release via hyperactivated RyR2 depolarizes diastolic membrane potential to induce triggered activity. In such cases, drugs that suppress RyR2 activity are expected to prevent the arrhythmias, but there is no clinically available RyR2 inhibitors at present. In this study, we searched for RyR2 inhibitors from a well-characterized compound library using a recently developed ER Ca2+-based assay, where the inhibition of RyR2 activity was detected by the increase in ER Ca2+ signals from R-CEPIA1er, a genetically encoded ER Ca2+ indicator, in RyR2-expressing HEK293 cells. By screening 1535 compounds in the library, we identified three compounds (chloroxylenol, methyl orsellinate, and riluzole) that greatly increased the ER Ca2+ signal. All of the three compounds suppressed spontaneous Ca2+ oscillations in RyR2-expressing HEK293 cells and correspondingly reduced the Ca2+-dependent [3H]ryanodine binding activity. In cardiomyocytes from RyR2-mutant mice, the three compounds effectively suppressed abnormal Ca2+ waves without substantial effects on the action-potential-induced Ca2+ transients. These results confirm that ER Ca2+-based screening is useful for identifying modulators of ER Ca2+ release channels and suggest that RyR2 inhibitors have potential to be developed as a new category of antiarrhythmic drugs. SIGNIFICANCE STATEMENT: We successfully identified three compounds having RyR2 inhibitory action from a well-characterized compound library using an endoplasmic reticulum Ca2+-based assay, and demonstrated that these compounds suppressed arrhythmogenic Ca2+ wave generation without substantially affecting physiological action-potential induced Ca2+ transients in cardiomyocytes. This study will facilitate the development of RyR2-specific inhibitors as a potential new class of drugs for life-threatening arrhythmias induced by hyperactivation of RyR2.

Ca2+ channels couple spiking to mitochondrial metabolism in substantia nigra dopaminergic neurons.

How do neurons match generation of adenosine triphosphate by mitochondria to the bioenergetic demands of regenerative activity? Although the subject of speculation, this coupling is still poorly understood, particularly in neurons that are tonically active. To help fill this gap, pacemaking substantia nigra dopaminergic neurons were studied using a combination of optical, electrophysiological, and molecular approaches. In these neurons, spike-activated calcium (Ca2+) entry through Cav1 channels triggered Ca2+ release from the endoplasmic reticulum, which stimulated mitochondrial oxidative phosphorylation through two complementary Ca2+-dependent mechanisms: one mediated by the mitochondrial uniporter and another by the malate-aspartate shuttle. Disrupting either mechanism impaired the ability of dopaminergic neurons to sustain spike activity. While this feedforward control helps dopaminergic neurons meet the bioenergetic demands associated with sustained spiking, it is also responsible for their elevated oxidant stress and possibly to their decline with aging and disease.

Cytosolic Ca2+-dependent Ca2+ release activity primarily determines the ER Ca2+ level in cells expressing the CPVT-linked mutant RYR2.

Type 2 ryanodine receptor (RYR2) is a cardiac Ca2+ release channel in the ER. Mutations in RYR2 are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT is associated with enhanced spontaneous Ca2+ release, which tends to occur when [Ca2+]ER reaches a threshold. Mutations lower the threshold [Ca2+]ER by increasing luminal Ca2+ sensitivity or enhancing cytosolic [Ca2+] ([Ca2+]cyt)-dependent activity. Here, to establish the mechanism relating the change in [Ca2+]cyt-dependent activity of RYR2 and the threshold [Ca2+]ER, we carried out cell-based experiments and in silico simulations. We expressed WT and CPVT-linked mutant RYR2s in HEK293 cells and measured [Ca2+]cyt and [Ca2+]ER using fluorescent Ca2+ indicators. CPVT RYR2 cells showed higher oscillation frequency and lower threshold [Ca2+]ER than WT cells. The [Ca2+]cyt-dependent activity at resting [Ca2+]cyt, Arest, was greater in CPVT mutants than in WT, and we found an inverse correlation between threshold [Ca2+]ER and Arest. In addition, lowering RYR2 expression increased the threshold [Ca2+]ER and a product of Arest, and the relative expression level for each mutant correlated with threshold [Ca2+]ER, suggesting that the threshold [Ca2+]ER depends on the net Ca2+ release rate via RYR2. Modeling reproduced Ca2+ oscillations with [Ca2+]cyt and [Ca2+]ER changes in WT and CPVT cells. Interestingly, the [Ca2+]cyt-dependent activity of specific mutations correlated with the age of disease onset in patients carrying them. Our data suggest that the reduction in threshold [Ca2+]ER for spontaneous Ca2+ release by CPVT mutation is explained by enhanced [Ca2+]cyt-dependent activity without requiring modulation of the [Ca2+]ER sensitivity of RYR2.

