Background/Objectives: The micronutrient iron is closely connected to inflammation and is among the complex factors contributing to beta-cell failure in diabetes. High levels of dietary iron increase the risk of developing type 2 diabetes, and excessive iron uptake by beta-cells can cause oxidative stress and inhibit function. Elevated levels of proinflammatory cytokines in obese individuals, such as interleukin (IL)-1beta and IL-6, increase the risk of developing type 2 diabetes, and there is evidence that these low levels of circulating cytokines can lead to islet dysfunction. Methods: In this study, gene microarray and other data were analyzed for expression differences in islets treated for 48 h with 10 pg/mL IL-1beta + 20 pg/mL IL-6 as a model of low-grade inflammation versus untreated. Results: Three iron-associated genes were among the most cytokine-sensitive in the mouse genome: Hamp, Steap4, and Lcn2. These proteins are all involved with increasing/retaining cellular iron. We hypothesized that increased cellular iron would lead to increased susceptibility to ferroptosis. Surprisingly, 24 h pre-exposure to low-grade inflammation, which upregulates this iron-gene network, prevented subsequent erastin-induced ferroptosis. We also found that Steap4 overexpression reduced islet dysfunction caused by high-dose proinflammatory cytokines (10× low-dose), suggesting an overall protective effect. Steap4 overexpression also upregulated Hamp and Lcn2, suggesting Steap4 regulates these cytokine-sensitive iron genes.; in contrast, ferritin and ferroportin gene expression, which are not sensitive to cytokines, were unchanged. Conclusions: These data suggest an inflammation-induced network of genes involved in cellular iron uptake and retention plays a protective role in islets against oxidative stress and ferroptosis.

The intricate relationship between the dopaminergic system and olfactory associative learning in Drosophila has been an intense scientific inquiry. Leveraging the formidable genetic tools, we conducted a screening of 57 dopaminergic drivers, leading to the discovery of DAN-c1 driver, uniquely targeting a pair of dopaminergic neurons (DANs) in the larval brain. While the involvement of excitatory D1-like receptors is well established, the role of D2-like receptors (D2Rs) remains underexplored. Our investigation reveals the expression of D2Rs in both DANs and the mushroom body (MB) of third-instar larval brains. Silencing D2Rs in DAN-c1 via microRNA disrupts aversive learning, further supported by optogenetic activation of DAN-c1 during training, affirming the inhibitory role of D2R autoreceptor. Intriguingly, D2R knockdown in the MB impairs both appetitive and aversive learning. These findings elucidate the distinct contributions of D2Rs in diverse brain structures, providing novel insights into the molecular mechanisms governing associative learning in Drosophila larvae.

The intricate relationship between the dopaminergic system and olfactory associative learning in Drosophila has been an intense scientific inquiry. Leveraging the formidable genetic tools, we conducted a screening of 57 dopaminergic drivers, leading to the discovery of DAN-c1 driver, uniquely targeting a pair of dopaminergic neurons (DANs) in the larval brain. While the involvement of excitatory D1-like receptors is well established, the role of D2-like receptors (D2Rs) remains underexplored. Our investigation reveals the expression of D2Rs in both DANs and the mushroom body (MB) of third-instar larval brains. Silencing D2Rs in DAN-c1 via microRNA disrupts aversive learning, further supported by optogenetic activation of DAN-c1 during training, affirming the inhibitory role of D2R autoreceptor. Intriguingly, D2R knockdown in the MB impairs both appetitive and aversive learning. These findings elucidate the distinct contributions of D2Rs in diverse brain structures, providing novel insights into the molecular mechanisms governing associative learning in Drosophila larvae.

