Regulation of the Intestinal Ca2+ Channels TRPV6

描述(申请人提供)：瞬时受体电位(Trp)通道是钙离子通道，在多种生物过程中发挥作用。尽管激活机制各不相同，但磷脂酰肌醇，特别是磷脂酰肌醇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才能激活。TRPV6是一种上皮性钙通道，负责肠道对钙的主动吸收。TRPV6的表达水平主要受维生素D3活性形式的调节。一旦表达，TRPV6就具有结构性活性，但其活性受到钙诱导的失活的限制。我们早些时候已经证明，该通道的活性依赖于PIP2的存在，而磷脂酶C(PLC)激活导致的这种脂质的耗尽在钙诱导的失活中起着主要作用。关于PIP2激活Trp通道的分子机制的了解非常有限。我们的假设是，PIP2通过与细胞质区域的正电荷残基结合来激活TRPV6，这种结合导致跨膜区6(TM6)的构象变化，从而导致通道的开放。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，它是结构性活性的，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正电荷氨基酸，以鉴定PIP2相互作用的残基。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。在目标2中，我们将使用半胱氨酸扫描突变来确定TRPV6中的门控结构，这些结构在PIP2结合时打开。细胞内ATP被认为与TRPV6直接结合，在全细胞膜片钳实验中，它的缺失与通道衰退有关。我们表明，在切除的斑块中，ATP只有在Mg2存在的情况下才能重新激活TRPV6。我们的假设是，镁三磷酸腺苷为脂蛋白激酶提供底物，从而允许PIP2的重新合成。我们将在目标3中通过在切除的斑块和平面脂质双层中的重组通道中应用含和不含镁的ATP的可水解性和不可水解性类似物来验证这一假设。我们还将测试切除贴片中的脂激酶抑制剂对镁三磷酸腺苷诱导的TRPV6活性的影响。钙调蛋白已被认为参与了钙诱导的TRPV6失活，但CaM的直接作用尚未在切除的斑块中得到证实。我们显示，在切除的斑块中，CaM对TRPV6具有强烈的钙依赖抑制作用。钙钙调素和肌醇磷脂的耗竭很可能协同作用，在胞浆内钙离子浓度升高时抑制通道活性。在目标4中，我们将结合电生理学、生物化学和分子生物学的方法，研究CaM和PIP2对TRPV6的调控关系。

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.