Дисертації з теми "Cyclic nucleotide-gated channel"
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1
Cukkemane, Abhishek. "Structural and functional studies of a prokaryotic cyclic nucleotide gated channel /." Jülich : Forschungszentrum, Zentralbibliothek, 2008. http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&doc_number=016779692&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA.
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Sunderman, Elizabeth R. "Single-channel kinetic analysis of the allosteric transition of rod cyclic nucleotide-gated channels /." Thesis, Connect to this title online; UW restricted, 1998. http://hdl.handle.net/1773/10526.
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Matulef, Kimberly Irene. "Cysteine-scanning mutagenesis of the ligand-binding domain of cyclic nucleotide-gated channels /." Thesis, Connect to this title online; UW restricted, 2002. http://hdl.handle.net/1773/5032.
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Lolicato, M. G. L. "STRUCTURAL STUDIES ON THE REGULATORY DOMAIN OF THREE HCN (HYPERPOLARIZATION-ACTIVATED CYCLIC NUCLEOTIDE-GATED) CHANNEL ISOFORMS." Doctoral thesis, Università degli Studi di Milano, 2012. http://hdl.handle.net/2434/168356.
Повний текст джерелаАнотація:
Hyperpolarization-activated cyclic nucleotide gated (HCN) chan- nels underlie the If /Ih cation currents that control pacemaker activity in heart and brain. HCN channels are dually ac- tivated by membrane hyperpolarization and binding of cAMP to their cyclic nucleotide binding domain (CNBD). Binding of cAMP shifts the activation curve of HCN2 and HCN4 by 17 mV, but that of HCN1 by only 2-4 mV. Tetramerization of the CNBD is seemingly part of the cAMP-induced allosteric con- formational changes that increase the open probability of the channel pore. We have obtained the crystal structures of the CNBD of the three isoforms, but the analysis revealed a very conserved structure between HCN1 versus HCN2 and HCN4, except for a loop of β-roll, previously shown to regulate the binding affinity of HCN4. We measured the binding affin- ity of the CNBD for the cAMP and the different propensity of the regulatory domain to tetramerize in absence or presence of the ligand. We confirm that tetramerization is the primary ef- fect of cAMP binding, and the first step in the transmission of this signal, that eventually removes the inhibition imposed by the CNBD on the channel. Accordingly, cAMP- binding releases HCN2 and HCN4 from inhibition, but has little or no effect on HCN1. Our data demonstrate that in HCN1 the CNBD is already tetrameric at basal cAMP concentrations contrary to HCN2 and HCN4. HCN1 shows this peculiar behavior despite its cAMP- binding affinity is in the same range of the affinity found in HCN2 and HCN4. This can be explained by two different
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affinity states (high and low). HCN1 is, at low cAMP concen- trations, already switched to the low affinity conformation, while the high affinity state is not measurable because the binding site is already occupied. Our results offer a logical explanation for the behavior of HCN1 and an experimental support to the leading hypothesis that ligand-induced tetramerization removes tonic inhibition from the pore. In addition, some more inter- esting information arose from the crystal structure, highlighting an additional electron density close to the tetramerization inter- face of the proteins. We investigated a range of molecules that could bind the proteins in that pocket and potentially alter the functionality of the channel.
5
Becirovic, Elvir. "Role of the CNGB1a Subunit of the Rod Cyclic Nucleotide-Gated Channel in Channel Gating and Pathogenesis of Retinitis Pigmentosa." Diss., lmu, 2010. http://nbn-resolving.de/urn:nbn:de:bvb:19-119088.
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Tanaka, Naoto. "A MISSENSE MUTATION IN CONE PHOTORECEPTOR CYCLIC NUCLEOTIDE-GATED CHANNELS ASSOCIATED WITH CANINE DAYLIGHT BLINDNESS OFFERS INSIGHT INTO CHANNEL STRUCTURE AND FUNCTION." Diss., Temple University Libraries, 2013. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/246634.
Повний текст джерелаАнотація:
BiologyPh.D.
Cone cyclic nucleotide-gated (CNG) channels are located in the retinal outer segments, mediating daylight color vision. The channel is a tetramer of A-type (CNGA3) and B-type (CNGB3) subunits. CNGA3 subunits are able to form homotetrameric channels, but CNGB3 exhibits channel function only when co-expressed with CNGA3. Mutations in the genes encoding these cone CNG subunits are associated with achromatopsia, an autosomal recessive genetic disorder which causes incomplete or complete loss of daylight and color vision. A missense mutation, aspartatic acid (Asp) to asparagine (Asn) at position 262 in the canine CNGB3 subunit (cB3-D262N), results in loss of cone function and therefore daylight blindness, highlighting the crucial role of this aspartic acid residue for proper channel biogenesis and/or function. Asp 262 is located in a conserved region of the second transmembrane segment containing three Asp residues designated the Tri-Asp motif. We exploit the conservation of these residues in CNGA3 subunits to examine the motif using a combination of experimental and computational approaches. Mutations of these conserved Asp residues result in a loss of nucleotide-activated currents and mislocalization in heterologous expression. Co-expressing CNGB3 Tri-Asp mutants with wild type CNGA3 results in functional channels, however, their electrophysiological characterization matches the properties of homomeric CNGA3 tetramers. This failure to record heteromeric currents implies that Asp/Asn mutations impact negatively both CNGA3 and CNGB3 subunits. A homology model of canine CNGA3 relaxed in a membrane using molecular dynamics simulations suggests that the Tri-Asp motif is involved in non-specific salt bridge pairings with positive residues of S3 - S4. We propose that the CNGB3-D262N mutation in daylight blind dogs results in the loss of these interactions and leads to an alteration of the electrostatic equilibrium in the S1 - S4 bundle. Because residues analogous to Tri-Asp residues in the voltage-gated Shaker K+ channel superfamily were implicated in monomer folding, we hypothesize that destabilizing these electrostatic interactions might impair the monomer folding state in D262N mutant CNG channels during biogenesis. Another missesnse sense mutation, Arginine (Arg) to tryptophan (Trp) at position 424 in the canine CNGA3 subunit (cA3-R424W), also results in loss of cone function. An amino acid sequence alignment with Shaker K+ channel superfamily indicates that this R424 residue is located in the C-terminal end of the sixth transmembrane segment. A3-R424W mutant channels resulted in no cyclic nucleotide-activated currents and mislocalization with intracellular aggregates. However, the localization of cA3-R424W mutant channels was not affected as severely as the Asp/Asn mutation in S2 Tri-Asp motif, showing a lot of cells with the proper localization of Golgi-like and membrane fluorescence. Moreover, the substitution of Arg 424 to Lysine (Lys), conserving the positive charge, preserved channel function in some cells, which is different from the results of the S2 Tri-Asp motif in which the Asp/Glu substitutions, conserving the negative charge, leads to loss of cyclic nucleotide-activated currents. Even though these missense mutations are both associated with canine daylight blindness, the Arg 424 residue might not be as critical for folding as the Tri-Asp residues in the S2 Tri-Asp motif and might be more of a problem in channel structure and function. The cA3 model relaxed with MD simulations indicated a possible interaction of Arg 424 with the Glu 304 residue in the S4-S5 linker. This hypothesis is supported by electrophysiological data in which the double mutation of reversing these residues, Glu 306 to Arg and Arg 424 to Glu (E306R-R424E) preserves channel function. In the model, this salt bridge appears to contribute to stabilization of the open pore state. The R424W mutation might disrupt the salt bridge formation, leading to deforming and closing the pore region.