Tau is an intracellular protein but also known to be released into the extracellular fluid. Tau release mechanisms have drawn intense attention as these are known to play a key role in Alzheimer's disease (AD) pathology. However, tau can also be released under physiological conditions although its physiological function and release mechanisms have been poorly characterized, especially in human neuronal cells. We investigated endogenous tau release in ReNCell VM, a human neuroprogenitor cell line, under physiological conditions and found that tau is spontaneously released from cells. To study activity-dependent release of endogenous tau, human ReNCell VM culture was stimulated by 100μM AMPA or 50mM KCl for one-hour, tau was actively released to the culture medium. The released tau was highly phosphorylated at nine phosphorylation sites (pSites) detected by phospho-specific tau antibodies including AT270 (T175/T181), AT8 (S202/T205), AT100 (T212/S214), AT180 (T231), and PHF-1 (S396/S404), showing that these pSites are important for activity-dependent tau release from human ReNCell VM. Intracellular tau showed various phosphorylation status across these sites, with AT270 and PHF-1 highly phosphorylated while AT8 and AT180 were minimally phosphorylated, suggesting that AT8 and AT180 pSites exhibit a propensity for secretion rather than being retained intracellularly. This activity-dependent tau release was significantly decreased by inhibition of GSK-3β, demonstrating that GSK3β-dependent phosphorylation of tau plays an important role in its release by neuronal activity. In this study, we showed that ReNCell VM serves as a valuable model for studying endogenous physiological tau release. Further, ReNCell model can be also used to study pathological release of human tau that will contribute to our understanding of the progression of AD and related dementias.

PDF file, 129KB, Blood analysis of WZB117-treated tumor-bearing mice.

PDF file, 70KB, WZB117 inhibited cell proliferation of cancer cells more than non-cancerous cells and exhibited synergy with anticancer drugs.

Synchrotron X-ray fluorescence microscopy (SXRF) presents a valuable opportunity to study the metallome of single cells because it simultaneously provides high-resolution subcellular distribution and quantitative cellular content of multiple elements. Different sample preparation techniques have been used to preserve cells for observations with SXRF, with a goal to maintain fidelity of the cellular metallome. In this case study, mouse pancreatic beta-cells have been preserved with optimized chemical fixation. We show that cell-to-cell variability is normal in the metallome of beta-cells due to heterogeneity and should be considered when interpreting SXRF data. In addition, we determined the impact of several immunofluorescence (IF) protocols on metal distribution and quantification in chemically fixed beta-cells and found that the metallome of beta-cells was not well preserved for quantitative analysis. However, zinc and iron qualitative analysis could be performed after IF with certain limitations. To help minimize metal loss using samples that require IF, we describe a novel IF protocol that can be used with chemically fixed cells after the completion of SXRF.

Parkinson's disease (PD) is a neurodegenerative disease showing uncontrollable motor symptoms that are primarily caused by the progressive loss of dopaminergic neurons in the brain. Currently no treatment exists to prevent PD progression. Therefore, discovery of new neuroprotective strategies still has great potential to benefit PD patients. A handful of studies show that activation of cAMP pathways is neuroprotective against PD progression. However, the neuroprotective role of this signaling cascade specifically in DA neurons has not been explored. In this study, fruit fly Drosophila melanogaster was used because of its sophisticated and powerful genetic approaches, especially with related to cAMP signaling pathway. We have investigated molecular mechanisms of neuroprotection in a fly larval model of PD by administering an environmental PD toxin rotenone. Increased cAMP signaling in the dunce mutant fly carrying defects in phosphodiesterase (PDE) gene, is neuroprotective against rotenone-induced locomotion deficits. Furthermore, the neuroprotective role of cAMP signaling specifically in DA neurons has been studied as it has not been explored. By using transgenic flies expressing designer receptors exclusively activated by designer drugs (DREADDs), we have shown that an increase of cAMP levels in DA neurons rescues rotenone-induced locomotion deficits. We also showed that this neuroprotection is mediated by activation of Gαs and PKA-C1 subunits. The results provide novel findings that expand our knowledge of neuroprotective mechanisms in DA neurons affecting PD progression, which could contribute to the development of new therapeutic treatments against PD. An important future study will explore downstream targets of cAMP-PKA signaling.

