No description available.

Dysregulation of low-voltage-activated calcium channels (CaV3.1-CaV3.3) underlies diseases including chronic pain, autism, and hypertension. As a major determinant of protein half-life, the ubiquitin-proteasome system (UPS) may not only cause abnormal CaV3 expression but also be targeted to control channel levels for therapy. There are >600 E3 ubiquitin ligases that catalyse the final step in ubiquitination. A crucial aspect of harnessing the UPS is knowing which E3 ligases regulate a given substrate, and whether their actions are redundant. We report that CaV3.1 and CaV3.2 are distinctively regulated by two NEDD4 family E3 ligases – NEDD4L and Smurf1. Reconstituted CaV3.1 currents were robustly suppressed by Smurf1 but not NEDD4L, whereas CaV3.2 was inhibited by both NEDD4L and Smurf1, concomitant with diminished channel surface density and expression. FRET experiments revealed NEDD4L and Smurf1 interact with distinct loci in CaV3.1 and CaV3.2. Nanobody-mediated targeting of NEDD4L or Smurf1, but not WWP1, HECT domains to CaV3.1 and CaV3.2 strongly suppressed currents through both channels. shRNA knockdown of either NEDD4L or Smurf1 in dorsal root ganglion (DRG) neurons substantially increased both low-voltage and high-voltage-activated calcium channel currents. The results reveal non-redundant regulation of CaV3 channels by NEDD4L and Smurf1; introduce Smurf1 as a potent determinant of ion channel expression; suggest a new mechanism for CaV3.2 up-regulation in chronic pain; and advance leveraging the UPS to control CaV3 expression for therapy.

<p> Voltage-gated potassium, sodium, and calcium channels play fundamental roles in cell physiology. They are targets for numerous drugs that are used to treat pain, cardiovascular, autoimmune, and other disorders. Atomic-resolution structures of ion channels and their complexes with ligands are necessary to understand the mechanisms of drug action of ligands. Electrophysiological and crystallographic studies have advanced our understanding of ion channels, but the binding sites, access pathways, and the mechanism of state-dependent action of medically important drugs remain unclear. During my graduate studies, I investigated the structure-function relationships of voltage-gated ion channels and their complexes with drugs by using energy calculations with experimental constraints. My work has helped resolve controversial interpretations of experiments addressing structural similarity between prokaryotic and eukaryotic K+ channels. Our model of the open Shaker K+ channel was confirmed by the later published X-ray structure of Kv1.2. Our Cav2.1 model reinterprets substituted-cysteine accessibility experiments, validates the proposed alignment between K+ and Ca2+ channels, and suggests a similar folding of voltage-gated K+ and Ca2+ channels. These results allowed me to model eukaryotic K+ and Na+ channels in the resting and open/slow-inactivated states, and to predict the binding sites of local anaesthetics, correolide, and chromanol 293B. In these studies, we proposed the involvement of metal ions in the binding of nucleophilic drugs and suggested that the deficiency of permeating ion(s) in the outer pore of the slow-inactivated channels stabilizes the ligands. Simultaneous studies of K+, Na+, and Ca2+ channels were advantageous because the information acquired from one family of ion channels was relevant to other families. My studies contributed to the growing knowledge about ion channels by offering structural information and suggesting mechanisms for the action of drugs. </p> / Thesis / Doctor of Philosophy (PhD)

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No description available.