Temple University--Theses
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Schünke, Sven Verfasser], Dieter [Akademischer Betreuer] [Willbold, and Lutz [Akademischer Betreuer] Schmitt. "NMR solution structures of the MloK1 cyclic nucleotide-gated ion channel binding domain / Sven Schünke. Gutachter: Lutz Schmitt. Betreuer: Dieter Willbold." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2011. http://d-nb.info/1015434975/34.
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Hundal, Sukhinder Paul Singh. "Molecular cloning, characterisation and function expression of cyclic nucleotide-gated ion channel genes expressed in sino-atrial node region of heart." Thesis, University of Leicester, 1994. http://hdl.handle.net/2381/35257.
Повний текст джерелаАнотація:
Pacemaker cells of the mammalian sino-atrial node (SAN) contain a hyperpolarization-activated, non-specific cationic current, If which is an important component involved in the initiation and neurotransmitter-mediated control of cardiac rhythm. cAMP can directly modulate If by a mechanism independent of phosphorylation, demonstrating that cyclic nucleotide-sensitive ion channel genes are expressed within cardiac pacemaker cells. Through a combination of library screening methods based on cross-hybridising cyclic nucleotide-gated channel probes and a PCR 'fingerprint' employing primers designed to sequences encoding an ion channel cyclic nucleotide-binding domain, partial cDNA clones were isolated from a prepared sino-atrial node regional-specific cDNA library, which were either homologues of previously identified ion channels shown to be expressed in sensory tissues or putative new channel clones. Isolate rscNGC 1 following retrieval of a full coding region by anchor-PCR, demonstrated 90.4% sequence identity to the a-subunit of the rod photoreceptor cGMP-gated channel. The PCR 'fingerprint' identified a SAN homologue of the olfactory neuron cAMP-gated channel within library aliquots. This was the first demonstration that two distinct cyclic nucleotide-gated ion channel genes were expressed in SAN region of heart. Heterologous expression of rscNGC 1 following micro-injection of capped cRNA in Xenopus oocytes, gave rise to cGMP-stimulated channel activity exhibiting electrophysiological properties similar to the characterised a-subunit of the rod photoreceptor cGMP-gated channel. A reconstituted second messenger-pathway mediating endogenous receptor coupling to heterologously expressed cAMP-gated ion channels - shown to be present within native nodal tissue - was attempted within MEL cells. However, the absence of endogenous receptors positively-coupled to adenylyl cyclase within MEL cells, and the inability to functional characterise cAMP-stimulated cationic conductances via electrophysiological methods, prevented such studies. Thus demonstrating the inappropriateness of the MEL cell, as a heterologous system for studying receptor-mediated second messenger coupling to cNG channels. Although cyclic nucleotide-gated ion channels are obligatorily coupled to intracellular signalling agonists commonly found in heart, they have yet to be described in functional terms within SAN or any other cardiac subregion. It is postulated that they may have a role in vasculature - underlying mechanisms of smooth muscle relaxation.
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Kimura, Koji. "Hyperpolarization-activated, cyclic nucleotide-gated HCN2 cation channel forms a protein assembly with multiple neuronal scaffold proteins in distinct modes of protein-protein interaction." Kyoto University, 2004. http://hdl.handle.net/2433/145287.
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Arrigoni, C. "MODULATION OF PORE GATING BY ¿SENSOR¿ DOMAINS IN VOLTAGE-GATED K+ CHANNELS." Doctoral thesis, Università degli Studi di Milano, 2013. http://hdl.handle.net/2434/215591.
Повний текст джерелаАнотація:
Many proteins in nature show a modular topology, because it is possible to recognize functional modules responsible of distinguishable functions in the protein. Voltage-gated can be considered modular proteins. The superfamily of voltage-gated channels is composed of channels in which a pore-module is in charge of generating an ion conductance across the cell membrane, and other “sensor”-modules perceive different stimuli and transmit them to the pore, readjusting the conductance in response to changes within the cell. The sensor module in voltage-gated channels is the voltage sensing domain. It is composed of four transmembrane segments and it is able to feel the electrical properties of the membrane, such as changes in potential and, through a mechanical load applied by a short linker, to affect the gating of the pore (i.e. opening or closure of the channel). A more sophisticated regulation is possible thanks to other modules fused to the same channel. Ligand-gated channels usually exhibit a C-terminal domain exposed in the cytoplasm, in contact with all the variable concentration of second messengers, able to modulate the activity of the channel. In this group, cyclic nucleotide-gated channels have a C-terminal domain, composed of a binding domain (CNBD) that respond to difference in concentration of cAMP or cGMP, and a C-linker region, connecting the CNBD to the pore. The CNBD acts as an allosteric domain, and modulate the channel opening upon cAMP binding.The idea that these domains evolved independently before fusing in a single protein, is strengthened by the fact that similar domains are found in a large variety of proteins, that don’t belong to channels family. For example, recently, a new enzyme was discovered, the voltage-sensing phosphatase (VSP) of Ciona intestinalis, whose voltage-sensor domain is fused to a phosphatase. The CNBD of the hyperpolarized cyclic nucleotide-activated channels (HCN), has a conserved structure compared to that of the cAMP-dependent protein kinases. My PhD thesis addresses two different topics, but in both cases I investigated how sensor-modules can give a sophisticated regulation of channel gating. In the first part, I approached the problem about how voltage-dependence originated in voltage-gated channels, in particular I obtained a voltage-gated K + channel fusing two unrelated protein modules: the voltage sensing domain of Ci-VSP and the pore-channel PBCV-1 Kcv. The fusion between a voltage sensor and a potassium channel with a quasi-ohmic behaviour generates a chimaeric protein called KvSynth1, an delayed outward rectifier potassium channel. KvSynth1 retains the pore properties of Kcv (selectivity and filter gating) and the voltage dependence of the Ci-VSP (half activation potential, slope factor, shift of activation curve due to mutations). Moreover, the quality of the rectification is dependent on the length of the
linker between the two modules. This highlights a mechanic role of the linker in transmitting the movement of the sensor to the pore, and shows that electromechanical coupling can occur without co-evolution of the two domains. In the second part, the allosteric modulation of the cyclic nucleotide binding domain of HCN channels has been studied on the basis of our findings in the crystal structure of the CNBD of the isoform 4 of HCN (hyperpolarized cyclic nucleotide-activated) channels. HCN are the molecular determinant of the If current, responsible of the autonomic regulation of the heart. In the structure of HCN4 CNBD a putative binding site for cyclic nucleotides in the C-linker region was found. Occupancy of this binding site by the prokaryote second messenger c-di-GMP can completely revert the effect of cAMP in the micromolar range. Docking a large set of molecules in the binding pocket, another compound was identified, (N’-biphenyl-2-yl-N-[1-(3-cyanobenzyl)piperidin-4-yl]-N-(pyridin-3-ylmethyl)urea), able to give the same effect on cAMP modulation. The effect of these molecules is restricted to HCN4; this isoform selectivity underlies that, although the C-terminus of the three isoforms is structured in a similar way, the modulation can be different. Some different features of HCN1, HCN2 and HCN4 were already analysed previously. These results highlight the presence of an second modulatory pathway in HCN channels, indicate a potential drug binding site for heart rate modulation and advance understanding of the mechanism of efficacy of cAMP binding in HCN channels.
