Regulation of the Intestinal Ca2+ Channels TRPV6
肠道 Ca2 通道 TRPV6 的调节
基本信息
- 批准号:8244980
- 负责人:
- 金额:$ 29.64万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2011
- 资助国家:美国
- 起止时间:2011-04-01 至 2015-03-31
- 项目状态:已结题
- 来源:
- 关键词:AffectAffinityAllyAmino AcidsBindingBinding SitesBiochemicalBiochemistryBiological ProcessCalciumCalcium ChannelCalmodulinCellsChargeChemicalsCholecalciferolCytoplasmic TailElectrophysiology (science)ElectrostaticsEpithelialFamilyIntestinesIon ChannelKidney CalculiKnowledgeLeadLipid BilayersLipidsMgATPModificationMolecularMolecular BiologyMutagenesisMutateMutationOsteoporosisOxidative StressPhosphatidylinositol 4,5-DiphosphatePhosphatidylinositolsPhospholipase CPhosphotransferasesPlayReagentRegulationRisk FactorsRoleScanningSignal Transduction PathwaySiteStimulusStructureTechniquesTestingTransmembrane DomainVanilloidabsorptionanalogcold temperatureinsightkinase inhibitormembernovelpatch clamppublic health relevancereceptorreconstitutionresearch studytoolwarm temperature
项目摘要
DESCRIPTION (provided by applicant): Transient Receptor Potential (TRP) channels are calcium permeable ion channels that play roles in a multitude of biological processes. Despite their diversity of activation mechanisms, phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate [PIP2] have emerged as common regulators of these ion channels. Most TRP channels have been shown to require PIP2 for activity. TRPV6 is an epithelial Ca2+ channel responsible for active Ca2+ absorption in the intestine. The expression level of TRPV6 is regulated mainly by the active form of vitamin D3. Once expressed, TRPV6 is constitutively active, but its activity is limited by Ca2+-induced inactivation. We have shown earlier that the activity of this channel depends on the presence of PIP2, and that depletion of this lipid by phospholipase C (PLC) activation plays a major role in Ca2+-induced inactivation. Knowledge on the molecular mechanism of PIP2 activation of TRP channels is very limited. Our hypothesis is that PIP2 activates TRPV6 through binding to positively charged residues in the cytoplasmic regions, and this binding causes a conformational change in transmembrane domain 6 (TM6) leading to opening of the channel. TRPV6 is an ideal candidate to study the mechanism of activation by PIP2, because, unlike other TRP channels, it is constitutively active; its activity only depends on PIP2. In aim 1 we will systematically mutate conserved positively charged amino acids in the cytoplasmic domains of TRPV6, to identify PIP2 interacting residues. We will test the effects of the mutations on the sensitivity of the channel to PIP2 using electrophysiological and biochemical techniques. In Aim 2 we will use Cys-scanning mutagenesis to identify gating structures in TRPV6 that open upon PIP2 binding. Intracellular ATP has been proposed to directly bind to TRPV6 and its absence has been associated with channel rundown in whole-cell patch clamp experiments. We show that in excised patches ATP re-activates TRPV6 only in the presence of Mg2+. Our hypothesis is that MgATP provides substrate for lipid kinases and thus allows PIP2 re-synthesis. We will test this hypothesis in aim 3 by applying hydrolysable and non-hydrolysable analogues of ATP with and without Mg2+ in excised patches and on reconstituted channels in planar lipid bilayers. We will also test the effects of lipid kinase inhibitors in excises patches on TRPV6 activity induced by MgATP. Calmodulin has been proposed to be involved in Ca2+-induced inactivation of TRPV6, but the direct effects of CaM have not been demonstrated in excised patches. We show robust calcium-dependent inhibition of TRPV6 by CaM in excised patches. It is likely that Ca-CaM and phosphoinositide depletion act in concert to inhibit channel activity upon increased cytoplasmic Ca2+ concentrations. In aim 4 we will study the relationship between CaM and PIP2 regulation of TRPV6, using the combination of electrophysiology, biochemistry and molecular biology.
PUBLIC HEALTH RELEVANCE: There are essentially no pharmacological tools available to modulate active absorption of Ca2+ in the intestines, in which TRPV6 plays a major role. Clinically, both enhancing and reducing Ca2+ absorption may be desirable, because increased absorption of Ca2+ is the most important risk factor for kidney stones and decreased Ca2+ absorption plays an important role in the pathomechanism of osteoporosis. The better understanding of how TRPV6 is regulated, could potentially lead to novel ways to pharmacologically enhance or reduce intestinal Ca2+ absorption.