In vivo Fluorescence Imaging of Extracellular ATP in the Mouse Cerebral Cortex with a Hybrid-type Optical Sensor.

Adenosine 5'-triphosphate (ATP) works as an extracellular signaling molecule for cells in the brain, such as neurons and glia. Cellular communication via release of ATP is involved in a range of processes required for normal brain functions, and aberrant communication is associated with brain disorders. To investigate the mechanisms underlying these cellular processes, various techniques have been developed for the measurement of extracellular ATP. To monitor the dynamics of extracellular ATP signaling with high spatiotemporal resolution, we recently developed a hybrid-type ATP optical sensor (ATPOS) that enables in vivo fluorescence imaging of extracellular ATP dynamics in the brain. ATPOS is synthesized by labeling an ATP-binding protein, Bacillus FoF1-ATP synthase ε subunit, with a small-molecular fluorescent dye Cy3. Injection of ATPOS into the cerebral cortex of living mice enables visualization of the wave-like propagation of extracellular ATP release in response to electrical stimulation. The protocol described here should be useful for visualizing ATP signaling in diverse processes involved in intercellular communication in the brain.

Reactive astrocyte nomenclature, definitions, and future directions.

Reactive astrocytes are astrocytes undergoing morphological, molecular, and functional remodeling in response to injury, disease, or infection of the CNS. Although this remodeling was first described over a century ago, uncertainties and controversies remain regarding the contribution of reactive astrocytes to CNS diseases, repair, and aging. It is also unclear whether fixed categories of reactive astrocytes exist and, if so, how to identify them. We point out the shortcomings of binary divisions of reactive astrocytes into good-vs-bad, neurotoxic-vs-neuroprotective or A1-vs-A2. We advocate, instead, that research on reactive astrocytes include assessment of multiple molecular and functional parameters-preferably in vivo-plus multivariate statistics and determination of impact on pathological hallmarks in relevant models. These guidelines may spur the discovery of astrocyte-based biomarkers as well as astrocyte-targeting therapies that abrogate detrimental actions of reactive astrocytes, potentiate their neuro- and glioprotective actions, and restore or augment their homeostatic, modulatory, and defensive functions.

Real-time in vivo imaging of extracellular ATP in the brain with a hybrid-type fluorescent sensor.

Adenosine 5' triphosphate (ATP) is a ubiquitous extracellular signaling messenger. Here, we describe a method for in-vivo imaging of extracellular ATP with high spatiotemporal resolution. We prepared a comprehensive set of cysteine-substitution mutants of ATP-binding protein, Bacillus FoF1-ATP synthase ε subunit, labeled with small-molecule fluorophores at the introduced cysteine residue. Screening revealed that the Cy3-labeled glutamine-105 mutant (Q105C-Cy3; designated ATPOS) shows a large fluorescence change in the presence of ATP, with submicromolar affinity, pH-independence, and high selectivity for ATP over ATP metabolites and other nucleotides. To enable in-vivo validation, we introduced BoNT/C-Hc for binding to neuronal plasma membrane and Alexa Fluor 488 for ratiometric measurement. The resulting ATPOS complex binds to neurons in cerebral cortex of living mice, and clearly visualized a concentrically propagating wave of extracellular ATP release in response to electrical stimulation. ATPOS should be useful to probe the extracellular ATP dynamics of diverse biological processes in vivo.