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Talke, Ina Nicola. "Cyclic nucleotide-gated channels in Arabidopsis thaliana." Thesis, University of York, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.274500.
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FLANNERY, RICHARD JOHN. "CLUSTERING OF CYCLIC-NUCLEOTIDE-GATED CHANNELS IN OLFACTORY CILIA." University of Cincinnati / OhioLINK, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1136913935.
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Chow, Sarah Sue Wen. "Energetic and structural impact of cyclic nucleotide binding to hyperpolarization-activated cyclic nucleotide-gated channels." Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/44980.
Повний текст джерелаАнотація:
Hyperpolarization-activated Cyclic Nucleotide-gated, HCN, channels contribute to the membrane potential of excitable cells including pacemaker cells of the heart and neurons in the brain. By binding to the inner side of the HCN channel, cAMP facilitates channel opening, but
the underlying mechanism has been mainly inferred from relating cAMP concentration to the degree of facilitation. Concentration-response relations reflect the tightly coupled process of cAMP binding and channel opening. The strength of binding and how it is linked to channel
opening is not known. Furthermore, cAMP facilitation is not equal among the four mammalian
HCN isoforms and the extent to which cAMP binding affinity contributes to these differences is
not known.My experiments support the conclusion that cAMP binds to one site of the isolated
tetrameric C-terminus of HCN2 and HCN4 with high affinity and to three sites with low affinity
revealing negative cooperativity. In contrast, only low affinity binding was observed in HCN1
with energetics of binding that were similar to those of the low affinity binding to HCN2. Cyclic
AMP enhanced oligomerization of the HCN2 C-terminus in solution, but had a negligible effect
on oligomerization of the HCN1 C-terminus. Oligomerization in solution is thought to reflect the formation of a gating ring in the intact channel that facilitates opening. Together, this suggests that HCN1 functions as though already disinhibited, explaining its easier opening in the absence of cAMP, its smaller facilitation of opening, and lack of negative cooperativity upon cAMP binding. Lysine substitution at residue 488 of HCN2, initially identified in an individual with
idiopathic generalized epilepsy, eliminated negative cooperativity and reduced oligomerization
of the isolated C-terminus upon cAMP binding. This likely reflects a decrease in its ability to form a gating ring in the intact channel and explains the reported inhibition of opening by this
mutation.The work presented in this thesis demonstrates the value of studying the C-terminus of the HCN channel in isolation to uncover the mechanism by which the HCN C-terminus and cAMP binding control channel opening that would otherwise be hidden by functional experiments.
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Adams, Nicolette. "Investigating the Role of Cyclic Nucleotide gated channels in Plant- Pathogen Interactions." Doctoral thesis, University of Cape Town, 2009. http://hdl.handle.net/11427/4236.
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Park, Graeme Jonathan. "A study of the physiological role of cyclic nucleotide gated channels in Arabidopsis thaliana." Thesis, University of York, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.440977.
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Weißgraeber, Stephanie [Verfasser], Kay [Akademischer Betreuer] Hamacher, and Gerhard [Akademischer Betreuer] Thiel. "Hyperpolarization-Activated cyclic nucleotide-gated channels - structure and evolution / Stephanie Weißgraeber. Betreuer: Kay Hamacher ; Gerhard Thiel." Darmstadt : Universitäts- und Landesbibliothek Darmstadt, 2015. http://d-nb.info/1112143858/34.
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Yamada, Rei. "Hyperpolarization-activated cyclic-nucleotide gated cation channels regulate auditory coincidence detection in nucleus laminaris of the chick." Kyoto University, 2005. http://hdl.handle.net/2433/144385.
Повний текст джерелаАнотація:
Kyoto University (京都大学)0048
新制・課程博士
博士(医学)
甲第11938号
医博第2920号
新制||医||911(附属図書館)
23727
UT51-2006-B117
京都大学大学院医学研究科脳統御医科学系専攻
(主査)教授 金子 武嗣, 教授 河野 憲二, 教授 伊藤 壽一
学位規則第4条第1項該当
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Frietsch, Sabine. "The role of Cyclic Nucleotide-Gated Channels (CNGC) in plant development and stress responses in Arabidopsis thaliana." [S.l. : s.n.], 2006.
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Hua, Li. "Phosphatidylinositol (4,5)-bisphosphate (PIP2) modulation of TRPV1 and functional interactions between A' helices in the C-linkers of open CNG channels /." Thesis, Connect to this title online; UW restricted, 2007. http://hdl.handle.net/1773/10545.
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Junor, Roderick Walter John. "Fetal and postnatal lung liquid transport : the role of cyclic nucleotide gated cation channels and pulmonary blood flow." Thesis, St George's, University of London, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.313811.
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Gonzalez, Amaliris. "EXPRESSION OF THE CNGB3 SUBUNIT IN RETINA OF ACHROMATOPSIA-AFFECTED DOGS." Diss., Temple University Libraries, 2015. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/337529.
Повний текст джерелаАнотація:
BiologyPh.D.
Light energy is converted into an electrical signal by a set of proteins in the phototransduction cascade in photoreceptors. In this work, I focus on two critical elements of the phototransduction cascade in canine, the opsin molecules and CNG channels of cone photoreceptors. Canines are dichromats possessing two types of cone photoreceptors with different opsin molecules that detect either long and medium wavelengths (L/M) or short wavelengths (S). The L/M- and S-opsin genes were cloned from an Alaskan Malamute and used to investigate key amino acids that are responsible for tuning the spectral properties of the 11-cis retinal chromophore. Cone CNG channels are composed of CNGA3 and CNGB3 subunits. I characterized antibodies to detect cone CNG channel subunits to investigate expression of mutations in CNGB3 subunit on two canine models for achromatopsia. One model contains a missense CNGB3 mutation D262N (CNGB3m/m) and the other is a complete deletion of all exons of the CNGB3 gene (CNGB3-/-). Studies presented in this thesis show CNGB3 is expressed later in cone during retinal development compared to CNGA3. It also presents evidence for the necessity of CNGB3 in cone outer segment targeting of CNGA3.
Temple University--Theses
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Nakashima, Noriyuki. "Hyperpolarisation-activated cyclic nucleotide-gated channels regulate the spontaneous firing rate of olfactory receptor neurons and affect glomerular formation in mice." Kyoto University, 2013. http://hdl.handle.net/2433/174812.
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Wongsamitkul, Nisa [Verfasser], Klaus [Akademischer Betreuer] Benndorf, Ingo [Akademischer Betreuer] Dahse, and Stephan [Akademischer Betreuer] Frings. "Translation of ligand binding to activation in olfactory cyclic nucleotide-gated (CNG) channels / Nisa Wongsamitkul. Gutachter: Klaus Benndorf ; Ingo Dahse ; Stephan Frings." Jena : Thüringer Universitäts- und Landesbibliothek Jena, 2015. http://d-nb.info/1079217932/34.
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Deutsch, Matthias [Verfasser], Sigrun [Gutachter] Korsching, Arnd [Gutachter] Baumann, and Guenter [Gutachter] Schwarz. "Role of Hyperpolarization activated and Cyclic Nucleotide gated (HCN) Channels in Hippocampal Neurons / Matthias Deutsch ; Gutachter: Sigrun Korsching, Arnd Baumann, Guenter Schwarz." Köln : Universitäts- und Stadtbibliothek Köln, 2020. http://d-nb.info/1207074314/34.
