Structural and Biophysical Studies of Kinase-Ion Channel Interactions

电信号到生化信号的转换需要离子在可兴奋细胞中从一个隔间移动到另一个隔间。因此，在正确的时间摄入适量的这些离子对正常的肌肉和神经功能至关重要。这些离子必须通过可以打开和关闭的特殊通道蛋白，从而决定它们的释放量和时间。蛋白激酶是一组调节蛋白质，通过在离子通道上添加一个小标签来修饰它，这反过来又可以改变通道的活动。这种标记通常是离子通道活性所必需的，这种标记被称为“磷酸化”，然而，当标记过多时，可能会导致紊乱。令人惊讶的是，人们对激酶参与离子通道调节知之甚少。只有几项研究表明哪个蛋白激酶针对哪个部位，而且更少的研究已经测量到了磷酸化速率。了解针对每个靶点的磷酸化速率具有重要的生理学意义，因为这种修饰改变了激发条件，如“战斗或逃跑”反应，其中离子通道开放的时机是关键的。我的研究项目重点是蛋白激酶如何利用生化和生物物理方法瞄准哺乳动物的离子通道及其相关蛋白。我的长期目标是用一种跨学科的方法来解开某些蛋白激酶是如何调节离子通道的。虽然这最终需要我们阐明涉及众多激酶和蛋白质的一系列信号事件，但我的短期目标是专注于两个蛋白激酶PKA和CaMKII，以及它们对电压门控钠通道(NAV)的调节。为此，我的实验室试图确定它们的催化亚基如何结合和调节参与信号连接形成的蛋白质和离子通道，从来自NAV的多肽底物开始，最终包括其他离子通道及其不同的异构体和辅助亚基。只有少数几个实验衍生的激酶-底物复合体。从长远来看，该计划旨在研究磷酸化在完全折叠通道而不是多肽的背景下的影响，并探索CaMKII和PKA等激酶是如何被招募到离子通道的。这将带来对它们的抑制的相伴的洞察和对这些激酶更好的机制的理解。最后，我们试图揭示其他调节蛋白是如何受到激酶的活性和作用的影响，因为这些已知的调节离子通道的方式依赖于磷酸化。这项工作不仅提高了我们对人类生理学的了解，而且最终可能有助于设计改变特定激酶-离子通道相互作用的药物。重要的是，拟议的研究利用了尖端的生化和生物物理方法，为加拿大的下一代科学人员和未来的研究创新提供了丰富的培训环境。

The transformation of electrical signals to biochemical ones requires the movement of ions from one compartment to another in excitable cells. Thus, having the right amount of these ions at the right time is critical for proper muscle and neuronal function. These ions must pass through special channel proteins that can open and close, thus determining the amount and timing of their release. Protein kinases are a group of regulatory proteins that modify ion channels by adding a little 'tag' to it, which in turn can alter the activity of the channel. This tagging, which is known as 'phosphorylation', is normally required for the activity of ion channels however, when the tagging is excessive, it can lead to disorders. Surprisingly, kinase involvement in ion channel regulation is poorly understood. Only a few studies show which protein kinase target which site, and fewer still have measured phosphorylation rates. Understanding the rate of phosphorylation toward each target site is physiologically important because this modification alters conditions of excitation, such as the "fight-or-flight" response, where timing of an ion channel opening is critical. My research program focuses on how protein kinases target mammalian ion channels and their associated proteins using biochemical and biophysical approaches. My long-term goal is to use an interdisciplinary approach to unravel how certain protein kinases regulate ion channels. While this ultimately this requires us to elucidate the cascade of signaling events involving numerous kinases and proteins, my short-term goal is to focus on two protein kinases PKA and CaMKII, and their regulation of the voltage gated sodium channels (NaVs). To this end, my lab seek to determine how their catalytic subunits bind and regulate proteins and ion channels involved in the formation of signaling junctions starting with peptide substrates derived from NaVs, and eventually including other ion channels, their different isoforms and ancillary subunits. There are only a handful of experimentally derived kinase-substrate complexes. Long-term, this program aims to study the affects of phosphorylation in context of fully folded channels as opposed to peptides and explore how kinases such as CaMKII and PKA are recruited to ion channels. This will bring concomitant insights into their inhibition and a better mechanistic understanding of these kinases. Finally, we seek to uncover how other regulatory proteins are affected by the activity and action of kinases, as these are known to regulate ion channels in a phosphorylation dependent manner. This work not only advance our knowledge of human physiology, but may eventually aid in design of drugs that alter specific kinase-ion channel interactions. Importantly, the proposed studies utilize cutting-edge biochemical and biophysical methods which provides a rich training environment for the next generation of scientific personnel and future research innovation in Canada.