Biologists often refer to a small molecule called adenosine triphosphate ­ or ATP for short ­ as 'the currency of life'. This molecule carries energy all through the body, and most cells and proteins require ATP to perform their various roles. Nerve cells (also known as neurons) in the brain release ATP when activated, and use this molecule to send signals to other active neurons or other cells in the brain. But ATP can also signal danger in the brain. A molecule derived from ATP is involved in transmitting the pain signals of migraines and severe headaches; and ATP levels can become imbalanced after strokes, when parts of the brain are deprived of blood. Despite its importance, ATP remains difficult to visualize in the body, and monitoring the molecule in the active brain in real time is challenging. To address this issue, Kitajima et al. designed an optical sensor that could monitor ATP in the healthy brain, and was sensitive enough to detect when and where it was released. First, Kitajima et al. made several potential sensors by attaching various fluorescent tags to different locations on a protein that binds ATP. Next each sensor was tested to determine whether it could bind ATP tightly and get bright upon binding. This is important because previous sensors could not detect ATP release in the brains of living animals. To illustrate the new sensors' potential, Kitajima et al. used the sensor to image ATP in the brains of live mice. A 'wave' of ATP was seen spreading through the brain after neurons were stimulated with a small electric pulse, mimicking a sudden migraine or stroke. The results confirm that this new sensor is suitable for imaging how ATP signals in the brain, and it may help resolve the underlying mechanisms of migraines and strokes. This sensor could also be used to understand other cellular process which rely on ATP to carry out their role.

Red fluorescent CEPIA indicators for visualization of Ca2+ dynamics in mitochondria.

Mitochondrial Ca2+ dynamics are involved in the regulation of multifarious cellular processes, including intracellular Ca2+ signalling, cell metabolism and cell death. Use of mitochondria-targeted genetically encoded Ca2+ indicators has revealed intercellular and subcellular heterogeneity of mitochondrial Ca2+ dynamics, which are assumed to be determined by distinct thresholds of Ca2+ increases at each subcellular mitochondrial domain. The balance between Ca2+ influx through the mitochondrial calcium uniporter and extrusion by cation exchangers across the inner mitochondrial membrane may define the threshold; however, the precise mechanisms remain to be further explored. We here report the new red fluorescent genetically encoded Ca2+ indicators, R-CEPIA3mt and R-CEPIA4mt, which are targeted to mitochondria and their Ca2+ affinities are engineered to match the intramitochondrial Ca2+ concentrations. They enable visualization of mitochondrial Ca2+ dynamics with high spatiotemporal resolution in parallel with the use of green fluorescent probes and optogenetic tools. Thus, R-CEPIA3mt and R-CEPIA4mt are expected to be a useful tool for elucidating the mechanisms of the complex mitochondrial Ca2+ dynamics and their functions.

Rational Design of a Near-infrared Fluorescence Probe for Ca2+ Based on Phosphorus-substituted Rhodamines Utilizing Photoinduced Electron Transfer.

Fluorescence imaging in the near-infrared (NIR) region (650-900 nm) is useful for bioimaging because background autofluorescence is low and tissue penetration is high in this range. In addition, NIR fluorescence is useful as a complementary color window to green and red for multicolor imaging. Here, we compared the photoinduced electron transfer (PeT)-mediated fluorescence quenching of silicon- and phosphorus-substituted rhodamines (SiRs and PRs) in order to guide the development of improved far-red to NIR fluorescent dyes. The results of density functional theory calculations and photophysical evaluation of a series of newly synthesized PRs confirmed that the fluorescence of PRs was more susceptible than that of SiRs to quenching via PeT. Based on this, we designed and synthesized a NIR fluorescence probe for Ca2+ , CaPR-1, and its membrane-permeable acetoxymethyl derivative, CaPR-1 AM, which is distributed to the cytosol, in marked contrast to our previously reported Ca2+ far-red to NIR fluorescence probe based on the SiR scaffold, CaSiR-1 AM, which is mainly localized in lysosomes as well as cytosol in living cells. CaPR-1 showed longer-wavelength absorption and emission (up to 712 nm) than CaSiR-1. The new probe was able to image Ca2+ at dendrites and spines in brain slices, and should be a useful tool in neuroscience research.

Visualization of astrocytic intracellular Ca2+ mobilization.