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Kondapuram, Mahesh [Verfasser], Klaus [Gutachter] Benndorf, Heinrich [Gutachter] Terlau, and Christoph [Gutachter] Fahlke. "Mechanisms underlying cAMP mediated gating in hyperpolarization-activated cyclic nucleotide-gated channels : interactions with-in HCN Channels / Mahesh Kondapuram ; Gutachter: Klaus Benndorf, Heinrich Terlau, Christoph Fahlke." Jena : Friedrich-Schiller-Universität Jena, 2021. http://d-nb.info/123814215X/34.
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Shah, Nikhil N. "SH3 AND MULTIPLE ANKYRIN REPEAT DOMAIN 3 (SHANK3) AFFECTS THE EXPRESSION OF HYPERPOLARIZATION-ACTIVATED CYCLIC NUCLEOTIDE-GATED (HCN) CHANNELS IN MOUSE MODELS OF AUTISM." VCU Scholars Compass, 2017. http://scholarscompass.vcu.edu/etd/4997.
Повний текст джерелаАнотація:
SH3 and multiple ankyrin repeat domains 3 (SHANK3) is a multidomain scaffold protein that is highly augmented in the postsynaptic density (PSD) of excitatory glutamatergic synapses within the central and peripheral nervous systems. SHANK3 links neurotransmitter receptors, ion channels, and other critical membrane proteins to intracellular cytoskeleton and signal transduction pathways. Mutations in SHANK3 are linked with a number neuropsychiatric disorders including autism spectrum disorders (ASDs). Intellectual disability, impaired memory and learning, and epilepsy are some of the deficits commonly associated with ASDs that result from mutations in SHANK3. Interestingly, these symptoms show some clinical overlap with presentations of human neurological disorders involving hyperpolarization-activated cyclin nucleotide-gated (HCN) channels. In fact, it has recently been demonstrated in human neurons that SHANK3 haploinsufficiency causes Ih-channel dysfunction, and that SHANK3 has a physical interaction with HCN channels via its ANKYRIN repeat domain. These insights suggest that SHANK3 may play important roles in HCN channel expression and function, and put forward the idea that HCN channelopathies may actually encourage some of the symptoms observed in patients with SHANK-deficiency related ASDs. In this study, we provide preliminary data that suggests the ANK domain of SHANK3 interacts with COOH portion of HCN1. We also exploited the differences between two mouse models of autism to show that a subset of SHANK3 isoforms may be involved in the proper expression and function of HCN channels. We found that HCN2 expression is significantly decreased in a mouse model lacking all major isoforms of SHANK3 (exons 13-16 deleted; Δ13-16), while HCN2 expression is unaltered in a mouse model only lacking SHANK3a and SHANK3b (exons 4-9 deleted; Δ4-9). Surprisingly, we also found that HCN4 expression is altered in SHANK3Δ13-16, but not SHANK3Δ4-9. Taken together, our results show HCN channelopathy as a major downstream carrier of SHANK3 deficiency.
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Smith, Trevor. "An investigation into the role of the hyperpolarisation-activated cyclic nucleotide-gated on channels in dorsal root ganglion neurons in rat models of chronic inflammatory and neuropathic pain." Thesis, University of Liverpool, 2012. http://livrepository.liverpool.ac.uk/9093/.
Повний текст джерелаАнотація:
Chronic pain {CP} is a major health problem that affects about 20% of adults worldwide. Unlike acute, physiological pain, which resolves promptly once the painful stimulus is removed, CP can last for months, years or even a lifetime. CP, which includes inflammatory pain {IP} that is associated with both tissue injury and the accompanying inflammation and peripheral neuropathic pain {NP} that is a direct consequence of a lesion or disease affecting the peripheral nervous system {PNS}, often is a constant burden, degrades peoples’ quality of life, and costs billions of pounds. Patients with CP usually complain of either: a) continuous or intermittent spontaneous, un-provoked pain; or b) hypersensitivity, due to either increased pain from a stimulus that normally provokes pain or pain due to a stimulus that does not normally provoke pain. Successful therapy for CP, particularly NP, remains a challenge because the currently available drugs are largely ineffective and many result in adverse side effects. Therefore, there is a pressing need to understand the pathophysiology of CP, in order to devise appropriate palliative and curative strategies. CP is believed to be due, at least partly, to increased excitability of normally quiescent dorsal root ganglion {DRG} neurons, which convey sensory information from the periphery to the central nervous system {CNS}. However, the underlying ionic and molecular mechanisms of this neuronal hyperexcitability and spontaneous activity {SA} are poorly understood. The aim of this research project was to examine the hypothesis that during peripheral CP states, hyperexcitability in DRG neurons could be due to increased expression of hyperpolarisation-activated cyclic nucleotide-gated {HCN} channels, possibly in combination with a change in their activation properties. This is because these channels, which are composed of 4 subunits {HCN1-4}, produce an excitatory inward current, termed the hyperpolarisation-activated {Ih} current in neurons, that depolarizes the membrane potential toward the threshold of action potential {AP} generation. To test this hypothesis, several integrated approaches, including behavioural pharmacology, in vivo electrophysiology, and immunofluorescent staining, were used in two rat models of CP that were compared to appropriate controls. The rat models of CP were: a) chronic inflammatory pain {CIP} model, which involved induction of hindlimb inflammation with complete Freund’s adjuvant {CFA}; and b) chronic neuropathic pain {CNP} model that involved L5 spinal nerve {SN} axotomy, in addition to loose ligation of the L4 SN with neuro-inflammation inducing chromic gut, referred to as modified SN Axotomy {mSNA}. The objectives of the current project were to: (i) evaluate, using behavioural pharmacology, the influence of modulating the HCN channels with the Ih-specific blocker, ZD7288, on pain hypersensitivity in both CIP and CNP. (ii) determine, using in vivo intracellular voltage and current recordings, the difference in AP parameters between normal and mSNA-treated L4 DRG neurons and the effect of ZD7288 on SA in the mSNA-treated L4 DRG neurons. (iii) determine, using immunofluoresence, the types of DRG neuron that express HCN1-HCN3 subunits in normal rats and whether expression of these subunits is altered in both CIP and CNP. The results showed: 1. In both CIP and CNP, peripheral administration of ZD7288 resulted in significant attenuation of mechanical hypersensitivity and a non-significant absence of spontaneous pain {SP}. 2. In the L4 DRG neurons of mSNA-treated animals with CNP, ZD7288 had no effect on the frequency of SA from low-threshold mechanoreceptors {LTM} and induced changes in hyperpolarisation-associated AP parameters in Aα/β-fibre DRG neurons. 3. In both CIP and CNP, an increased proportion of small and medium sized DRG neurons express HCN2, but not HCN1 or HCN3, channel protein. Taken together, the findings suggest that HCN channels, particularly HCN2, in specific sub-populations of DRG neurons contribute to the development!of CIP and CNP.
28
Abdel, Hamid Huda. "Structural - functional Analysis of Plant Cyclic Nucleotide Gated Ion Channels." Thesis, 2013. http://hdl.handle.net/1807/35761.
