Muscle contraction and relaxation are controlled by the intracellular free Ca²⁺ concentration. The sarcoplasmic reticulum (SR) is an intracellular membrane system which regulates this internal free Ca²⁺ concentration. Responding to an electrical excitation of the cell surface membrane, the SR releases Ca²⁺ through a specific Ca²⁺ release channel, thus elevating the Ca²⁺ concentration inside muscle cell and causing the muscle to contract. Subsequent sequestration of Ca²⁺ by the SR Ca²⁺ pumps restores the resting state of the muscle cell. This research focuses on the Ca²⁺ release channel from skeletal muscle SR. The planar lipid bilayer technique was used to study the channel at the single channel level. The SR Ca²⁺ release channel was identified and isolated via its interaction with specific sulfhydryl oxidizing agents. This protein of a molecular mass of 106 kDa was then incorporated into a planar lipid bilayer membrane (BLM). In an asymmetrical Ca²⁺ solution, the channel protein demonstrates a single channel conductance of 107 ± 13 pS and a permeability ratio of Ca²⁺ versus Tris⁺ of 7.4 ± 3.3. In a symmetrical 250 mM NaCl solution, the channel protein displays a large single channel conductance of 400 ± 20 pS, and a weak voltage-dependence. The channel is activated by millimolar ATP and inhibited by micromolar ruthenium red. Nanomolar concentrations of ryanodine modify the channel by changing it from a rapidly gating full conductance state to a long-lived subconductance state. These results demonstrate that the isolated 106 kDa protein channel has properties similar to those observed following fusion of SR vesicles to a BLM. The bilayer system was also used to examine the effect of Ag⁺ on the SR Ca²⁺ release channel. Ag⁺ (0.2-1. 0 μM ) activates the SR Ca²⁺ release channel. Activation by Ag⁺ does not require the presence of Ca²⁺, Mg²⁺, or ATP. Ag⁺ activates the channel by increasing the open probability Po. Ag⁺ activation is always followed by a spontaneous inactivation. The channel is still sensitive to ruthenium red inhibition after exposure to Ag⁺. Isolated SR vesicles were fused to a BLM to study the effect of the photooxidizing dye, rose bengal, on the gating characteristics of the reconstituted SR Ca²⁺ release channel. Rose bengal activates the Ca²⁺ release channel in the presence of light by increasing the channel open probability and leaving the single channel conductance unchanged. This photoactivation is independent of the myoplasmic Ca²⁺ concentration, and can be achieved from either side of the membrane. In addition, the effect is inhibited by addition of 10-20 μM ruthenium red. When modified to its subconducting state by ryanodine, subsequent addition of rose Bengal reactivates the channel to a rapidly fluctuating full conducting state. These studies carried out at the single channel level utilizing the planar lipid bilayer technique have not only enhanced our understanding of the Ca²⁺ release mechanism of skeletal muscle SR, but also provided information about the toxic effects on biological membrane systems caused by heavy metals and oxidizing agents.

Calcium ions are important mediators in the mechanism of contraction and relaxation of muscle fibers. Depolarization of sarcolemma and transverse tubule causes an increase of myoplasmic ca2+ concentration which induces contraction of the myofibrils. In skeletal muscle fibers, the intracellular Ca2+ concentraton is regulated by an extensive membrane system, the sarcoplasmic reticulum (SR). Ca2+-release from SR is initiated by depolarization of the transverse tubule via a process referred to as excitation-contraction coupling. The Ca2+ - release channel located in the junctional SR plays an important role in this mechanism.

No description available.

Plant cells contain multiple mobile Golgi bodies. Golgi bodies receive cargo from specialized subdomains of the endoplasmic reticulum (ER), so-called ER export sites (ERES). How ERES operate in plant cells is largely uncharacterized. <p>In mammals and yeast, the commonly recognized ER-to-Golgi transport model asserts that protein transport between these two organelles is mediated by vesicles. Formation of these vesicles is interceded by COPII and COPI coat complexes. COPII coat proteins assemble at ERES. The minimal components of the COPII coat comprise the following proteins: the GTPase Sar1, and two large heterodimeric complexes, Sec23/24 and Sec13/31. COPII vesicles are responsible for forward (anterograde) protein traffic from the ER to the Golgi apparatus. Proteins are constantly recycled from the Golgi back to the ER through a conserved backward (retrograde) pathway mediated by COPI coat proteins. Fusion of the anterograde and retrograde carriers with target membranes is mediated by a subset of specialized proteins called soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs). Studies conducted in mammalian and yeast systems also concluded that ER-to-Golgi SNARE proteins and membrane cargo proteins are concentrated into COPII vesicles through a direct interaction and binding with the pre-budding complex Sec23/24-Sar1. <p>The COPII component distribution and their biological function in plant cells are largely uncharacterized. Therefore, through the study of the COPII protein Sec24, this work aimed (i) to investigate where and how protein transport between ER and Golgi occurs in plant cells, and (ii) to establish the importance of the anterograde and retrograde transport equilibrium in regulating the ER protein export. To do so, live cell imaging of a fluorescent protein fusion of Sec24 was used and the dynamics of this protein chimaera were followed in tobacco leaf epidermal cells. The imaging investigations were complemented by mutagenesis studies and biochemical analyses. <p>The obtained results indicate that in plant cells Sec24 is localized at specific regions of the ER that represent mobile units continuously joined to the Golgi apparatus. From this study the importance of the balance between the anterograde and retrograde transport in protein ER export has also emerged. I have shown in fact, that blockage of the retrograde pathway using Arf1 mutants and COPI chemical inhibitor determines the collapse of the anterograde protein trafficking from the ER to the Golgi. Moreover, this study has shown that Sec24 is capable of an interaction with the SNAREs Sed5 and Sec22. This is a forward step in our understanding of the role of Sec24 in the mechanism of cargo selection and recruitment.