Astrocytes generate robust intracellular Ca2+ concentration changes (Ca2+ signals), which are assumed to regulate astrocytic functions that play crucial roles in the regulation of brain functions. One frequently used strategy for exploring the role of astrocytic Ca2+ signalling is the use of mice deficient in the type 2 inositol 1,4,5-trisphosphate receptor (IP3 R2). These IP3 R2-knockout (KO) mice are reportedly devoid of Ca2+ mobilization from the endoplasmic reticulum (ER) in astrocytes. However, they have shown no functional deficits in several studies, causing a heated debate as to the functional relevance of ER-mediated Ca2+ signalling in astrocytes. Recently, the assumption that Ca2+ mobilization from the ER is absent in IP3 R2-KO astrocytes has been re-evaluated using intraorganellar Ca2+ imaging techniques. The new results indicated that IP3 R2-independent Ca2+ release may generate Ca2+ nanodomains around the ER, which may help explain the absence of functional deficits in IP3 R2-KO mice.

Store-operated Ca2+ entry-dependent Ca2+ refilling in the endoplasmic reticulum in astrocytes.

Astrocytes regulate various brain functions, for which Ca2+ release from the endoplasmic reticulum (ER) often play crucial roles. Because astrocytic ER Ca2+ release is robust and frequent, the ER Ca2+ refilling mechanism should be critical for ongoing Ca2+ signaling in astrocytes. In this study, we focused on the putative functional significance of store-operated Ca2+ entry (SOCE) in ER Ca2+ refilling. We expressed the ER luminal Ca2+ indicator G-CEPIA1er in astrocytes in acute cortical slices to directly monitor the decrease and recovery of ER Ca2+ concentration upon spontaneous or norepinephrine-induced Ca2+ release. Inhibition of SOCE significantly slowed the recovery of ER Ca2+ concentration after Ca2+ release in astrocytes. This delayed recovery resulted in a prolonged decrease in the ER Ca2+ content in astrocytes with periodic spontaneous Ca2+ release, followed by the attenuation of cytosolic Ca2+ responses upon Ca2+ release. Therefore, our results provide direct evidence for the physiological significance of SOCE in ER Ca2+ refilling after ER Ca2+ release.

Insights into channel modulation mechanism of RYR1 mutants using Ca2+ imaging and molecular dynamics.

Type 1 ryanodine receptor (RYR1) is a Ca2+ release channel in the sarcoplasmic reticulum in skeletal muscle and plays an important role in excitation-contraction coupling. Mutations in the RYR1 gene cause severe muscle diseases such as malignant hyperthermia (MH), which is a disorder of CICR via RYR1. Thus far, >300 mutations in RYR1 have been reported in patients with MH. However, owing to a lack of comprehensive analysis of the structure-function relationship of mutant RYR1, the mechanism remains largely unknown. Here, we combined functional studies and molecular dynamics (MD) simulations of RYR1 bearing disease-associated mutations at the N-terminal region. When expressed in HEK293 cells, the mutant RYR1 caused abnormalities in Ca2+ homeostasis. MD simulations of WT and mutant RYR1s were performed using crystal structure of the N-terminal domain (NTD) monomer, consisting of A, B, and C domains. We found that the mutations located around the interdomain region differentially affected hydrogen bonds/salt bridges. Particularly, mutations at R402, which increase the open probability of the channel, cause clockwise rotation of BC domains with respect to the A domain by alteration of the interdomain interactions. Similar results were also obtained with artificial mutations that mimic alteration of the interactions. Our results reveal the importance of interdomain interactions within the NTD in the regulation of the RYR1 channel and provide insights into the mechanism of MH caused by the mutations at the NTD.

Inositol 1,4,5-trisphosphate receptor type 2-independent Ca2+ release from the endoplasmic reticulum in astrocytes.

Accumulating evidence indicates that astrocytes are actively involved in the physiological and pathophysiological functions of the brain. Intracellular Ca2+ signaling, especially Ca2+ release from the endoplasmic reticulum (ER), is considered to be crucial for the regulation of astrocytic functions. Mice with genetic deletion of inositol 1,4,5-trisphosphate receptor type 2 (IP3 R2) are reportedly devoid of astrocytic Ca2+ signaling, and thus widely used to explore the roles of Ca2+ signaling in astrocytic functions. While functional deficits in IP3 R2-knockout (KO) mice have been found in some reports, no functional deficit was observed in others. Thus, there remains a controversy regarding the functional significance of astrocytic Ca2+ signaling. To address this controversy, we re-evaluated the assumption that Ca2+ release from the ER is abolished in IP3 R2-KO astrocytes using a highly sensitive imaging technique. We expressed the ER luminal Ca2+ indicator G-CEPIA1er in cortical and hippocampal astrocytes to directly visualize spontaneous and stimulus-induced Ca2+ release from the ER. We found attenuated but significant Ca2+ release in response to application of norepinephrine to IP3 R2-KO astrocytes. This IP3 R2-independent Ca2+ release induced only minimal cytosolic Ca2+ transients but induced robust Ca2+ increases in mitochondria that are frequently in close contact with the ER. These results indicate that ER Ca2+ release is retained and is sufficient to increase the Ca2+ concentration in close proximity to the ER in IP3 R2-KO astrocytes.