Among the complex factors contributing to beta‐cell failure is inflammation. Elevated levels of proinflammatory cytokines in obese individuals, such as interleukin (IL)‐1beta and IL‐6, increases the risk of developing type‐2 diabetes (T2D), and there is evidence that these low levels of circulating cytokines lead to islet dysfunction. Iron is closely connected to both the inflammatory response and diabetes. High levels of dietary iron increase risk of developing T2D, and excessive iron uptake by beta‐cells can cause oxidative stress and inhibit function. In this study, islets were treated for 48h with 10 pg/mL IL‐1beta + 20 pg/mL IL‐6 as a model of low‐grade inflammation. Analysis of gene microarray data identified three iron‐associated genes among the most cytokine‐sensitive: HAMP, STEAP4, and LCN2. These proteins are all involved directly or indirectly with increasing and/or sequestering cellular iron. RT‐PCR following exposure to various stressors known to induce beta‐cell failure revealed upregulation of HAMP, STEAP4, and LCN2 to be cytokine‐specific. Overexpression of STEAP4 induced upregulation of HAMP and LCN2, while ferritin and ferroportin expression were unaffected. Beta‐cells scanned by synchrotron X‐Ray fluorescence provided data showing cytokine exposure alters iron distribution. Iron was found in discrete structures (area ~0.15‐0.45 µm2) that were significantly smaller and more iron‐dense in cytokine‐treated beta‐cells, consistent with the sequestering function of identified iron‐associated genes. These data suggest a network of iron‐regulating genes in beta‐cells plays a role in sequestering iron in response to low‐grade inflammation.

Neuronal activity can enhance tau release and thus accelerate tauopathies. This activity-dependent tau release can be used to study the progression of tau pathology in Alzheimer's disease (AD), as hyperphosphorylated tau is implicated in AD pathogenesis and related tauopathies. However, our understanding of the mechanisms that regulate activity-dependent tau release from neurons and the role that tau phosphorylation plays in modulating activity-dependent tau release is still rudimentary. In this study, Drosophila neurons in primary culture expressing human tau (hTau) were used to study activity-dependent tau release. We found that hTau release was markedly increased by 50 mM KCl treatment for 1 h. A similar level of release was observed using optogenetic techniques, where genetically targeted neurons were stimulated for 30 min using blue light (470 nm). Our results showed that activity-dependent release of phosphoresistant hTauS11A was reduced when compared with wildtype hTau. In contrast, release of phosphomimetic hTauE14 was increased upon activation. We found that released hTau was phosphorylated in its proline-rich and C-terminal domains using phosphorylation site-specific tau antibodies (e.g., AT8). Fold changes in detectable levels of total or phosphorylated hTau in cell lysates or following immunopurification from conditioned media were consistent with preferential release of phosphorylated hTau after light stimulation. This study establishes an excellent model to investigate the mechanism of activity-dependent hTau release and to better understand the role of phosphorylated tau release in the pathogenesis of AD since it relates to alterations in the early stage of neurodegeneration associated with increased neuronal activity.

Pancreatic beta-cells secrete insulin, a hormone that maintains blood glucose homeostasis. The dysfunction of beta-cells leads to development of type2 diabetes (T2D). The causes of beta-cell dysfunction in T2D are complex. In obese individuals, increases in circulating proinflammatory cytokines IL-6 and IL-1beta are linked to a higher risk of developing diabetes, suggesting that these cytokines can be diabetes-promoting. When used at circulating concentrations found in obese individuals, these cytokines have been shown to alter calcium homeostasis in mouse beta-cells. In addition, changes in homeostasis of zinc and iron have been reported in diabetic individuals. The central hypothesis of this study: exposure to diabetes-promoting cytokines, at concentrations found in obese individuals, leads to changes in metal intracellular concentrations or distributions in beta-cells. Mouse primary beta-cells were exposed for 48 hours to cytokines: 10pg/mL IL-1beta and 20pg/mL IL-6. After cytokine exposure the cells were chemically fixed and synchrotron X-Ray fluorescence (SXRF) was employed, to investigate intracellular distributions and concentrations of zinc, calcium and iron. SXRF estimated the total cellular iron content to be 30.44 ± 12.18 (fg), and after acute exposure to cytokines the iron content was slightly increased to 47.21 ± 36.44 (fg) (mean ± S.D.). High concentrations of iron were localized in puncta structures, which were observed throughout the cytosol in all beta-cells and further analysis of the iron puncta revealed that iron was found at higher density in puncta of cells after they were exposed to cytokines, suggesting iron accumulation in these structures. The total cellular zinc content was 158.69 ± 57.68 (fg) in control cells and was significantly decreased to 65.73 ± 29.65 (fg) in cells after exposure to cytokines (mean ± S.D.) (Welch's t-test, t(28.97) = 6.655, P<0.0001). Zinc was localized to a perinuclear space of the cells in control cells and was evenly distributed after exposure to cytokines, suggesting that cytokines had a significant effect on zinc homeostasis in beta-cells. Calcium was also observed in perinuclear space of beta-cells before cytokine exposure and was localized to similar cellular compartments as zinc. Synchrotron X-ray fluorescence estimated total cellular calcium to be 216.10 ± 67.37 femtograms (fg) control cells and was significantly decreased to 154.28 ± 68.66 (fg) after the exposure to cytokines (mean ± S.D.), (Two sample t-test, t(43)=3.040, P=0.0040). In conclusion, the synchrotron X-ray fluorescence identified significant changes to zinc, calcium and iron metallomes of pancreatic beta-cells after exposure to cytokines, which warrant further investigation.

