3-Hydroxy-3-Methylglutaryl Coenzyme A (HMG CoA) reductase is a 97 kDa glycoprotein located in the endoplasmic reticulum responsible for cholesterol biosynthesis in mammalian liver and intestine. HMG CoA reductase inhibitors (statins) (e.g. simvastatin, mevastatin and parvastatin) are used clinically to treat and prevent coronary artery diseases by reducing plasma LDL-cholesterol level. Recent studies have demonstrated that statins can provide beneficial effects (pleiotropic effects) beyond its lipid-lowering activity. However, the modulatory effects of statins on ion channels activities have not been fully explored. Hence, this study is designed to demonstrate the existence of the HMG CoA reductase in various human isolate cardiovascular preparations and the modulatory effect(s) of simvastatin on both large-conductance calcium-activated (BKCa) and ATP-sensitive (IKATP) potassium channels of porcine isolated coronary vascular smooth muscle cells. / In conclusion, our results demonstrated the biochemical existence of HMG CoA reductase in various human isolated cardiovascular preparations and porcine isolated coronary artery. Simvastatin modulates the BKCa and IKATP channels of the porcine isolated coronary artery via different and multiple cellular mechanisms. / In this study, we demonstrated the biochemical existence of the HMG CoA reductase in various human isolated cardiovascular preparations and porcine isolated coronary artery. In addition, we demonstrated that simvastatin modulates both the BKCa channels and IKATP channels of porcine isolated coronary artery via different mechanisms. Acute application of simvastatin (100 nM) slightly enhanced whereas simvastatin (≥ 1 muM) inhibited the BKCa amplitude of porcine coronary artery smooth muscle cells. The classical HMG CoA reductase-mevalonate cascade is important in mediating the inhibitory effect of simvastatin observed at low concentrations (1 and 3 muM), whereas an increased PKC-delta protein expression and activation is important in simvastatin (10 muM)-mediated inhibition of BKCa channels. In contrast, the basal activity of the IKATP channels was not affected by simvastatin (1, 3 and 10 muM). However, acute application of simvastatin (1, 3 and 10 muM) inhibited the opening of the IKATP channels by cromakalim and pinacidil in a PP2A-dependent manner (sensitive to okadaic acid, a PP2A inhibitor). The okadaic acid-sensitive, simvastatin-mediated inhibitory effect on IKATP channel is mediated by an activation of AMPK in a Ca2+-dependent manner. Activation of AMPK probably increased the activity of the Na+/K+ ATPase and subsequently caused an influx of glucose via the SGLT1 down the Na + concentration gradient for the ouabain-sensitive, glucose-dependent activation of PP2A. / Seto, Sai Wang. / Adviser: Yiu-Wa Kwan. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3456. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 221-254). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

In recent years, the determination of several high-resolution structures of transient receptor potential (TRP) channels has led to significant progress within this field. The primary focus of this dissertation is to elucidate the structural characterization of TRPML1 and TRPP2. Mutations in TRPML1 cause mucolipidosis type IV (MLIV), a rare neurodegenerative lysosomal storage disorder. We determined the first high-resolution crystal structures of the human TRPML1 I-II linker domain using X-ray crystallography at pH 4.5, pH 6.0, and pH 7.5. These structures revealed a tetramer with a highly electronegative central pore which plays a role in the dual Ca2+/pH regulation of TRPML1. Notably, these physiologically relevant structures of the I-II linker domain harbor three MLIV-causing mutations. Our findings suggest that these pathogenic mutations destabilize not only the tetrameric structure of the I-II linker, but also the overall architecture of full-length TRPML1. In addition, TRPML1 proteins containing MLIV-causing mutations mislocalized in the cell when imaged by confocal fluorescence microscopy. Mutations in TRPP2 cause autosomal dominant polycystic kidney disease (ADPKD). Since novel technological advances in single-particle cryo-electron microscopy have now enabled the determination of high-resolution membrane protein structures, we set out to solve the structure of TRPP2 using this technique. Our investigations offer valuable insight into the optimization of TRPP2 protein purification and sample preparation procedures necessary for structural analysis.

