Intracellular Ca signals reach their greatest intensity and highest frequencies in striated muscle. Sustaining them is calsequestrin, Casq, notable for its high Ca-binding capacity, convenient affinity and for two unique properties demonstrated in vitro: a marked dependence of its ability to bind Ca on how much free Ca is present and a Ca-driven tendency to polymerize. In addition to these Ca storing properties, there are evidences for a gating function, whereby Casq senses the surrounding Ca concentration and accordingly induces the Ca release channel, RyR, to open or close, through a mechanical link presumably provided by triadin, Tr. The relevance of these functions becomes obvious in view of multiple mutations in Casq that are linked to grave diseases. Our goal is to define the operation in vivo of these unique properties demonstrated in vitro. To this end we joined a lab that has driven the study of Casq's physical chemistry and one that pioneered the quantification of Casq's functions in adult muscle. The plan includes the testing, in vitro and in vivo, of three hypotheses: (1) the extent and type of polymerization of Casq in cells changes as [Ca] depletes inside the cellular store (SR). (2) Changes in [Ca2+]SR are translated, via Casq and Tr, to gating changes in the RyR. (3) Mutations in Casq2 linked to the disease CPVT (catecholaminergic polymorphic ventricular tachycardia), as well as M87T, a variant in Casq1 present in a sizable percentage of patients tested for the disease MH (malignant hyperthermia), cause the disease phenotype through mechanisms (1) and (2). To test (1) we will examine the EM structure of Casq1 in Ca-depleted cells, test the ability of a non-polymerizing mutated Casq1 to restore function in Casq-null cells, and carry out in vitro measurements of isotopic Ca diffusion, probing whether the presence of Casq alters Ca diffusion, and how the effects depends on Casq polymerization. For (2) we will explore the effects on Ca signaling of eliminating the putative link provided by Tr in (a) Tr-null mice and (b) cells acutely deprived of the link by expression of decoys, the oligopeptides that bind Casq in the Tr sequence. For (3) we will characterize the Ca-dependent physical chemistry of 11 known mutants of Casq2, the homologous mutants of Casq1 and its M87T variant. We will then test the ability of these mutants to restore function in Casq-null mouse fibers. While understanding how this iconic biobuffer works will be a main reward, the long-term goal is to build a basic science foundation translatable to rational strategies that address human diseases, namely: find the causative mutation?generate the protein?characterize its function in vitro ? then in vivo specify the pathogenic functional gain or loss. Successful completion of these stages will allow us in future iterations of the project to ?design possible rescue strategies?test their efficacy.
Intracellular calcium signals drive a myriad of cellular phenomena. The signals are made by calcium release from intracellular deposits. Calsequestrin evolved to provide efficient storage and delivery of this calcium in the tissues (muscles) where the movements are greatest. It is also present in other tissues, where its functions are not well understood. Calsequestrin's individual molecules appear to work much better when they cooperate to form a complex polymeric network. Mutations of the molecule lead to diseases of the heart, and perhaps skeletal muscles as well. We test here the idea that the disease brought about by the mutations largely stems from failure to form the network. The rules that govern calcium binding and network formation are best studied in solution, but the consequences of failure must be studied within cells. We, a lab that studies the physical chemistry of calsequestrin, and one specialized in demonstrating its properties within cells, have joined forces in a team configured specifically to define the mechanisms in solution and demonstrate them inside living cells. The study will include the diseased mutant protein, so that a picture will emerge of the causative changes in the molecular properties and the consequences of these changes in living individuals. The study of mutant molecules has two-way relevance, to understand the malfunctioning mechanisms and eventually remediate the disease, and also because the mutant molecules are ready-made constructs to test the functional relevance of different stretches within the molecular sequencethe so-called structure-function relationship, a holy grail of basic science that eventually acquires relevance to public health. Finally, this project proposes the first systematic study of signals in human subjects with a naturally occurring modification of calsequestrin that is likely to alter the functions of skeletal muscle. Because disease consequences have not been demonstrated yet, the modification is dubbed a variant rather than a mutation. This last goal can then be simply put as: Is this variant a mutation? Note: this section is the same as in the original.
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