Повний текст джерелаАнотація:
The Arabidopsis thaliana genome encodes twenty putative cyclic nucleotide-gated channel (CNGC) genes. Studies on A. thaliana CNGCs so far have revealed their ability to selectively transport cations that play a role in various stress responses and development, however, the regulation of plant CNGCs is not yet fully understood. Thus, in this study I have attempted to analyze the structure-function relationship of AtCNGCs, mainly by using suppressor mutants of the rare gain-of function mutant, cpr22.
The A. thaliana mutant cpr22 resulted from an approximately 3kb deletion that fused the 5’ half and the 3’ half of two CNGC-encoding genes, AtCNGC11 and AtCNGC12, respectively. The expression of this chimeric CNGC, the AtCNGC11/12 gene confers easily detectable characteristics such as stunted morphology with curly leaves and hypersensitive response-like spontaneous lesion formation. Through a suppressor screen, twenty nine new alleles were identified in AtCNGC11/12. Since the cytosolic C-terminal region contains important regulatory domains, such as a cyclic-nucleotide binding domain, eleven cytosolic C-terminal mutants, S17, S35, S81, S83, S84, S100, S135, S136, S137, S140 and S144, were analyzed. A detailed analysis of two mutants, S100 (AtCNGC11/12:G459R) and S137 (AtCNGC11/12:R381H), suggested that G459 and R381 are important for basic channel function rather than channel regulation. Site-directed mutagenesis and fast protein liquid chromatography (FPLC) showed that these two amino acids influence both intra- and inter-subunit interactions that are involved in stabilizing the tertiary structure of the channel.
In addition, calmodulin binding domain(s) (CaMBD) and cyclic nucleotide binding domain(s) (CNBD) of some of AtCNGCs were studied using computational modeling and biophysical analyses. The data indicated that AtCNGC12 has two CaMBDs in both N- and C- cytosolic termini, whereas AtCNGC11 has only one CaMBD located in the N-terminal region of the channel. In addition, a thermal shift assay suggested that AtCNGC12 has higher affinity to bind cAMP over cGMP.
Taken together, the current study contributes to identify key residues for channel function and provides new insights into CaMBD and CNBD in plant CNGCs.
29
Dose, Andrea Christina. "Molecular characterization of the cyclic nucleotide-gated cation channel of bovine rod outer segments." Thesis, 1995. http://hdl.handle.net/2429/4785.
Повний текст джерелаАнотація:
The cyclic nucleotide-gated (CNG) cation channel of rod photoreceptors plays an
important role in the perception of light. The channel gating is the final step in the visual
transduction cascade. The cloning (Kaupp et al., 1989) of what is now known to be the
CNG channel α subunit facilitated its molecular characterization. A heterologous
expression system was developed to examine molecular aspects of the α and β subunits
of the CNG channel. Expression in mammalian cells helped resolve confusion regarding
the JVL of the rod CNG channel a subunit. Although the cDNA codes for an 80 kDa
protein, the a subunit exists in the rod outer segment as a 63 kDa protein suggesting the
existence of a processing mechanism. PCR was used to construct cDNA clones which
code for both the 63 and 80 kDa forms of the a subunit of the rod CNG channel. A
polyclonal antibody, generated to specifically label the 80 kDa α subunit, was used in
conjunction with a monoclonal antibody specific for the 63 kDa α subunit in labeling
studies. These antibodies were shown to be mono-specific, capable of differentiating
between the two size forms of the a subunit. Immunohistochemical studies
demonstrated that the 63 kDa α subunit is the predominant species in the rod outer
segment while the 80 kDa form is present in very low quantities. Rod outer segment
(ROS) purification on sucrose gradients also showed that the 80 kDa form co-sediments
with the 63 kDa α subunit indicating that the two size forms coexist in the outer
segment. Examining the α subunits of the photoreceptors of another species
demonstrated similar processing of the CNG α subunit polypeptides, however, an
olfactory a subunit did not appear to undergo similar processing.
The cloned CNG channel β subunit cDNA codes for a protein with a predicted
molecular mass of 155 kDa (Korschen et al, 1995). Heterologous expression of the β
subunit cDNA yielded a 240 kDa protein, positively identifying the 240 kDa protein in
ROS which co-purifies with the a subunit as the CNG β subunit. Construction and expression of a truncated form of the (3 subunit demonstrated that it is the glutamic acidrich
N-terminal portion of the (3 subunit that is responsible for its anomalous migration
on an SDS gel. Co-expression of both subunits in mammalian cells indicated that the 3
subunit was not responsible for the processing of the α subunit. As seen for the subunits
of the native CNG channel the heterologously expressed α and β subunits coimmunoprecipitated.
The subunit interaction was not dependent on the 92 N terminal
amino acids of the 80 kDa α subunit or on the glutamic acid-rich portion of the P
subunit. A method for reconstitution of the heterologously expressed CNG channel was
also developed. The expressed a subunit reconstituted alone did not generate functional
cGMP-gated channels. Reconstitution of the heterologously expressed channel complex
comprising the 80 kDa α and complete β subunits generated functional channels.
Compared to the native CNG channel, 10% of the heterologously expressed channel
complex exhibited cGMP-gated channel activity. This is the first example of
reconstitution of a heterologously expressed cation channel into lipid vesicles for Ca²⁺ efflux measurements. The system presented here will be useful to further define CNG α
and β subunit interactions and to carry out structure-function studies on the channel
using a biochemical efflux assay.
30
Baxter, Joyce. "Identification of a functionally essential amino acid for an Arabidopsis cyclic nucleotide-gated ion channel." 2007. http://link.library.utoronto.ca/eir/EIRdetail.cfm?Resources__ID=788863&T=F.
Повний текст джерела
31
Doheny, Jason. "Trigeminal neuropathic pain in rats: a role for thalamic hyperpolarization-activated cyclic nucleotide-gated channel activity." Thesis, 2020. https://hdl.handle.net/2144/41212.
Повний текст джерелаАнотація:
Trigeminal neuropathic pain (TNP) is a condition that occurs when one or more branches of the trigeminal nerve are insulted. Trigeminal neuropathic pain has been shown to be refractory to treatment. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels regulate neuronal excitability in both the peripheral and central nerve systems. Emerging evidence indicates that HCN channels are involved in the development and maintenance of chronic pain, however, the impact of thalamic HCN channel activity on TNP has yet to be elucidated. In this report, we used a chronic constriction of the distal infraorbital nerve (dIoN-CCI) to induce TNP in rats. By infusing HCN channel blockers into the ventral posteromedial (VPM) nucleus of the thalamus in dIoN-CCI rats, we demonstrated that inhibition of HCN channel activity ameliorated TNP. We found that the HCN blocker ZD7288 and the clinical drug ivabradine dose-dependently attenuated both evoked and none-evoked nociceptive behaviors in dIoN-CCI rats. Electrophysiological measurements showed the expression of HCN current (Ih) in the thalamocortical neurons in the VPM was sensitive to the HCN channel modulator cyclic adenosine monophosphate (cAMP), suggesting a contribution of the HCN2 subunit in thalamic HCN current. In the thalamus, surface expression of the HCN2 subunit was increased in dIoN-CCI rats. Taken together, we propose that an increase in HCN channel activity in the thalamus in the ascending nociceptive pathway contributed to trigeminal neuropathic pain.