Proper functioning of the voltage gated sodium channel, NaV1.5, is essential for maintenance of normal cardiac electrophysiological properties. Changes to the biophysical properties of sodium channels can take many forms and can affect the peak component of current carried during phase zero of the action potential; the “persistent” or “late” current component conducted during the repolarizing phases of the action potential; the availability of the channel as seen by changes in window current; and the kinetics of channel transitions between closed, opened and inactivated states. Mutations in NaV1.5 that alter these parameters of channel function are linked to a number of cardiac diseases including arrhythmias such as atrial fibrillation. In addition, mutations in many of the auxiliary proteins that form part of the sodium channel macromolecular complex have likewise been associated with diseases of the heart. Mutations in regions of the sodium channel responsible for interactions with these auxiliary proteins have also been linked to various dysfunctional cardiac states. Indeed, a large number of disease causing mutations are localized to the C-terminal domain of NaV1.5, a hotspot for interacting proteins. Using a transgenic mouse model, we show that expression of a mutant sodium channel with gain-of-function properties conferring increased persistent current, is sufficient to cause both structural and electrophysiological abnormalities in the heart driving the development of spontaneous and prolonged episodes of atrial fibrillation. The sustained and spontaneous atrial arrhythmias, an unusual if not unique phenotype in mice, enabled explorations of mechanisms of atrial fibrillation using in vivo (telemetry), ex vivo (optical voltage mapping), and in vitro (cellular electrophysiology) techniques. Since persistent sodium current was the driver of the structural and electrophysiological abnormalities leading to atrial fibrillation, we subsequently pursued studies exploring the mechanisms of persistent sodium current. Prior work of heterologously expressed sodium channels identified calmodulin as a regulator of persistent current. Mutation of the calmodulin binding site in the C-terminus of the cardiac sodium channel caused increased persistent current when the channel was expressed heterologously. The role of calmodulin in the regulation of the sodium channel in cardiomyocytes has not been definitively determined. We created transgenic mice expressing human sodium channels harboring a mutation of the calmodulin binding site. Using whole cell patch clamping, we found, in contrast to previously reported findings, that ablation of the calmodulin binding site did not induce increased persistent sodium current. Instead, loss of calmodulin binding stabilized the inactivated state by shifting the V50 for steady-state inactivation in the hyperpolarizing direction. Furthermore, loss of calmodulin binding sped up the transition to the inactivated state demonstrated by a significantly shortened tau of inactivation. In contrast to studies performed in heterologous expression systems, our findings thus suggest that in heart cells, calmodulin binding increases availability, similar to its role in regulating NaV1.4 channels. The studies were then expanded to explore the role of other interacting proteins, fibroblast growth factor (FGF) homologous factors (FHF), in the presence and absence of calmodulin binding. Using whole cell patch clamping, we found that a mutation (H1849R) of the sodium channel causing decreased FHF binding affinity leads to a rightward shift in steady-state inactivation and a slowed rate of inactivation of INa. A third mutant channel, with concurrent decreased FHF and calmodulin binding affinity similarly results in a rightward shift in steady-state inactivation suggesting a dominant effect of the H1849R mutation. Persistent current was not elevated in either of these mutant channels. Importantly, the methodology that we report enables us and other groups to carry out studies of human sodium channels in the native environment of NaV1.5. Our investigation into calmodulin’s role, which yielded conclusions distinct from prior findings in heterologous expression systems, demonstrates the value of this approach.

Dans ce travail, nous présentons l’obtention de canaux permanents synthétiques, à base de cyclodextrines amphiphiles, en utilisant une méthode de polymérisation divergente d’oxiranes. Des modifications sélectives de cyclodextrines sont développées de manière à générer de nouveaux amorceurs de polymérisation anionique d’oxyde d’éthylène. Dans les conditions de synthèse utilisées, la démonstration du contrôle de la polymérisation est réalisée et l’obtention de molécules à 14 branches de POE, de longueur variable, est montrée. Parmi différentes applications envisageables, nous développons ici la possibilité d’utiliser ces molécules en étoile pour former des canaux ioniques permanents avec des temps de résidence de l’ordre de l’heure, ouvrant la voie par exemple à la translocation de molécules et de macromolécules. / In this work, we present the design of artificial permanent cyclodextrin-based channels, obtained by divergent polymerization. Selective modifications of cyclodextrins have been developed to generate original initiators of ethylene oxide ring-opening polymerization. Considering the experimental conditions used, the demonstration of controlled polymerization was performed, leading to molecules with 14 PEO arms having various molar masses. Among various applications, we focused on the possibility to use this new class of star-polymer architectures as permanent ionic channels exhibiting long residence time (hour scale), paving the way to translocation of molecules and macromolecules for example.

No description available.

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.

The perspective that asymmetrical power relationship on vertical channel would lead to dominating partner exploitation with the use of coercive power is examined within the context of horizontal channel exchange relationship between department stores and tenant retailers. A power structure-action-outcome framework is offered to study casual links between dependence (structural aspects of power), use of coercive and noncoercive power (action), and three aspects of outcome - economic and social satisfaction, commitment and strategic performance. Data was collected through personal interview with 302 small, medium size retail tenants of department stores in PR China. Analysis of findings provided support to the model and the hypotheses. Results confirmed that dependence and use of power were only weakly related. The findings demonstrate the contextual influence of Chinese collectivistic culture on coercive power which involves the use of punishment or penalty. To further explore the relationship between dependence and power, investigation was carried out to examine if dependence moderates the effect of coercive power on economic satisfaction, with a positive result. On the other hand, use of non-coercive power showed positive results for economic and social satisfaction, which are positively related to commitment and strategic performance. It can be concluded that use of non-coercive power is strongly recommended for store-tenant relationship in the retail sector of China. Once again, the importance of channel context must not be ignored in the study of distribution channel issues. / This research project contributes to the channel literature by adding to the contemporary state of knowledge on the use of power in horizontal channel relationships with power asymmetry, and constructs were brought together that have not been empirically investigated within a single study. It is unique in that the model was tested in non-American setting, namely PR China.

ClC-1 is the major skeletal muscle chloride channel and is essential for re-establishing the resting membrane potential of muscle cells after an action potential has occurred. Many mutations throughout the CLCN1 gene, which codes for the CIC-1 protein, have been demonstrated via characterisation in heterologous expression systems, to be causative mutations for either Dominant Myotonia Congenita or Recessive Generalised Myotonia. Recently, increasing numbers of myotonic mutations have been found in the carboxyl tail of CIC-1, which demonstrates its importance as a domain that is essential for the normal function of CIC-1 channels. Previous studies in our laboratory defined a region of 18 amino acids in the immediate post D13 segment of rat CIC-1, essential for the expression of functional channels. / thesis (PhDBiomedicalScience)--University of South Australia, 2002.