Endoplasmic reticulum (ER) stress has been implicated in pancreatic beta-cell loss contributing to diabetes mellitus, however the molecular mechanisms of ER stress-induced apoptosis are unclear. In the first project of this thesis, the contribution of ER stress in proinflammatory cytokine-mediated beta-cell dysfunction and apoptosis is examined. Although exogenous cytokine treatment did induce unfolded protein response (UPR) genes, increased chaperone capacity had no effect on apoptosis induction, insulin biosynthesis and insulin secretion. Thus, ER stress is most likely not an important pathway in cytokine toxicity under our experimental system. The second project develops a pathophysiological model of ER stress based on the mutant misfolded insulin of the Akita mouse. Microarray analysis was conducted and we observed early induction of ER chaperone and ER-associated degradation (ERAD) genes, followed by a large increase in pro-apoptotic genes with mutant insulin expression. A detailed analysis of the ER stress response in this system is presented.

Endoplasmic reticulum (ER) stress has been implicated in pancreatic beta-cell loss contributing to diabetes mellitus, however the molecular mechanisms of ER stress-induced apoptosis are unclear. In the first project of this thesis, the contribution of ER stress in proinflammatory cytokine-mediated beta-cell dysfunction and apoptosis is examined. Although exogenous cytokine treatment did induce unfolded protein response (UPR) genes, increased chaperone capacity had no effect on apoptosis induction, insulin biosynthesis and insulin secretion. Thus, ER stress is most likely not an important pathway in cytokine toxicity under our experimental system. The second project develops a pathophysiological model of ER stress based on the mutant misfolded insulin of the Akita mouse. Microarray analysis was conducted and we observed early induction of ER chaperone and ER-associated degradation (ERAD) genes, followed by a large increase in pro-apoptotic genes with mutant insulin expression. A detailed analysis of the ER stress response in this system is presented.

Plant cells contain multiple mobile Golgi bodies. Golgi bodies receive cargo from specialized subdomains of the endoplasmic reticulum (ER), so-called ER export sites (ERES). How ERES operate in plant cells is largely uncharacterized. <p>In mammals and yeast, the commonly recognized ER-to-Golgi transport model asserts that protein transport between these two organelles is mediated by vesicles. Formation of these vesicles is interceded by COPII and COPI coat complexes. COPII coat proteins assemble at ERES. The minimal components of the COPII coat comprise the following proteins: the GTPase Sar1, and two large heterodimeric complexes, Sec23/24 and Sec13/31. COPII vesicles are responsible for forward (anterograde) protein traffic from the ER to the Golgi apparatus. Proteins are constantly recycled from the Golgi back to the ER through a conserved backward (retrograde) pathway mediated by COPI coat proteins. Fusion of the anterograde and retrograde carriers with target membranes is mediated by a subset of specialized proteins called soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs). Studies conducted in mammalian and yeast systems also concluded that ER-to-Golgi SNARE proteins and membrane cargo proteins are concentrated into COPII vesicles through a direct interaction and binding with the pre-budding complex Sec23/24-Sar1. <p>The COPII component distribution and their biological function in plant cells are largely uncharacterized. Therefore, through the study of the COPII protein Sec24, this work aimed (i) to investigate where and how protein transport between ER and Golgi occurs in plant cells, and (ii) to establish the importance of the anterograde and retrograde transport equilibrium in regulating the ER protein export. To do so, live cell imaging of a fluorescent protein fusion of Sec24 was used and the dynamics of this protein chimaera were followed in tobacco leaf epidermal cells. The imaging investigations were complemented by mutagenesis studies and biochemical analyses. <p>The obtained results indicate that in plant cells Sec24 is localized at specific regions of the ER that represent mobile units continuously joined to the Golgi apparatus. From this study the importance of the balance between the anterograde and retrograde transport in protein ER export has also emerged. I have shown in fact, that blockage of the retrograde pathway using Arf1 mutants and COPI chemical inhibitor determines the collapse of the anterograde protein trafficking from the ER to the Golgi. Moreover, this study has shown that Sec24 is capable of an interaction with the SNAREs Sed5 and Sec22. This is a forward step in our understanding of the role of Sec24 in the mechanism of cargo selection and recruitment.

Thesis (Ph. D.)--Cornell University, January, 2007. / Vita. Includes bibliographical references (leaves 100-109).

Thesis (Ph. D.)--University of Chicago, Dept. of Biochemistry and Molecular Biology, December 2003. / Includes bibliographical references. Also available on the Internet.

Thesis--University of Helsinki. / Includes bibliographical references.