Ryanodine receptor studies using genetically engineered mice

AbstractRyanodine receptors (RyR) regulate intracellular Ca2+ release in many cell types and have been implicated in a number of inherited human diseases. Over the past 15years genetically engineered mouse models have been developed to elucidate the role that RyRs play in physiology and pathophysiology. To date these models have implicated RyRs in fundamental biological processes including excitation–contraction coupling and long term plasticity as well as diseases including malignant hyperthermia, cardiac arrhythmias, heart failure, and seizures. In this review we summarize the RyR mouse models and how they have enhanced our understanding of the RyR channels and their roles in cellular physiology and disease

Analysis of Cardiac Ion Channels to Understand Arrhythmias Which Lead to Sudden Cardiac Death

There are 300,000-400,000 fatalities attributed to sudden cardiac death every year in the U.S. due to a lack of sufficient research on mechanisms causing arrhythmias1. Malfunctions with the ion channels in the heart may lead to lethal arrhythmias. The purpose of this work is to study ion channels and evaluate malfunctions relative to normally functioning hearts. Plasmid insertion in E. coli assayed whether functional ion channels reach the membrane, and confocal fluorescent microscopy was used to illuminate cellular functionality. In addition, genetic analysis was used to determine the extent of hereditary factors in sudden cardiac death. Genes that encode for the voltage-gated sodium, potassium, and calcium ion channels were analyzed at the genetic level using isolated DNA samples and traditional Sanger sequencing methods to identify mutations that may be responsible for sudden cardiac death syndromes. For example, Long QT syndrome, Short QT syndrome, and Brugada syndrome are caused by mutations in these ion channels. Once these mutations are identified, genetic engineering techniques can be used in the generation of new heart cells from the stem cells found in somatic tissue. Generation of such heart cells is important because it could lead to the development of personalized treatment for degenerative diseases such as heart failure in the future. Rubart, M. et al., Mechanisms of Sudden Cardiac Death, 2005. J. clin. invest. 115(9):2305-2315

Leaky ryanodine receptors in β-sarcoglycan deficient mice: a potential common defect in muscular dystrophy

Disruption of the sarcolemma-associated dystrophin-glycoprotein complex underlies multiple forms of muscular dystrophy, including Duchenne muscular dystrophy and sarcoglycanopathies. A hallmark of these disorders is muscle weakness. In a murine model of Duchenne muscular dystrophy, mdx mice, cysteine-nitrosylation of the calcium release channel/ryanodine receptor type 1 (RyR1) on the skeletal muscle sarcoplasmic reticulum causes depletion of the stabilizing subunit calstabin1 (FKBP12) from the RyR1 macromolecular complex. This results in a sarcoplasmic reticular calcium leak via defective RyR1 channels. This pathological intracellular calcium leak contributes to reduced calcium release and decreased muscle force production. It is unknown whether RyR1 dysfunction occurs also in other muscular dystrophies. To test this we used a murine model of Limb-Girdle muscular dystrophy, deficient in β-sarcoglycan (Sgcb−/−). Skeletal muscle RyR1 from Sgcb−/− deficient mice were oxidized, nitrosylated, and depleted of the stabilizing subunit calstabin1, which was associated with increased open probability of the RyR1 channels. Sgcb−/− deficient mice exhibited decreased muscle specific force and calcium transients, and displayed reduced exercise capacity. Treating Sgcb−/− mice with the RyR stabilizing compound S107 improved muscle specific force, calcium transients, and exercise capacity. We have previously reported similar findings in mdx mice, a murine model of Duchenne muscular dystrophy. Our data suggest that leaky RyR1 channels may underlie multiple forms of muscular dystrophy linked to mutations in genes encoding components of the dystrophin-glycoprotein complex. A common underlying abnormality in calcium handling indicates that pharmacological targeting of dysfunctional RyR1 could be a novel therapeutic approach to improve muscle function in Limb-Girdle and Duchenne muscular dystrophies

Unitary Ca2+ current through recombinant type 3 InsP3 receptor channels under physiological ionic conditions

The ubiquitous inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) channel, localized primarily in the endoplasmic reticulum (ER) membrane, releases Ca2+ into the cytoplasm upon binding InsP3, generating and modulating intracellular Ca2+ signals that regulate numerous physiological processes. Together with the number of channels activated and the open probability of the active channels, the size of the unitary Ca2+ current (iCa) passing through an open InsP3R channel determines the amount of Ca2+ released from the ER store, and thus the amplitude and the spatial and temporal nature of Ca2+ signals generated in response to extracellular stimuli. Despite its significance, iCa for InsP3R channels in physiological ionic conditions has not been directly measured. Here, we report the first measurement of iCa through an InsP3R channel in its native membrane environment under physiological ionic conditions. Nuclear patch clamp electrophysiology with rapid perfusion solution exchanges was used to study the conductance properties of recombinant homotetrameric rat type 3 InsP3R channels. Within physiological ranges of free Ca2+ concentrations in the ER lumen ([Ca2+]ER), free cytoplasmic [Ca2+] ([Ca2+]i), and symmetric free [Mg2+] ([Mg2+]f), the iCa–[Ca2+]ER relation was linear, with no detectable dependence on [Mg2+]f. iCa was 0.15 ± 0.01 pA for a filled ER store with 500 µM [Ca2+]ER. The iCa–[Ca2+]ER relation suggests that Ca2+ released by an InsP3R channel raises [Ca2+]i near the open channel to ∼13–70 µM, depending on [Ca2+]ER. These measurements have implications for the activities of nearby InsP3-liganded InsP3R channels, and they confirm that Ca2+ released by an open InsP3R channel is sufficient to activate neighboring channels at appropriate distances away, promoting Ca2+-induced Ca2+ release