32
Chin, Kimberley. "Investigation of Structure-function and Signal Transduction of Plant Cyclic Nucleotide-gated Ion Channels." Thesis, 2013. http://hdl.handle.net/1807/43505.
Повний текст джерелаАнотація:
Cyclic nucleotide-gated channels (CNGCs) are non-selective cation channels that were first identified in vertebrate photosensory and olfactory neurons. Although the physiological roles and biophysical properties of animal CNGCs have been well studied, much less is known about these channels in plants. The Arabidopsis genome encodes twenty putative CNGC subunits that are postulated to form channel complexes that mediate various physiological processes involving abiotic and biotic stress responses, ion homeostasis and development.
The identification of Arabidopsis autoimmune CNGC mutants, such as defense no death class (dnd1 and dnd2), and the constitutive expressor of pathogenesis related genes 22 (cpr22) implicate AtCNGC2, 4, 11 and 12 in plant immunity. Here, I present a comprehensive study of the molecular mechanisms involved in CNGC-mediated signaling pathways with emphasis on pathogen defense. Previously, a forward genetics approach aimed to identify suppressor mutants of the rare gain-of-function autoimmune mutant, cpr22, identified key residues that are important for CNGC subunit interactions and channel function.
First, I present a structure-function analysis of one of these suppressor mutants (S58) that revealed a key residue in the cyclic nucleotide binding domain involved in the stable regulation of CNGCs. Second, I present a new suppressor screen using AtCNGC2 T-DNA knockout mutants that specifically aimed to identify novel downstream components of CNGC-mediated pathogen defense signaling. In this screen, I successfully isolated and characterized the novel Arabidopsis mutant, repressor of defense no death 1 (rdd1), and expanded this study to demonstrate its involvement in AtCNGC2 and AtCNGC4-mediated signal transduction. Additionally, I demonstrated for the first time, the physical interaction of AtCNGC2 and AtCNGC4 subunits in planta.
The findings presented in this thesis broaden our current knowledge of CNGCs in plants, and provide a new foundation for future elucidation of the structure-function relationships and signal transduction mediated by these channels.
33
Liu, Kao-Chao, and 劉高超. "The stability of Calmodulin and the effect of Calmodulin binding site of cyclic-nucleotide-gated olfactory ion channel." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/00576445818853029884.
Повний текст джерелаАнотація:
碩士國立東華大學
化學系
90
Abstract Calmodulin (CaM) is a 148-residue protein which serves as the promary receptor for intracellular Ca2+. The structure of CaM contains two globular domains and a long sentral helix. In the first part of the thesis, we have performed the chemical denaturation experiments to determine the stability of wild type, three point-mutated, and the N-terminal domain of CaMs. Based on the two-state and three-state unfolding model, the free energy of unfolding (ΔGo) and the transition midpoint of CaM and its mutants have been calculated. The stability of apo and holo Y99W, Y138W was almost unchanged compared to wild type CaM. Interestingly, the stability of Y99WY1338W was decreased slightly in the holo form however, increased by about 0.5 kcal/mol compared to wild type CaM and Y99W and Y138W. The N-terminal domain of CaM is more stable than the C-domain in their apo-forms. However, upon binding to Ca2+, the C-terminal domain is more stable than the N-terminal one. In the second part of the thesis, we have investigated the structural change and the binding stoichiometric ratio of CaM with the binding domain of a nucleotide-gated ion channel. We have found that the peptide adopted α-helical structure upon binding to CaM and that the binding stoichiometric ratio is 1:1.
34
Guo, Jhao-Heng, and 郭肇恆. "Structural Study of Calmodulin Complexed with Calmodulin Binding Domain of Mutated Cyclic Nucleotide-Gated Ion Channel by NMR." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/48416763339931417372.
Повний текст джерела
35
Becirovic, Elvir [Verfasser]. "Role of the CNGB1a subunit of the rod cyclic nucleotide gated channel in channel gating and pathogenesis of retinitis pigmentosa / Elvir Becirovic." 2010. http://d-nb.info/1006625305/34.
Повний текст джерела
36
Huang, Po-Chaung, and 黃柏川. "Purification, crystallization and preliminary x-ray crystallographic analysis of a hybrid molecule of calmodulin and calmodulin binding domain of olfactory cyclic nucleotide-gated ion channel." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/18787375825063015418.
Повний текст джерелаАнотація:
碩士國立東華大學
化學系
92
Calmodulin (CaM) is a ubiquitous calcium sensor, which binds to and regulates many proteins in a Ca2+-dependent manner. Calmodulin has been shown to bind directly to the olfactory channels and to modulate their sensitivity to cyclic nucleotides and appears to play a role in the termination of the signal-transduction pathway in olfactory neurons. In this study, a hybrid molecule, which consists of CaM and CaM-binding domain of olfactory nucleotide gated ion channel, was purified and crystallized. Preliminary crystallographic analysis was achieved. The crystals diffracted to a maximum resolution of 1.85 Å at a cryogenic temperature (100K) using X-rays from a rotating copper tube (1.54 Å wavelength). The crystal belonged to a monoclinic system, and the space group was C2 with unit cell dimensions of a=64.76 Å, b=36.23 Å, c=70.96 Å, α=γ=90˚, β=109.4˚. Each asymmetric unit contained one CaM-OLFp hybrid molecule, which gave rise to a specific volume (VM) of 1.94 Å3Da-1 based on the calculated molecular mass of 20 kDa for a CaM-OLFp hybrid molecule and an estimated solvent content of 36.42%, indicating that our crystal is suitable for further structure analysis.
37
Weißgraeber, Stephanie. "Hyperpolarization-Activated cyclic nucleotide-gated channels - structure and evolution." Phd thesis, 2015. http://tuprints.ulb.tu-darmstadt.de/4212/1/diss_tuprints2015.pdf.
Повний текст джерелаАнотація:
Computational models can shed light on protein function and the underlying mechanisms, where experimental approaches reach their limit. We developed an in silico mechanical model to analyze the process of cAMP-induced modulation in hyperpolarization-activated
cyclic nucleotide-gated (HCN) channels, which conduct cations across the membrane of mammalian heart and brain cells. The structural analysis revealed a quaternary twist of the four subunits of the HCN channel tetramer. This motion has previously been shown to be part of the voltage-gating mechanism of other ion channels.
The insight gained from the mechanical approach was supported by results of analyses of intramolecular coevolution: Covariation of amino acids is induced by compensating mutations that maintain vital functions of a protein. Therefore, these covariations can be
used to locate positions relevant for protein function. We found long-range coevolutionary relationships in HCN that suggest the existence of large domain rearrangements like the ones we found for the allosteric conformational change upon cAMP binding.
This thesis can be divided into two approaches: one based on structural data and another which analyzes sequence information. Together these results contribute to a deeper understanding of the gating mechanism of HCN channels.
• Mechanics of the HCN channel
– A homology model of the transmembrane domain of the HCN4 channel was
developed and joined with the crystal structure of the C-terminal domain to create a combined model of HCN4.
– Release of cAMP from the binding pocket was simulated using an elastic network model and linear response theory to study the resulting conformational change.
– The displacement from this allosteric change was compared to intrinsic low frequency modes of the protein structure.
– Contacts were switched off one by one to identify key players of the observed motion.