Chemical Landscape for Tissue Clearing Based on Hydrophilic Reagents.

We describe a strategy for developing hydrophilic chemical cocktails for tissue delipidation, decoloring, refractive index (RI) matching, and decalcification, based on comprehensive chemical profiling. More than 1,600 chemicals were screened by a high-throughput evaluation system for each chemical process. The chemical profiling revealed important chemical factors: salt-free amine with high octanol/water partition-coefficient (logP) for delipidation, N-alkylimidazole for decoloring, aromatic amide for RI matching, and protonation of phosphate ion for decalcification. The strategic integration of optimal chemical cocktails provided a series of CUBIC (clear, unobstructed brain/body imaging cocktails and computational analysis) protocols, which efficiently clear mouse organs, mouse body including bone, and even large primate and human tissues. The updated CUBIC protocols are scalable and reproducible, and they enable three-dimensional imaging of the mammalian body and large primate and human tissues. This strategy represents a future paradigm for the rational design of hydrophilic clearing cocktails that can be used for large tissues.

Efficient Gene Transfer to Myelinating Schwann Cells in the Rodent Sciatic Nerve Using In Vivo Electroporation.

To study the signaling mechanism of the development and maintenance of myelinating Schwann cells (SCs) in the peripheral nervous system, in vivo techniques for SC-selective gene manipulation are useful. The present protocol describes an in vivo electroporation method that allows efficient transfection of myelinating SCs in rodent sciatic nerves. This technique allows us to introduce the genes of interest into myelinating SCs by simply applying electric pulses to the sciatic nerve after plasmid DNA injection.

Efficient High-Throughput Screening by Endoplasmic Reticulum Ca2+ Measurement to Identify Inhibitors of Ryanodine Receptor Ca2+-Release Channels.

Genetic mutations in ryanodine receptors (RyRs), Ca2+-release channels in the sarcoplasmic reticulum essential for muscle contractions, cause various skeletal muscle and cardiac diseases. Because the main underlying mechanism of the pathogenesis is overactive Ca2+ release by gain-of-function of the RyR channel, inhibition of RyRs is expected to be a promising treatment of these diseases. Here, to identify inhibitors specific to skeletal muscle type 1 RyR (RyR1), we developed a novel high-throughput screening (HTS) platform using time-lapse fluorescence measurement of Ca2+ concentrations in the endoplasmic reticulum (ER) ([Ca2+]ER). Because expression of RyR1 carrying disease-associated mutation reduces [Ca2+]ER in HEK293 cells through Ca2+ leakage from RyR1 channels, specific drugs that inhibit RyR1 will increase [Ca2+]ER by preventing such Ca2+ leakage. RyR1 carrying the R2163C mutation and R-CEPIA1er, a genetically encoded ER Ca2+ indicator, were stably expressed in HEK293 cells, and time-lapse fluorescence was measured using a fluorometer. False positives were effectively excluded by using cells expressing wild-type (WT) RyR1. By screening 1535 compounds in a library of well characterized drugs, we successfully identified four compounds that significantly increased [Ca2+]ER They include dantrolene, a known RyR1 inhibitor, and three structurally different compounds: oxolinic acid, 9-aminoacridine, and alexidine. All the hit compounds, except for oxolinic acid, inhibited [3H]ryanodine binding of WT and mutant RyR1. Interestingly, they showed different dose dependencies and isoform specificities. The highly quantitative nature and good correlation with the channel activity validated this HTS platform by [Ca2+]ER measurement to explore drugs for RyR-related diseases.

A three-dimensional single-cell-resolution whole-brain atlas using CUBIC-X expansion microscopy and tissue clearing.