Physiologic β-adrenergic activation of PKA during the sympathetic “fight-or-flight” response increases calcium influx through CaV1.2 in cardiomyocytes, leading to increased cardiac contractility. The molecular mechanisms of β-adrenergic regulation of CaV1.2 in cardiomyocytes are incompletely known, but activation of PKA is required for this process. The second chapter of this dissertation describes our investigation of the functional PKA phosphorylation target for β-adrenergic regulation of CaV1.2. Recent data confirm that β-adrenergic regulation of CaV1.2 does not require any combination of PKA phosphorylation sites on α1C or β2B subunits. Proximity proteomic labeling methods led us to other potential PKA targets near the CaV1.2 complex, including Rad, a calcium channel inhibitor that changes its position within the calcium channel neighborhood after β-adrenergic stimulation. With expression of α1C, β2B, and Rad in a heterologous expression system, we reconstituted forskolin-PKA regulation of CaV1.2. By mutating potential PKA phosphorylation sites on Rad, we identified specific residues that are critical for this mechanism to occur and validated Rad as the functional PKA target for regulation of CaV1.2. In the third chapter, we probe the contribution of both CaV1.2 α1C and β subunits in β-adrenergic regulation. Previous results have shown that binding between α1C and β subunits is required for adrenergic stimulation of the calcium channel in the heart. Using transgenic mouse models, we demonstrate that this phenomenon requires a rigid IS6-AID linker in the α1C subunit, as introduction of glycines in this region increased flexibility of the linker and abolished a response to adrenergic agonists even though α1C was able to bind to β. The fourth chapter examines the role of the auxiliary β subunit in β-adrenergic regulation of CaV1.2. Binding of Rad to the β subunit is also necessary for this mechanism to occur. Although the β2B isoform is the predominant subunit in the heart, we show that transgenic mice with β3 or β4 replacing β2B in the heart are viable and still have normal β-adrenergic regulation of CaV1.2, indicating that this mechanism is universal to other voltage gated calcium channels that bind to β subunits and RGK proteins. The fifth chapter verifies that Rad is the PKA target in the heart. Using a knock-in mouse model with four PKA phosphorylation sites mutated to alanine, we definitively show that phosphorylation of Rad is necessary for β-adrenergic regulation of CaV1.2 in the heart. We investigate the importance of Rad phosphorylation on many levels. First, we study Rad’s role in regulating the calcium channel. Second, we observe the effect phosphorylation of Rad has on the calcium transient using isolated cardiomyocytes. Third, we examine cardiovascular function in vivo using radiotelemetry and echocardiograms. Finally, we assess the “fight-or-flight” response in an animal model with exercise capacity testing. Together, these findings conclusively show that in the heart, phosphorylation of Rad is the essential mechanism for the sympathetic nervous system control of calcium influx in both atrial and ventricular cardiomyocytes. Additionally, Rad modulates both heart rate and contractility in vivo. In the sixth chapter, we explore the mechanism of Rad modulation of CaV1.2 in depth using a flow-cytometry Förster resonance energy transfer (FRET) two-hybrid assay. We closely examine the roles of phosphorylation sites on both Rad’s N-terminus and C-terminus. By creating phosphomimetic mutations on Rad, we uncover the importance of phosphorylating the C-terminus for release of Rad from both the membrane and the β subunit. Taken together, these findings elucidate the mechanism behind β-adrenergic regulation of CaV1.2 in the heart – a longstanding query for over forty years in the cardiovascular ion channel field. At baseline, Rad “tunes” the amount of calcium influx into the cell by inhibiting a population of channels as a functional reserve. Upon adrenergic stimulation, Rad is phosphorylated, lessening its interaction with the membrane, and releasing inhibition of the calcium channel. The enhanced local calcium influx allows for additional calcium release into the cytoplasm through ryanodine receptors leading to increased contractility of the heart, a notable characteristic of the evolutionary survival mechanism— “fight-or-flight.”

The plant glutamate receptors (GLRs) are homologs of mammalian ionotropic glutamate receptors (iGluRs) and are hypothesized to be potential amino acid sensors in plants. Since their first discovery in 1998, the members of plant GLRs have been implicated in diverse processes such as C/N ratio sensing, root formation, pollen germination and plant-pathogen interaction. However, the exact properties of these channels, such as the spectrum of ligands, ion specificities, and subunit compositions are still not well understood. It is well established that animal iGluRs form homo- or hetero-tetramers in order to form ligand-gated cation channels. The first aspect of this research was to determine if plant GLRs likewise require different subunits to form functional channels. A modified yeast-2-hybrid system approach was initially taken and applied to 14 of the 20 AtGLRs to identify a number of candidate interactors in yeast. Forster resonance energy transfer (FRET), which measures the transfer of energy between interacting molecules, was performed in mammalian cells to confirm interaction between a few of those candidates. Interestingly, despite an abundance of overlapping co-localization between heteromeric combinations, only homomeric interactions were identified between GLRs 1.1 and 3.4 in HEK293 cells. Further, amino acids have been implicated in signaling between plants and microbes, but the mechanisms for amino acid perception in defense responses are far from being understood. Recently it was demonstrated that calcium responses initiated by bacterial and fungal microbe-associated molecular patterns (MAMPs) were diminished in seedlings treated with known agonists and antagonists of mammalian iGluRs, suggesting potential roles of GLRs in pathogen responses. Analysis of publicly available microarray data shows altered gene expression of a sub-fraction of GLRs in response to pathogen infection and bacterial elicitors. Thus, the second goal of my PhD research was aimed at determining whether GLRs are involved in the interaction between plants and pathogens. Gene expression changes of a number of candidate GLRs as well as pathogen growth was examined in response to the plant pathogen Pseudomonas syringae pv. tomato DC3000. Interestingly, single gene and multi-gene deficient plants responded differently with regards to pathogen susceptibility, likely as a result of functional compensation between GLRs. / Ph. D.