• Intramolecular coevolution of HCN channels
– Parameter sets for multiple sequence alignments were analyzed with a visual analytics approach to improve alignment quality prior to coevolutionary analysis.
– Graph measures of the coevolutionary network of HCN were compared to four other proteins and two null models.
– We identified pairwise relationships that show long-range coevolution between the transmembrane region and the C-terminal domain.
– Three-dimensional mutual information revealed coevolving groups of residues at the interface between neighboring subunits of the tetramer.
38
Rato, Claudia. "Regulation of pollen tube growth by calmodulin, cyclic nucleotides and cyclic nucleotide-gated channels." Doctoral thesis, 2007. http://sibul.reitoria.ul.pt/F/?func=item-global&doc_library=ULB01&type=03&doc_number=000511486.
Повний текст джерелаАнотація:
Tese de doutoramento em Biologia (Biologia Celular), apresentada à Universidade de Lisboa através da Faculdade de Ciências, 2007Pollen tube growth plays an essential role in fertilization of flowering plants. Several signalling pathways were identified during pollen tube growth, including Ca2+, CaM and 3',5'-cyclic adenosine monophosphate (cAMP). These constitute a complex web of signalling networks that intersect at various levels (reviewed in Chapter I). In order to elucidate the role of CaM we mapped its activity in growing pollen tubes. We found that CaM activity exhibits a tip-focused gradient, similar to the distribution of cytosolic free Ca2+ ([Ca2+]c), and that it oscillates with a period similar to [Ca2+]c . Moreover, we show that CaM is also involved in the guidance mechanism and has its activity strongly modulated by intracellular changes in cAMP. A putative target of the crosstalk between CaM and cAMP is the secretory machinery as observed in pollen tubes loaded with the FM 1-43 dye. Our data thus suggest that pollen tube growth and orientation depends on an intricate crosstalk between multiple signalling pathways in which CaM is a key element (Chapter II). In Chapter III we study two possible targets of CaM, CNGC7 and CNGC8. These proteins are both expressed in pollen and show strong homology, suggesting functional redundancy. To gain insights into the biological function of CNGC7 and CNGC8, we used reverse-genetics. Neither CNGC7 nor CNGC8 was found to be essential, but CNGC7 appears to play a role in fertilization. To further address a possible redundancy between CNGC7 and CNGC8, we generated a double-mutant and the double knockout was found to be gametophyte lethal. These results provide the first genetic evidence for redundancy among CNGCs in fertilization. Plant CNGCs contain a C-terminal cyclic nucleotide binding domain with an overlapping CaM binding site. Cyclic nucleotides induce channel opening, while CaM mediates the feed-back inhibition of the channel. To gain insights into the structure-function relationship of plant CNGCs, we used a site-directed mutagenesis technique coupled with complementation assays of cngc18 mutants. Amino acid substitutions E520A, F565A and F565W provided partial complementation. Taken together, these results show that F565 and E520 are key amino acid residues for cyclic nucleotide binding and that changes in the cyclic nucleotide binding domain are capable of disrupting protein function (Chapter IV). In Chapter V all results obtained were discussed.
39
Ullmer, Wendy Elizabeth. "Expression and sub-cellular localization of cyclic nucleotide-gated ion channels in Arabidopsis thaliana." Thesis, 2007. http://hdl.handle.net/10125/20744.
Повний текст джерела
40
RESTA, FRANCESCO. "Hyperpolarization-activated Cyclic Nucleotide gated (HCN) channels as promising new target for neuropathic pain treatment." Doctoral thesis, 2016. http://hdl.handle.net/2158/1049658.
Повний текст джерела
41
Chiang, Wei-Che, and 蔣惟哲. "NMR structural study of binding specificity of calmodulin to mutant olfactory cyclic nucleotide-gated ion channels." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/60411804119080908559.
Повний текст джерелаАнотація:
碩士國立東華大學
化學系
102
Calmodulin (CaM), the primary intracellular Ca2+ receptor, regulates a large number of key enzymes and controls a wide spectrum of important biological responses. The structure of CaM and its target sequence in rat olfactory nucleotide gated ion channel (OLFp) was characterized by NMR spectroscopy. Our previous data indicated that distinct CaM/OLFp complexes (mode I and II) existed simultaneously with stable structures that were not inter-exchangeable within the NMR time scale. The NMR structures of these two CaM/OLFp complexes showed that the helical OLFp with C2 symmetry bound to CaM in two orientations and the palindromic sequence of OLFp (F1QRIVRLVGVIRDW14) is crucial for its targeting orientation to CaM. In order to further support this hypothesis, we investigated the interaction of CaM with a series of mutated OLFp with less palindromic feature by NMR. We found that the Valine residue in the position 5 is the most important residue for the orientation-specific targeting to CaM. Only one complex structure was detected when CaM in association with the mutated OLFp_V5A. The complex structure of CaM/OLFp_V5A was determined. It shows that the subtle change of the side chain strongly affects the stability of the weak interactions between OLFp and CaM.
42
El-Kholy, Wasim. "The role of hyperpolarization activated cyclic nucleotide modulated and voltage gated potassium channels in pancreatic beta-cell function." 2007. http://link.library.utoronto.ca/eir/EIRdetail.cfm?Resources__ID=478866&T=F.
Повний текст джерела
43
Frietsch, Sabine [Verfasser]. "The role of cyclic nucleotide gated channels (CNGC) in plant development and stress responses in Arabidopsis thaliana / vorgelegt von Sabine Frietsch." 2006. http://d-nb.info/995451265/34.
Повний текст джерела
44
"Expressions of cyclic nucleotide-gated ionic conductances in rat cerebellar purkinje neurons =: 大鼠小腦浦肯野細胞環核苷酸門控離子通道的表達". 2005. http://library.cuhk.edu.hk/record=b5892479.
Повний текст джерелаАнотація:
Tsoi Sze Man.Thesis (M.Phil.)--Chinese University of Hong Kong, 2005.
Includes bibliographical references (leaves 82-104).
Text in English; abstracts in English and Chinese.
Tsoi Sze Man.