Creating the Cahokian Community: the Power of Place in Early Mississippian Sociopolitical Dynamics

This study is an examination of how sociopolitical change occurs, particularly the formation of large scale polities from culturally diverse populations. Drawing on Benedict Anderson’s concept of “imagined communities” and recent developments in archaeological theory, particularly agency and practice theory, I contend that the social construction of space and community identities at multiple scales were instrumental in the creation of the Cahokia polity in the American Bottom region of southwestern Illinois around A.D. 1050. In this study, I employ a multi-scalar perspective that includes detailed analyses of material culture, architecture, and spatial organization at five sites located in the American Bottom floodplain near the monumental Mississippian site of Cahokia. All five sites include occupations dating to the Mississippian Transition (A.D. 975–1100) which spans the Terminal Late Woodland Lindeman and Edelhardt phases (A.D. 1000–1050) and the early Mississippian Lohmann phase (A.D. 1050–1100). The mapping, geophysical survey, excavation, and material analyses for each of these sites combined with regional comparisons using a Geographic Information System provide evidence for changes in the construction of space, movement of people into and around the region, and the simultaneous dissolution of local communities and the construction of a large–scale community identity centered on Cahokia

Regulation of Type-2 and Type-3 Inositol (1,4,5)-Trisphosphate Receptors by cAMP-Dependent Protein Kinase Phosphorylation and ATP Binding

Thesis (Ph. D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Pharmacology and Physiology, 2008.Hormones, neurotransmitters, growth factors and pharmacological agents bind to cellular receptors that initiate the production of the second messenger inositol (1,4,5)-trisphosphate (InsP3). InsP3 induces the release of Ca2+ from endoplasmic reticulum (ER) stores via the activation of InsP3 receptors (InsP3R), which are Ca2+ release channels localized to the ER. Three isoforms of InsP3R (InsP3R1, InsP3R2, InsP3R3) are expressed in mammalian cells, but the relative abundance of each isoform differs greatly among different cell types. InsP3R are also regulated by molecular interactions in addition to InsP3 binding. Key regulatory mechanisms of InsP3R include Ca2+ binding, phosphorylation, ATP binding and interactions with other proteins. These and other factors differentially modulate the activities of the three InsP3R isoforms, thus playing a major role in shaping the temporal and spatial characteristics of intracellular Ca2+ signals. Much of the work on InsP3R regulation has been done using InsP3R1 as the model. Exocrine glands such as the pancreas and salivary glands, however, express very little InsP3R1. In fact, severe deficiencies in these tissues occurred when InsP3R2 and InsP3R3 were knocked out indicating a vital role for these isoforms in exocrine secretion. The main goal of the work presented in this thesis, therefore, was to identify the molecular mechanisms behind regulation of InsP3R2 and InsP3R3. An additional goal was to determine what impact these regulatory mechanisms have on Ca2+ signaling in exocrine acinar cells. Regulation of InsP3R2 and InsP3R3 by cAMP-dependent protein kinase (PKA) and ATP binding were explored in detail. These regulatory mechanisms were chosen because major differences in the responses of these two isoforms to PKA phosphorylation and ATP binding were thought to occur. Specifically, PKA was thought to enhance InsP3R2 but inhibit InsP3R3 and ATP was known to positively regulate InsP3R3 but thought to have no effect on InsP3R2 function. Furthermore, even though these regulatory interactions are likely to impact exocrine physiology, the molecular mechanisms behind the effects are unknown. Stable cell lines expressing InsP3R2 and InsP3R3 on a null background were created and used as the basis for study. These cell lines provided the unique advantage of allowing analysis of isoform-specific regulation in cells expressing a single wild type or mutant InsP3R in isolation. The effects of PKA phosphorylation and ATP binding on the function of these isoforms were determined using digital Ca2+ imaging in intact and permeabilized cells. The effects of mutating putative regulatory sites in InsP3R2 and InsP3R3 were tested. Ca2+ imaging experiments in pancreatic and parotid acinar cells isolated from InsP3R2 knockout mice were also performed to probe the role of InsP3R2 regulation in an endogenous setting. The findings presented in this thesis establish that InsP3R2 and InsP3R3 are both positively regulated by PKA phosphorylation. The specific residues phosphorylated by PKA were, however, found to be different in the two isoforms. Furthermore, similar to the other isoforms, InsP3R2 can be enhanced by ATP binding. ATP enhanced InsP3R2 activity exclusively at low levels of stimulation and with a high sensitivity (EC50 of 40 M). In comparison, InsP3R3 activity was enhanced by ATP at all levels of stimulation, but with a 10-fold lower sensitivity. The molecular determinants of ATP modulation were also different for the two isoforms. While ATP regulation of InsP3R2 required the presence of a conserved putative ATP-binding domain, mutation of the same site in InsP3R3 had no effect on ATP modulation. The high sensitivity of InsP3R2 to ATP was also the predominant effect in pancreatic acinar cells. The sensitivity of InsP3-induced Ca2+ release to ATP was significantly reduced in cells from InsP3R2 knockout mice. These results establish a unique role for InsP3R2 in setting the sensitivity of InsP3-induced Ca2+ release to ATP. In total, the results presented in this thesis provide novel insights into the molecular determinants and physiological implications of the regulation of InsP3R2 and InsP3R3 by PKA phosphorylation and ATP binding