Biophysical elucidation of ion channel complex function

总体计划的基本原理和重点：* 离子通道通过使关键过程发生来调节生物功能的基本方面，这些过程可以与大脑或心脏中的电信号和细胞体积控制一样多样化。电压依赖性门控导致电压激活离子通道的传导或开放状态，并随后导致其失活和关闭的分子事件在许多情况下仍然定义不清。我们的长期目标是更好地了解钾离子正常门控的生物物理机制，（IKr，IKs）和钠（INa）离子通道，这将通过解决四个相关的短期目标的研究来实现：*#1）IKr电压门控的动力学结构：我们假设离子通道电压传感器域内部电荷相互作用控制延迟的Kv11.1离子通道激活/失活和电压传感器域-炮塔快速失活。创新的门控电流分析、甲烷硫代磺酸盐暴露研究和荧光探针的遗传掺入将用于精确绘制亚基间运动。2和#3）INa失活和IKs亚基间相互作用的结构基础的实时研究：我们假设a）Nav1.5结构域的瞬时分子构象定义了失活的稳定性，B）外部裂缝中KCNQ 1和KCNE 1 - 5之间的特异性和独特的物理关联赋予门控特性，以及c）控制亚基化学计量。遗传编码的光激活桥、MS分析和全细胞和单通道补丁记录将用于绘制这些相互作用。4)药物对IKr动力学影响的分子机制：我们假设a）通过hERG的S6氨基酸残基的阳离子-Pi相互作用对于孔阻断至关重要，F656和Y652处的氟化UAA系列将用于检查环电荷分布和阻断的决定因素。**培训内容：我的实验室和教师的研究小组通过先进的技术实验，研究生课程，实验室会议，期刊俱乐部和科学会议上的演讲创造了一个严格而多样化的学习环境。学员获得必要的实验，演讲和写作技能，以实现独立和专业的职业目标，并在生物物理学领域产生新的领导者。重大影响：我们对IKr、IKs和INa门控的深入研究将揭示这些生物学看门人打开和关闭的分子事件，这反过来将提高我们对控制细胞电活动机制的理解。这一知识将通过在高影响力期刊上发表文章、应邀参加会议来传播，此外，预计这项工作将带来更多与国家能源和环境研究中心合作的机会，例如，IRAP更广泛地利用信息并创造更多的经济机会。

Overall program rationale and focus: ***Ion channels regulate essential aspects of biological function by enabling critical processes to occur that can be as diverse as electrical signaling in the brain or heart and cell volume control. The molecular events underlying voltage-dependent gating that lead to the conducting or open state of voltage-activated ion channels, and subsequently to their inactivation and closure are still poorly defined in many situations.***Our long-term objective is to understand better the biophysical mechanisms of normal gating in potassium (IKr, IKs) and sodium (INa) ion channels, and this will be achieved by research addressing four related shorter-term goals:******#1) The kinetic structure of IKr voltage gating: We hypothesize that ion channel voltage sensor domain internal charge interactions control delayed Kv11.1 ion channel activation/deactivation and voltage sensor domain-turret rapid inactivation. Innovative gating current analysis, methane thio-sulphonate exposure studies, and genetic incorporation of fluorescent probes will be used to precisely map inter-subunit movements.******#2 and #3) Real-time studies of the structural basis of INa inactivation and IKs inter-subunit interactions: We hypothesize that a) transient molecular conformations of Nav1.5 domains define stability of inactivation, b) specific and unique physical associations between KCNQ1 and KCNE1-5 in exterior clefts confer gating properties, and c) control subunit stoichiometry. Genetically-encoded photo-activated bridges, MS analysis and whole cell and single channel patch recording will be used to map these interactions.******#4) Molecular mechanisms of drug effects on IKr kinetics: We hypothesize that a) cation-Pi interactions via S6 amino acid residues of hERG are critical for pore block, and fluorinated UAA series at F656 and Y652 will be used to examine ring charge distribution and determinants of block.******Training: My laboratory and the research group of faculty create a rigorous and diverse learning environment through advanced technical experimentation, graduate courses, lab meetings, journal clubs, and presentation at scientific meetings. Trainees acquire necessary experimental, presentation and writing skills to achieve independent and professional career goals, and give rise to new leaders in this field of biophysics.******Significant impact: Our in-depth studies of IKr, IKs and INa gating will uncover molecular events underlying opening and closing of these biological gatekeepers, which in turn will improve our understanding of mechanisms that control cell electrical activity. This knowledge will be disseminated via publication in high-impact journals, invited conference, and additionally, this work is anticipated to lead to further NSERC-partnered opportunities, e.g. IRAP to more widely utilize information and generate further economic opportunities.**