Chapter Chapter 1 --- Introduction --- p.1
Chapter 1.1 --- Overview of study --- p.1
Chapter 1.2 --- Cerebellum --- p.2
Chapter 1.2.1 --- General Structure of cerebellum --- p.3
Chapter 1.2.2 --- Cell types of cerebellar cortex --- p.4
Chapter 1.2.2.1 --- Basket cells --- p.5
Chapter 1.2.2.2 --- Stellate cells --- p.6
Chapter 1.2.2.3 --- Purkinje cells --- p.6
Chapter 1.2.2.4 --- Granule cells --- p.7
Chapter 1.2.2.5 --- Golgi cells --- p.8
Chapter 1.2.2.6 --- Unipolar brush cells --- p.9
Chapter 1.2.2.7 --- Deep cerebellar nuclear neurons --- p.11
Chapter 1.2.3 --- The neuronal circuitry of the cerebellum --- p.12
Chapter 1.2.4 --- Cerebellar function --- p.14
Chapter 1.3 --- Cyclic nucleotide-gated (CNG) channels --- p.16
Chapter 1.3.1 --- Molecular characterization of CNG channels --- p.16
Chapter 1.3.2 --- Functional properties of CNG channels --- p.19
Chapter 1.3.3 --- Expression of CNG channels in brain --- p.21
Chapter 1.3.4 --- CNG channel and neuronal plasticity --- p.23
Chapter 1.4 --- Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels --- p.26
Chapter 1.4.1 --- Molecular characterization of HCN channels --- p.27
Chapter 1.4.2 --- Functional properties of HCN channels and Ih current --- p.29
Chapter 1.4.3 --- Modulation by cyclic nucleotides --- p.31
Chapter 1.4.4 --- Expression of HCN channels in brain --- p.33
Chapter 1.4.5 --- Physiological roles of Ih current in central nervous system --- p.35
Chapter 1.5 --- Aims of study --- p.38
Chapter Chapter 2 --- Material and Methods --- p.39
Chapter 2.1 --- Immunohistochemistry Experiments --- p.39
Chapter 2.1.1 --- Animal preparation --- p.39
Chapter 2.1.2 --- Tissue preparation --- p.39
Chapter 2.1.3 --- Primary and secondary antibodies --- p.40
Chapter 2.1.4 --- Immunofluroescence staining --- p.41
Chapter 2.1.5 --- Confocal laser scanning microscopy and data processing --- p.41
Chapter 2.2 --- Whole cell patch clamp recordings --- p.42
Chapter 2.2.1 --- Brain slice preparation and identification of the cerebellar Purkinje neurons --- p.42
Chapter 2.2.2 --- Whole cell voltage- and current-clamp recordings --- p.43
Chapter 2.2.3 --- Drug solutions and delivery --- p.44
Chapter 2.2.4 --- Statistical analysis --- p.45
Chapter Chapter 3 --- Expression of Various Cyclic Nucleotide-Gated (CNG) Channel Subunits in Rat Cerebellum --- p.46
Chapter 3.1 --- Introduction --- p.46
Chapter 3.2 --- Results --- p.46
Chapter 3.2.1 --- Immunoreactivity of CNGA1 in cerebellum --- p.46
Chapter 3.2.2 --- Immunoreactivity of CNGA2 in cerebellum --- p.47
Chapter 3.2.3 --- Immunoreactivity of CNGA3 in cerebellum --- p.47
Chapter 3.3 --- Discussion --- p.48
Chapter Chapter 4 --- Expression of Various Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) Channel Subunits in Rat Cerebellum --- p.53
Chapter 4.1 --- Introduction --- p.53
Chapter 4.2 --- Results --- p.53
Chapter 4.2.1 --- Immunoreactivity of HCN 1 in cerebellum --- p.53
Chapter 4.2.2 --- Immunoreactivity of HCN2 in cerebellum --- p.55
Chapter 4.2.3 --- Immunoreactivity of HCN3 in cerebellum --- p.55
Chapter 4.2.4 --- Immunoreactivity of HCN4 in cerebellum --- p.55
Chapter 4.3 --- Discussion --- p.55
Chapter Chapter 5 --- Electrophysiological Recordings of Cyclic Nucleotide-Gated Ionic Conductance in Rat Cerebellar Purkinje Neurons --- p.59
Chapter 5.1 --- Introduction --- p.59
Chapter 5.2 --- Results --- p.59
Chapter 5.2.1 --- Effect of cyclic nucleotides on the membrane potential of cerebellar Purkinje neurons --- p.59
Chapter 5.2.2 --- Ionic conductance of the cyclic nucleotide-induced inward current --- p.61
Chapter 5.2.3 --- The mechanism of the cyclic nucleotide-induced inward current --- p.61
Chapter 5.2.3.1 --- Site of action --- p.62
Chapter 5.2.3.2 --- Involvement of CNG channels and HCN channels --- p.63
Chapter 5.2.3.3 --- Involvement of protein kinase A (PKA) and protein kinase G (PKG) --- p.65
Chapter 5.2.3.4 --- Involvement of inwardly rectifying potassium (Kir) channels and transient receptor potential (TRP) channels --- p.65
Chapter 5.2.4 --- Effect of cyclic nucleotides on Ih current in Purkinje neurons --- p.67
Chapter 5.3 --- Discussion --- p.68
Chapter Chapter 6 --- Concluding remarks References --- p.78
References --- p.82
45
Urquhart, William. "Characterization of AtCNGC11/12-induced Cell Death and the Role of AtCNGC11 and AtCNGC12 in Ca2+ Dependent Signalling Pathways." Thesis, 2011. http://hdl.handle.net/1807/29897.
Повний текст джерелаАнотація:
The Arabidopsis cyclic nucleotide-gated ion channels (AtCNGCs) form a large family consisting of 20 members. It has been suggested that CNGCs contribute to a wide array of biological functions such as pollen tube growth and pathogen defence signalling. However, the precise mechanisms by which AtCNGCs act, and the extent of their biological roles, have yet to be fully elucidated.
AtCNGC11/12, the chimeric CNGC that resulted from the fusion of AtCNGC11 and 12, induces a number of pathogen defence related phenotypes in the Arabidopsis mutant cpr22. Spontaneous lesion formation is one such phenotype. Interestingly, when AtCNGC11/12 is transiently expressed in N. benthamiana it causes cell death which was characterized in this study. Also, AtCNGC11/12 was used to investigate the structural features responsible for the proper function and regulation of AtCNGCs. Electron microscopic analysis of the AtCNGC11/12-induced cell death showed similar characteristics to programmed cell death (PCD), such as plasma membrane shrinkage and vesicle formation. Interestingly caspase-1 inhibitors and the silencing of vacuolar processing enzyme, a plant enzyme with caspase-1 activity, suppressed the induction of cell death. Additionally, pharmacological analyses indicated that the AtCNGC11/12-indiced cell death was also dependent on Ca2+. Furthermore, 3 amino acid residues, R190, A225, and G287, were demonstrated to be essential for AtCNGC11/12-induce cell death. Taken together, these results indicate that the cell death that develops in the cpr22 mutant is indeed PCD and that AtCNGC11/12, is at the point of, or up-stream of, the Ca2+ signal necessary for the development of HR. Furthermore, the functionality of AtCNGC11/12 as a model for AtCNGC structure-function analyses was demonstrated by the identification of several amino acids necessary for cell death development.
Yoshioka et al. (2006) demonstrated that the loss of AtCNGC11 or 12 results in decreased resistance to avirulent isolates of the oomycete pathogen, H. arabidopsidis. Thus, the present biological role suggested for AtCNGC11 and 12 is in pathogen defence, specifically within effector triggered immunity (ETI). Like AtCNGC11 and 12, AtCNGC2 has been demonstrated to contribute to pathogen defence signalling but has also been implicated in other physiological responses such as ion stress and senescence. To better understand the roles of AtCNGC11 and 12 in both pathogen defence and other Ca2+ dependent signalling processes, I have investigated promoter:GUS reporter lines, as well as, AtCNGC11 and 12 KO and RNAi silenced lines subjected to various treatments. From this work, I have demonstrated that AtCNGC11 and 12 have similar expression patterns during pathogen defence, development, and dark-induced senescence. Additionally, the findings presented here further characterize AtCNGC11 and 12 as contributors to ETI rather than PAMP triggered immunity. Furthermore, I demonstrated that AtCNGC11 and 12 are likely involved in the endogenous movement of Ca2+, contributing to a range of Ca2+ associated signalling pathways including gravitropism and senescence. Taken together, these results have greatly improved the characterization of AtCNGC11 and 12; significantly contributing to the understanding of a large and increasingly important channel family.