Regulation of the Intestinal Ca2+ Channels TRPV6

肠道 Ca2 通道 TRPV6 的调节

• 批准号: 8784840
• 负责人: Tibor Rohacs
• 金额: $ 20.2万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 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: https://reporter.nih.gov/project-details/8784840
• 关键词: Regulation; Intestinal; Ca2+; Channels; TRPV6

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.

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。尽管激活机制多种多样，但磷酸肌醇，尤其是磷脂酰肌醇 4,5-二磷酸 [PIP2] 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 PIP2 才能活动。 TRPV6 是一种上皮 Ca2+ 通道，负责肠道中的主动 Ca2+ 吸收。 TRPV6 的表达水平主要受维生素 D3 活性形式的调节。 TRPV6 一旦表达，即具有组成型活性，但其活性受到 Ca2+ 诱导的失活的限制。我们之前已经表明，该通道的活性取决于 PIP2 的存在，并且磷脂酶 C (PLC) 激活导致的这种脂质的消耗在 Ca2+ 诱导的失活中起着重要作用。关于 PIP2 激活 TRP 通道的分子机制的知识非常有限。我们的假设是，PIP2 通过与细胞质区域带正电荷的残基结合来激活 TRPV6，这种结合会导致跨膜结构域 6 (TM6) 发生构象变化，从而导致通道打开。 TRPV6 是研究 PIP2 激活机制的理想候选者，因为与其他 TRP 通道不同，它具有组成型活性；其活性仅取决于PIP2。在目标 1 中，我们将系统地突变 TRPV6 细胞质结构域中保守的带正电荷的氨基酸，以鉴定 PIP2 相互作用残基。我们将使用电生理学和生化技术测试突变对 PIP2 通道敏感性的影响。在目标 2 中，我们将使用 Cys 扫描诱变来识别 TRPV6 中在 PIP2 结合时打开的门控结构。细胞内 ATP 已被提议直接与 TRPV6 结合，并且它的缺失与全细胞膜片钳实验中的通道耗尽有关。我们发现，在切除的斑块中，只有在 Mg2+ 存在的情况下，ATP 才会重新激活 TRPV6。我们的假设是 MgATP 为脂质激酶提供底物，从而允许 PIP2 重新合成。我们将在目标 3 中通过在切除的斑块和平面脂质双层的重建通道上应用含或不含 Mg2+ 的 ATP 的可水解和不可水解类似物来测试这一假设。我们还将测试切除贴片中的脂质激酶抑制剂对 MgATP 诱导的 TRPV6 活性的影响。钙调蛋白被认为参与 Ca2+ 诱导的 TRPV6 失活，但 CaM 的直接作用尚未在切除的斑块中得到证实。我们在切除的斑块中显示出 CaM 对 TRPV6 的强烈钙依赖性抑制。 Ca-CaM 和磷酸肌醇消耗很可能协同作用，在细胞质 Ca2+ 浓度增加时抑制通道活性。在目标 4 中，我们将结合电生理学、生物化学和分子生物学研究 TRPV6 的 CaM 和 PIP2 调节之间的关系。