Pancreatic beta-cells synthesize and secrete insulin maintaining an organism's energy homeostasis. In humans, beta-cell dysfunction and death contribute to the pathogenesis of type 2 diabetes (T2D). Although the causes of beta-cell dysfunction are complex, obesity-induced low-grade systemic inflammation plays a role. For example, obese individuals exhibiting increased levels of proinflammatory cytokines IL-6 and IL-1beta have a higher risk of beta-cell dysfunction and T2D. Interestingly, obesity-induced inflammation changes the expression of several cellular metal regulating genes, prompting this study to examine changes in the beta-cell metallome after exposure to proinflammatory-cytokines. Primary mouse beta-cells were exposed to a combination of IL-6 and IL-1beta for 48 hours, were chemically fixed and imaged by synchrotron X-ray fluorescent microscopy. Quantitative analysis showed a surprising 2.4-fold decrease in the mean total cellular content of zinc from 158 ± 57.7 femtograms (fg) to 65.7 ± 29.7 fg; calcium decreased from 216 ± 67.4 to 154.3 ± 68.7 fg (control vs. cytokines, respectively). The mean total cellular iron content slightly increased from 30.4 ± 12.2 to 47.2 ± 36.4 fg after cytokine treatment; a sub-population of cells (38%) exhibited larger increases of iron density. Changes in the subcellular distributions of zinc and calcium were observed after cytokine exposure. Beta-cells contained numerous iron puncta that accumulated still more iron after exposure to cytokines. These findings provide evidence that exposure to low levels of cytokines is sufficient to cause changes in the total cellular content and/or subcellular distribution of several metals known to be critical for normal beta-cell function.

The microtubule-associated protein tau (τ) is a phosphoprotein that is crucial for regulating microtubule dynamics. Tau is highly enriched in neurons, where it functions by binding tubulin and stabilizing axonal microtubules. Phosphorylation of tau within its microtubule-binding repeat (R) domains significantly reduces its affinity for tubulin, leading to a loss in microtubule stability. In neurons, dysregulated kinase activity often results in the formation of hyper-phosphorylated tau isoforms that remain permanently detached from microtubules. If left untreated, hyper-phosphorylated tau can aggregate into insoluble, prion-like oligomers that contribute to the pathogenesis of neurodegenerative disease. Consequently, there is considerable interest in developing inhibitors that reduce levels of hyper-phosphorylated tau in neurons. In this study, we have generated a synthetic peptide mimetic (tR1) of the tau R1 domain as an inhibitor of microtubule-affinity regulating kinase 2 (MARK2). In vitro assays showed that tR1 inhibits the MARK2-mediated phosphorylation of tau within its R1 domain at Ser262, a residue critical for favorable tau-tubulin interactions. We also demonstrate that tR1 peptides are > 90% stable up to 24 h in neurobasal medium and RPMI media supplemented with human serum. Uptake experiments in cultured rat primary cortical neurons indicate that tR1 is internalized through an energy-dependent mechanism and can be delivered to the cytoplasm when co-treated with bafilomycin A1 or chloroquine. Furthermore, we show tR1 inhibits phosphorylation of endogenous tau at Ser262 in cultured neurons following activation of intracellular kinases. This inhibitory effect was selective for kinases that phosphorylate tau at Ser262, as tR1 did not inhibit tau phosphorylation at Thr231. Collectively, these results establish tR1 as a highly-stable, peptide-based kinase inhibitor that reduces the level of phosphorylated tau proteins in neurons.