Du, Juan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 110-134). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Kwan Hiu Yee. / "June 2006." / 2+ in the title is superscript. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 131-150). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

Attempts were made to determine the pore properties, such as permeability, rectification and voltage-dependent block, of the putative TRPV4-TRPC1 channel. We demonstrated that this putative TRPV4-TRPC1 heterotetrameric channels displays distinct property different (although not drastically different) from TRPV4 homotetrameric channel with regard to I-V relation, kinetics of cation current, cations permeability and rectification properties. Together, the data from FRET and functional studies both suggest that heterologous expression of TRPV4 and TRPC1 can produce functional TRPV4-TRPC1 heterotetrameric channel. / Hemodynamic blood flow is one of most important physiological factors that control vascular tone. Flow shear stress acts on the endothelium to stimulate the release of vasodilators such as nitric oxide (NO), prostacyclin and endothelium-derived hyperpolarizing factors, causing endothelium-dependent vascular relaxation. In many cases, a key early signal in this flow-induced vascular dilation is Ca2+ influx in endothelial cells in response to flow. There is intense interest in searching for the molecular identity of the channels that mediate flow-induced Ca2+ influx. The present study aimed at identifying an interaction of TRPV4 with TRPC1, and investigating functional role of such a complex in flow-induced Ca2+ influx / In functional study, flow elicited a [Ca2+]i rise in TRPV4-expressing HEK cells. Co-expression of TRPC1 with TRPV4 markedly prolonged this [Ca2+]i transient, and it also enabled this [Ca2+]i transient to be negatively modulated by protein kinase G (PKG). Furthermore, this [Ca2+]i rise was inhibited by an anti-TRPC1 blocking antibody T1E3 and a dominant negative construct TRPC1Delta567-793. Physical interaction of TRPV4 with TRPC1 and functional role of such a complex were also found in the primary cultured rat mesenteric artery endothelial cells (MAECs) and human umbilical vein endothelial cells (HUVECs). A TRPC 1-specific siRNA was used to knock-down TRPC1 protein levels in HUVECs. Interestingly, this siRNA not only reduced the magnitude of flow-induced [Ca2+]i rise, but also accelerated the decay of flow-induced [Ca2+]i transient. Pressure myograph was used to investigate the functional role of such a complex in flow-induced vascular dilation. T1E3 also decreased flow-induced vascular dilation. Thogether, the data from endothelial cells are consistent with those in overexpressed HEK cells, supporting the notion that TRPC 1 interacts with TRPV4 to prolong the flow-induced[Ca2+]i transient, and that TRPV4-TRPC1 complex plays an important role in flow-induced vascular dilation. / In summary, my study demonstrated that TRPV4 is capable of assembling with TRPC1 to form a functional TRPV4-TRPC1 heteromeric channel. TRPV4-TRPC1 heteromeric channel can rapidly translocate to the plasma membrane after Ca 2+ depletion in intracellular stores. This TRPV4-TRPC1 heteromeric channel plays an important role in flow-induced endothelial Ca2+ influx and its associated vascular relaxation. / Ion channels are delivered to the plasma membrane via vesicle trafficking. Thus the vesicle trafficking is a key mechanism to control the amount of TRP channel proteins in the plasma membrane, where they perform their function. TRP channels in vivo are often composed of heteromeric subunits. However, up to the present, there is lack of knowledge on trafficking of heteromeric TRP channels via vesicular translocation. In the present study, we examined the effect of Ca2+ store depletion on the translocation of TRPV4-TRPC1 heteromeric channels to the plasma membrane. Experiments using total internal fluorescence reflection microscopy (TIRFM) and biotin surface labeling showed that depletion of intracellular Ca2+ stores triggered a rapid translocation of TRPV4-TRPC1 channel proteins into the plasma membrane. Fluorescent Ca2+ measurement and patch clamp studies demonstrated that store Ca2+ depletion augmented several TRPV4-TRPC1 complex-related functions, which include store-operated Ca2+ influx and cation current as well as 4alpha-PDD-stimulated Ca2+ influx and cation current. The translocation required stromal interacting molecule 1 (STIM1). Furthermore, TRPV4-TRPC1 complex is more favorably translocated to the plasma membrane than TRPC1 or TRPV4 homomers. Similar mechanisms were identified in native endothelial cells, where the TRPV4-TRPC I complex is a key component mediating flow-induced Ca2+ influx and subsequent vascular relaxation. / With the use of fluorescence resonance energy transfer (FRET), co-immunoprecipitation and subcellular colocalization methods, it was found that TRPC1 interacts physically with TRPV4 to form a heteromeric channel complex. In addition, our experimental results indicate that C-terminal and N-terminal domains of both channels are required for their interaction. / Ma, Xin. / Adviser: Yao Xiaodiang. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 109-121). / 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 Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.