描述(由申请人提供):瞬时受体电位(TRP)通道是钙渗透性离子通道,在多种生物过程中发挥作用。尽管其激活机制的多样性,磷酸肌醇,特别是磷脂酰肌醇4,5-二磷酸[PIP 2]已成为这些离子通道的常见调节剂。大多数TRP通道已被证明需要PIP 2的活动。TRPV 6是一种上皮钙通道,负责肠内活性钙吸收。TRPV 6的表达水平主要由活性形式的维生素D3调节。一旦表达,TRPV 6是组成型活性的,但其活性受到Ca 2+诱导的失活的限制。我们先前已经表明,该通道的活性取决于PIP 2的存在,并且磷脂酶C(PLC)激活对该脂质的消耗在Ca 2+诱导的失活中起着重要作用。关于PIP 2激活TRP通道的分子机制的知识非常有限。我们的假设是PIP 2通过与胞质区域中带正电荷的残基结合来激活TRPV 6,并且这种结合引起跨膜结构域6(TM 6)的构象变化,从而导致通道的打开。TRPV 6是研究PIP 2激活机制的理想候选者,因为与其他TRP通道不同,它是组成型活性的;它的活性仅取决于PIP 2。在目标1中,我们将系统地突变TRPV 6胞质结构域中保守的带正电荷的氨基酸,以鉴定PIP 2相互作用残基。我们将使用电生理学和生物化学技术测试突变对通道对PIP 2的敏感性的影响。在目标2中,我们将使用Cys扫描诱变来鉴定TRPV 6中在PIP 2结合时打开的门控结构。已经提出细胞内ATP直接结合TRPV 6,并且其缺失与全细胞膜片钳实验中的通道下降相关。我们发现,在切除的补丁ATP重新激活TRPV 6只有在Mg 2+的存在下。我们的假设是MgATP为脂质激酶提供底物,从而允许PIP 2再合成。我们将在目标3中通过在切除的贴片和平面脂质双层中的重建通道上应用具有和不具有Mg 2+的ATP的可水解和不可水解类似物来测试这一假设。我们还将测试脂质激酶抑制剂对MgATP诱导的TRPV 6活性的影响。钙调素已被提出参与钙离子诱导的TRPV 6失活,但钙调素的直接作用尚未在离体斑块中得到证实。我们显示出强大的钙依赖性抑制TRPV 6的钙调素在切除的补丁。这是可能的,Ca-CaM和磷酸肌醇消耗一致行动,以抑制通道活性后,细胞质Ca 2+浓度增加。目的4:利用电生理、生物化学和分子生物学相结合的方法,研究CaM与PIP 2对TRPV 6的调控关系。
公共卫生相关性:基本上没有药理学工具可用于调节肠中Ca 2+的主动吸收,其中TRPV 6起主要作用。在临床上,增加和减少Ca 2+吸收可能是可取的,因为Ca 2+吸收增加是肾结石的最重要的危险因素,而Ca 2+吸收减少在骨质疏松症的病理机制中起着重要作用。更好地了解TRPV 6是如何调节的,可能会导致新的方法来增强或减少肠道Ca 2+的吸收。
项目成果
期刊论文数量(0)
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Tibor Rohacs其他文献
Tibor Rohacs的其他文献
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{{ truncateString('Tibor Rohacs', 18)}}的其他基金
Regulation of the Intestinal Ca2+ Channels TRPV6
肠道 Ca2 通道 TRPV6 的调节
- 批准号:
8452114 - 财政年份:2011
- 资助金额:
$ 29.64万 - 项目类别:
Regulation of the Intestinal Ca2+ Channels TRPV6
肠道 Ca2 通道 TRPV6 的调节
- 批准号:
8634799 - 财政年份:2011
- 资助金额:
$ 29.64万 - 项目类别:
Regulation of the Intestinal Ca2+ Channels TRPV6
肠道 Ca2 通道 TRPV6 的调节
- 批准号:
8102559 - 财政年份:2011
- 资助金额:
$ 29.64万 - 项目类别:
Regulation of the Intestinal Ca2+ Channels TRPV6
肠道 Ca2 通道 TRPV6 的调节
- 批准号:
8784840 - 财政年份:2011
- 资助金额:
$ 29.64万 - 项目类别:
Regulation of the Intestinal Ca2+ Channels TRPV6
肠道 Ca2 通道 TRPV6 的调节
- 批准号:
9275766 - 财政年份:2011
- 资助金额:
$ 29.64万 - 项目类别:
Lipid Regulation of Transient Receptor Potential Channels
瞬时受体电位通道的脂质调节
- 批准号:
7623470 - 财政年份:2007
- 资助金额:
$ 29.64万 - 项目类别:
Regulation of sensory TRP channels by phospholipids and G-proteins
磷脂和 G 蛋白对感觉 TRP 通道的调节
- 批准号:
10405595 - 财政年份:2007
- 资助金额:
$ 29.64万 - 项目类别:
Lipid Regulation of Transient Receptor Potential Channels
瞬时受体电位通道的脂质调节
- 批准号:
7317216 - 财政年份:2007
- 资助金额:
$ 29.64万 - 项目类别:
Lipid Regulation of Transient Receptor Potential Channels
瞬时受体电位通道的脂质调节
- 批准号:
8581450 - 财政年份:2007
- 资助金额:
$ 29.64万 - 项目类别:
Regulation of sensory TRP channels by phospholipids and G-proteins
磷脂和 G 蛋白对感觉 TRP 通道的调节
- 批准号:
10166960 - 财政年份:2007
- 资助金额:
$ 29.64万 - 项目类别:
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