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INIZIO_TESTO_DA_INDICIZZARE

RESEARCH PROGRAM

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Keywords
RYANODINE RECEPTORS, CALCIUM SIGNALLING, SORCIN, CREATIN KINASE 2, CARDIAC CONTRACTION

Supramolecular complexes of sorcin in the generation and regulation of Calcium-dependent cellular functions

Università degli Studi di Siena
Abstract
Ca2+ is a second messenger that regulates several cellular functions by activating specific enzymatic activities and gene expression (Berridge, 1993). Ca2+ signalling is based on the ability of cells to keep resting levels of free Ca2+ in the cytosol in the range of 100 nM through the activity of Ca2+ transporters both on the plasma membrane and on the endoplasmic reticulum and to regulate transient increases of Ca2+ in the µM range by allowing Ca2+ to enter from extracellular fluids or to be released from internal stores on the endo/sarcoplasmic reticulum (Pozzan et al., 1994). Key elements in deciphering Ca2+ signalling are many Ca2+-binding proteins that, following Ca2+ binding, become able to interact with their specific targets and thus regulate the function of the interacting proteins. Most Ca2+-binding proteins contain characteristic EF hand motif and CaM is probably the most known of these proteins.

Sorcin is a ubiquitous 22 kDa Ca2+-binding protein of the penta EF-hand family of proteins that following Ca2+ binding undergoes a conformational change that allows reversible interaction with target proteins. In the heart, sorcin is involved in the regulation of the excitation-contraction-relaxation processes, where it plays an important role in the transition between contraction and relaxation cycles.. In particular, sorcin has an important role in the transition between contraction and relaxation. Sorcin has at least three different function: 1) inhibits >>>

Principal Investigator
Vincenzo Sorrentino Università degli Studi di SIENA
Research Objectives
The study of sorcin and of its target/interacting proteins represents a common point in the research of the three units participating to this project. Unit Colotti has a long lasting experience in studying both structural and functional properties of sorcin. Conversely, Unit Sorrentino has a research record on genetics and function of ryanodine receptors and Unit Ruzzene can supply relevant support in studying protein phosphorylation patterns. Given this background we expect that the coordinated activity of the three units present in this project can contribute to extend our knowledge on the interaction occurring between sorcin and RyR2 and between sorcin and CK2. The molecular basis of the formation of sorcin NCX will also be studied. Both RyR2 and NCX represent key interactors of sorcin in the context of regulation of cardiac contraction: RyR2 is involved essentially in the excitation-contraction phase, and its opening determines the increase of cytoplasmic calcium concentration, while NCX mostly contributes to the subsequent phase of decontraction. As to the interaction between sorcin and CK2, we expect to know whether this interaction may result in the phosphorylation of sorcin and, should this be the case, we will verify the predicted sites as the actual targets of CK2, and to verify whether phosphylation of sorcin may affect its binding to and/or its regulation of RyR2.

Planned activities are expected also to provide direct information on whether RyR2 >>>

First Results
The study of sorcin and of its target/interacting proteins represents a common point in the research of the three units participating to this project. The dynamic establishment of complexes with ionic channels such as the ryanodine receptor (RyR2), L-type voltage dependent channel, Na+-Ca2+ exchanger (NCX) and the sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) in the heart, allows sorcin to contribute to their regulation. Overall, sorcin participates in lowering cytoplasmic Ca2+ concentration, operating in at least three different modalities: it inhibits calcium release from SR by inhibiting RyR2, increases calcium entry from cytosol to SR and favours its extrusion throughout the sarcolemma, by activating SERCA2a and NCX, respectively. The knowledge of the molecular basis of the interaction of sorcin with the two channels will permit the comprehension of functional alterations in the channels, when they are mutated. Based on the previous experience of the participating units we expect, at the end of the first year, to have defined the regions important for the interaction between RyR2 and sorcin and between the NCX cytoplasmic loop and sorcin.
The characterization of the topology of sorcin-RyR2 and sorcin-NCX interaction will be important to unveil the molecular mechanisms at the basis of the excitation-contraction-decontraction cycle in the myocyte. The knowledge of the molecular details of the supramolecular complexes studied will have an impact for the comprehension >>>

Timescale
24 months
National and international background
Ca2+ regulates a number of functions in eukaryotic cells
Ca2+-dependent cellular functions include a wide range of cellular processes, such as secretion, gene expression, muscle contraction, egg fertilization, cell division, apoptosis in virtually all eukaryotic cell types (Berridge, 2006; Rizzuto and Pozzan, 2006). Ca2+ ions have proven to be particularly suitable to act as an intracellular messenger and regulator of cellular function because the very high gradient between the extra-cellular and the cytosolic concentrations. This would allow rapid and reversible changes of its concentration inside the cell, a property that is of key importance for a biological messenger to work properly. In addition, the specific combination of size and charge of Ca2+ allows it to reversibly bind to ligands having sufficient structural complexity to ensure specificity of interaction. The cytosolic concentration of Ca2+ is regulated by transport across plasma and intracellular membranes. Various channels, transport ATPases, uniporters, and antiporters in the plasma membrane, endoplasmic and sarcoplasmic reticulum, and mitochondria are responsible for the transport of Ca2+ (Brini and Carafoli, 2000). Advancements in methods for measuring Ca2+ have revealed that changes in the intracellular Ca2+ concentration occur following specific spatial and temporal patterns. Ca2+ transients may be organized into regular oscillations, whose frequency can be finely modulated to encode specific >>>