A three-dimensional single-cell-resolution mammalian brain atlas will accelerate systems-level identification and analysis of cellular circuits underlying various brain functions. However, its construction requires efficient subcellular-resolution imaging throughout the entire brain. To address this challenge, we developed a fluorescent-protein-compatible, whole-organ clearing and homogeneous expansion protocol based on an aqueous chemical solution (CUBIC-X). The expanded, well-cleared brain enabled us to construct a point-based mouse brain atlas with single-cell annotation (CUBIC-Atlas). CUBIC-Atlas reflects inhomogeneous whole-brain development, revealing a significant decrease in the cerebral visual and somatosensory cortical areas during postnatal development. Probabilistic activity mapping of pharmacologically stimulated Arc-dVenus reporter mouse brains onto CUBIC-Atlas revealed the existence of distinct functional structures in the hippocampal dentate gyrus. CUBIC-Atlas is shareable by an open-source web-based viewer, providing a new platform for whole-brain cell profiling.

Role of Endoplasmic Reticulum-Mediated Ca2+ Signaling in Neuronal Cell Death.

SIGNIFICANCE: Properly controlled intracellular Ca2+ dynamics is crucial for regulation of neuronal function and survival in the central nervous system. The endoplasmic reticulum (ER), a major intracellular Ca2+ store, plays a critical role as a source and sink for neuronal Ca2+. Recent Advances: Accumulating evidence indicates that disrupted ER Ca2+ signaling is involved in neuronal cell death under various pathological conditions, providing novel insight into neurodegenerative disease mechanisms. CRITICAL ISSUES: We summarize current knowledge concerning the relationship between abnormal ER Ca2+ dynamics and neuronal cell death. We also introduce recent technical advances for probing ER intraluminal Ca2+ dynamics with unprecedented spatiotemporal resolution. FUTURE DIRECTIONS: Further studies on ER Ca2+ signaling are expected to provide progress for unmet medical needs in neurodegenerative disease. Antioxid. Redox Signal. 29, 1147-1157.

Schwann cell mitochondria as key regulators in the development and maintenance of peripheral nerve axons.

Formation of myelin sheaths by Schwann cells (SCs) enables rapid and efficient transmission of action potentials in peripheral axons, and disruption of myelination results in disorders that involve decreased sensory and motor functions. Given that construction of SC myelin requires high levels of lipid and protein synthesis, mitochondria, which are pivotal in cellular metabolism, may be potential regulators of the formation and maintenance of SC myelin. Supporting this notion, abnormal mitochondria are found in SCs of neuropathic peripheral nerves in both human patients and the relevant animal models. However, evidence for the importance of SC mitochondria in myelination has been limited, until recently. Several studies have recently used genetic approaches that allow SC-specific ablation of mitochondrial metabolic activity in living animals to show the critical roles of SC mitochondria in the development and maintenance of peripheral nerve axons. Here, we review current knowledge about the involvement of SC mitochondria in the formation and dysfunction of myelinated axons in the peripheral nervous system.

Genotype-Phenotype Correlations of Malignant Hyperthermia and Central Core Disease Mutations in the Central Region of the RYR1 Channel.

Type 1 ryanodine receptor (RYR1) is a Ca2+ release channel in the sarcoplasmic reticulum of skeletal muscle and is mutated in some muscle diseases, including malignant hyperthermia (MH) and central core disease (CCD). Over 200 mutations associated with these diseases have been identified, and most mutations accelerate Ca2+ -induced Ca2+ release (CICR), resulting in abnormal Ca2+ homeostasis in skeletal muscle. However, it remains largely unknown how specific mutations cause different phenotypes. In this study, we investigated the CICR activity of 14 mutations at 10 different positions in the central region of RYR1 (10 MH and four MH/CCD mutations) using a heterologous expression system in HEK293 cells. In live-cell Ca2+ imaging, the mutant channels exhibited an enhanced sensitivity to caffeine, a reduced endoplasmic reticulum Ca2+ content, and an increased resting cytoplasmic Ca2+ level. The three parameters for CICR (Ca2+ sensitivity for activation, Ca2+ sensitivity for inactivation, and attainable maximum activity, i.e., gain) were obtained by [3 H]ryanodine binding and fitting analysis. The mutant channels showed increased gain and Ca2+ sensitivity for activation in a site-specific manner. Genotype-phenotype correlations were explained well by the near-atomic structure of RYR1. Our data suggest that divergent CICR activity may cause various disease phenotypes by specific mutations.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation.

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.