Regulation of the Intestinal Ca2+ Channels TRPV6

描述（由申请人提供）：瞬时受体电位（TRP）通道是钙离子渗透通道，在多种生物过程中发挥作用。尽管磷脂酰肌醇的激活机制多种多样，但它们，尤其是磷脂酰肌醇4,5-二磷酸[PIP2]，已成为这些离子通道的共同调节剂。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，负责肠道内活跃的Ca2+吸收。TRPV6的表达水平主要受维生素D3活性形式的调控。一旦表达，TRPV6具有组成性活性，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）激活对这种脂质的消耗在Ca2+诱导的失活中起主要作用。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通过与细胞质区带正电的残基结合激活TRPV6，这种结合导致跨膜结构域6 （TM6）的构象改变，从而打开通道。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，它具有组成活性；其活性仅依赖于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的氨基酸，以鉴定PIP2相互作用残基。我们将使用电生理和生化技术测试突变对通道对PIP2敏感性的影响。在Aim 2中，我们将使用cys扫描诱变技术来鉴定TRPV6中在PIP2结合时打开的门控结构。在全细胞膜片钳实验中，细胞内ATP被认为可以直接与TRPV6结合，其缺失与通道减少有关。我们发现，在切除的斑块中，ATP仅在Mg2+存在的情况下才能重新激活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构通道中应用含和不含Mg2+的可水解和不可水解ATP类似物来验证这一假设。我们还将测试运动贴片中脂激酶抑制剂对MgATP诱导的TRPV6活性的影响。钙调素被认为参与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发现CaM对TRPV6的钙依赖性抑制作用。很可能Ca-CaM和磷酸肌苷耗竭协同作用，在细胞质Ca2+浓度增加时抑制通道活性。在aim 4中，我们将结合电生理学、生物化学和分子生物学来研究CaM与PIP2调控TRPV6之间的关系。

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.