The belief that the vertebrate brain functions normally without classical lymphatic drainage vessels has been held for many decades. On the contrary, new findings show that functional lymphatic drainage does exist in the brain. The brain lymphatic drainage system is composed of basement membrane-based perivascular pathway, a brain-wide glymphatic pathway, and cerebrospinal fluid (CSF) drainage routes including sinus-associated meningeal lymphatic vessels and olfactory/cervical lymphatic routes. The brain lymphatic systems function physiological as a route of drainage for interstitial fluid (ISF) from brain parenchyma to nearby lymph nodes. Brain lymphatic drainage helps maintain water and ion balance of the ISF, waste clearance, and reabsorption of macromolecular solutes. A second physiological function includes communication with the immune system modulating immune surveillance and responses of the brain. These physiological functions are influenced by aging, genetic phenotypes, sleep-wake cycle, and body posture. The impairment and dysfunction of the brain lymphatic system has crucial roles in age-related changes of brain function and the pathogenesis of neurovascular, neurodegenerative, and neuroinflammatory diseases, as well as brain injury and tumors. In this review, we summarize the key component elements (regions, cells, and water transporters) of the brain lymphatic system and their regulators as potential therapeutic targets in the treatment of neurologic diseases and their resulting complications. Finally, we highlight the clinical importance of ependymal route-based targeted gene therapy and intranasal drug administration in the brain by taking advantage of the unique role played by brain lymphatic pathways in the regulation of CSF flow and ISF/CSF exchange.

The microtubule-associated protein tau stabilizes microtubules by interacting with tubulins in neurons. Tau that is post-translationally phosphorylated can dissociate from tubulin, leaving polymeric microtubule structures in highly dynamic states. Tau phosphorylation at specific amino acids is precisely regulated by tau kinases and tau phosphatases. When this machinery is dysregulated, tau can become hyper-phosphorylated. Prolonged dissociation of hyper-phosphorylated tau induces microtubule collapse and disrupts axonal transport. More critically, phosphorylated tau can form prion-like oligomers, which have been implicated in the pathogenesis of age-dependent neurodegenerative diseases such as Alzheimer's disease (AD). Elevated activity of tau kinases, including microtubule affinity regulating kinase 2 (MARK2), has been demonstrated during AD pathogenesis. MARK2 is known to phosphorylate tau in one of four repeat (R) domains. Our objective was to develop synthetic peptide mimetics of human tau R domains as non-cytotoxic, cell-permeable ligands to inhibit MARK2-mediated phosphorylation of endogenous tau. A series of human tau R domain mimetics were synthesized and showed minimal effects on viability when added to rat primary cortical neurons at concentrations up to 100 μM. Antibody-based fluorescence polarization assays were used to demonstrate that our hTau R1 peptide mimetic was a ligand of MARK2 in vitro. We observed that fluorescent hTau R1 was internalized by cortical neurons and were present throughout the cell body, neurites and puncta, suggesting internalization by an endocytotic pathway. Treating cortical neurons with phenylarsine oxide (PAO) to upregulate MARK2 activity increased endogenous tau Ser262 phosphorylation 290 ± 23.9% over non-treated controls. Interestingly, co-treatment of PAO-treated cells with hTau R1 and endosome disruptor bafilomycin A1 reduced Ser262 phosphorylation levels to 200 ± 17.1% over non-treated controls. PAO also significantly reduced phosphorylation of tau at Thr231 through an unknown mechanism. Notably, co-treatment of PAO-treated cells with hTau R1 and bafilomycin A1 did not change the level of phosphorylated tau at Thr231. Furthermore, a six-day pre-incubation of cortical neurons with 10 μM hTau R1 significantly reduced the okadaic acid-induced tau phosphorylation at Ser262, but did not have a measurable effect on the level of phosphorylation at Thr231. Taken together, these data suggest that R1 peptides are internalized by cortical neurons and inhibit MARK2-dependent phosphorylation of endogenous tau at Ser262 when delivered to the cytosol. Importantly, these results show that hTau R1 peptide mimetics are capable of selectively inhibiting tau phosphorylation at MARK2-dependent sites. We anticipate that hTau R1 peptide mimetics will serve as strong leads in the development peptide-based AD therapeutics and as tools to study the complex nature of tau biology. Support or Funding Information This work was supported in part by the Department of Chemistry and Biochemistry, the Edison Biotechnology Institute, College of Arts and Sciences and the Vice President for Research at Ohio University. Additional funding for this research came from the Ohio Musculoskeletal and Neurological Institute (OMNI) at Ohio University (project# GA017434G). Uptake of R1 peptides in rat cortical neurons. Fluorescently-labeled R1 peptides were incubated with primary neuronal cultures for 6 days. Arrows show regions where peptide has accumulated in punctae, presumably endosomes. R1 peptide in neuronal processes are shown in circled regions.

Zinc is an essential trace element and its critical role in the normal function and pathophysiology of the nervous system is firmly established. However, the study of zinc in neurons is still in its infancy when considering the many intracellular processes that may be involved in maintaining homeostatic control of zinc, possible zinc signaling pathways, and dysfunction in disease. A complete understanding of these key homeostatic and signaling mechanisms relies on accurate and reproducible measurement of the dynamics of both intracellular total and free zinc, as well as the subcellular distribution of zinc in the cytosol and organelles. The methods available to researchers to study and quantify intracellular zinc dynamics in neurons have advanced considerably over the past 10-15 years. Several quantitative methods are available that can reliably report the total levels of zinc in a neuron as well as methods that can detect and monitor the intracellular distribution and free concentration in the cytosol and various organelle compartments. In this chapter, the authors wish to bring the reader up-to-date on methods used to quantify levels and changes in intracellular concentrations of zinc in cultured neurons. Here, we will be focusing on: (1) synchrotron radiation X-ray fluorescence (SRXRF) for total cellular zinc and topographic analysis, (2) population spectrofluorometric analysis of the dynamics of cytosolic zinc, and (3) Forster resonance energy transfer (FRET) microscopic analysis of genetically encoded sensors, which can report the dynamics of free concentrations of zinc in the cytosol and organelles.

A co-culture, or cell culture containing two separate populations of cell types, enables the study of cell-cell interactions or complex multicellular systems. They hold largely untapped potential for industrial, environmental and translational biomedical sciences (Goers, Freemont and Polizzi, 2014). Many neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease, are hypothesized to involve the cell-to-cell propagation of characteristic misfolded proteins (Costanzo and Zurzolo, 2013). The development of a co-culture, or hybrid culture, model of neurodegenerative disease would not only permit the in vitro study of the mechanism of protein propagation, but it would also provide a system in which to test potential therapeutic compounds. The hybrid culture in this thesis project consists of two types of neurons: primary cortical rat neurons and primary embryonic fly neurons. Genetic control of the Drosophila (fruit fly) genome permits the restricted expression of the protein of interest to the fly neurons. Thus, with clearly identifiable preand post-synaptic neurons, the characteristics of the protein propagation from the fly neuron to the rat neuron can be investigated. This honors thesis aims to develop and apply a hybrid culture model to study the propagation of the protein tau in Alzheimer’s disease (AD). To do so, it was first necessary to create, optimize and characterize a primary hybrid cell culture including rat and fly neurons. The environment for optimal hybrid culture survival was identified by systematically manipulating media conditions and evaluating cell

Woodier et al. (1) report that the cardiac type 2 ryanodine receptor channel (RyR2) shows two modes (sites) of regulation by zinc. When calcium is activating the channel, zinc increases channel Po around 100 pM (high affinity), whereas in the absence of calcium, zinc must reach 100 nM (low affinity) before channel Po is increased (compare Fig. 1 with Fig. 4, model in Fig.10). We would like to offer an alternate interpretation of these data. In order to achieve nominally 0 free calcium, 1 mM BAPTA (2,2 -(ethylenedioxy)dianiline-N,N,N ,N -tetraacetic acid) was added. We believe the primary reason that higher zinc (100 nM) must be added to modulate RyR2 gating in 0 free calcium is that zinc is chelated by BAPTA as well. It is not possible to use BAPTA to selectively chelate calcium without similarly affecting free zinc concentrations because Zn binds to BAPTA (Kd 7.9 nM) with greater affinity than Ca (Kd 110 nM) (2– 4). Using these values, one can estimate free zinc concentrations under the conditions given in Fig. 4 (with the addition of 1 mM BAPTA). With 1 nM zinc added, the free zinc concentration will be 0.1 fM, and with 100 M zinc added, the free zinc concentration will be 1 nM. It is noteworthy that 1 nM free zinc is approximately the same concentration range that modulates gating with calcium present (Fig. 1). Thus, the simplest interpretation of these data is that RyR2 has a single high affinity (1 nM) zinc binding site that functions to both modulate calcium-activated gating and activate channel opening when calcium is absent.