SLIP-MODE CONDUCTANCE IN HEART

Ca2+通过ttx敏感的Na+通道进入心脏细胞是由PI发现的[154]，称为“滑模电导”。在蛋白激酶A （PKA）的激活下，Ca2+相对于Na+的通透性（P/Ca/P/Na）从接近零增加到约1.0。虽然在表达心脏α亚基的HEK293细胞中证实了滑模传导，但β亚基必须与[38]共表达，并且来自脑和骨骼肌的α亚基都不能取代心脏（见初步结果）。本研究将研究心脏Na+通道的滑模电导，量化其生理特性并确定其分子基础。计划中的实验将测试滑模电导在生理条件下提供显著Ca2+内流的假设。利用共聚焦Ca2+成像和膜片钳方法，PI将通过解决两个实验问题，在心肌细胞和表达Na+通道的HEK293细胞中测试这一假设。（1）. 心脏中瞬态的[Ca2+]有多少是由于滑移模电导引起的？将定量检查滑模电导，以确定Ca2+内流，并确定它是如何受到心脏中Ca2+信号的生理调节剂的影响（例如pH值，SR蛋白激酶C中Ca2+的量）。将比较大鼠、小鼠和人类心脏的心肌细胞。来自转基因和基因敲除小鼠的心脏细胞将使a激酶锚定蛋白（AKAPs）和β亚基在滑模电导中的研究成为可能。（2）. 为什么心脏Na+通道会被滥用来激活滑模电导？α亚基的心脏-骨骼肌嵌合体将用于确定心脏α亚基的哪一部分是滑模传导所必需的。影响通道动力学的突变（例如快速失活）将用于检查通道门控对滑模电导的影响。提出的实验应该扩大我们对心脏Ca2+信号的理解。我们应该确定Ca2+渗透心脏Na+通道的分子基础，并表征其生理重要性。计划中的工作因此支持了PI扩大我们对心脏功能的理解的长期计划。

Ca2+ entry though TTX-sensitive Na+ channels was discovered by the PI in heart cells [154] and called "slip-mode conductance". Activated by protein kinase A (PKA), Ca2+ permeability relative to Na+ (P/Ca/P/Na) increased from near zero to approximately 1.0. While slip-mode conductance was confirmed in HEK293 cells expressing cardiac alpha subunits, beta subunits had to be co-expressed [38] and alpha subunits from neither brain nor skeletal muscle could replace cardiac (see Preliminary Results). This proposal will examine slip-mode conductance of the cardiac Na+ channel, quantify its physiological and determine its molecular basis. The planned experiments will test the hypothesis that slip-mode conductance provides significant Ca2+ influx under physiological conditions. Using confocal Ca2+ imaging and patch clamp methods, the PI will test the hypothesis in cardiac myocytes and in HEK293 cells expressing Na+ channels by addressing two experimental questions. (1). What fraction of the [Ca2+]i transient in heart that is due to slip mode conductance? Slip-mode conductance will be examined quantitatively to determine Ca2+ influx and establish how it is affected by physiologic modulators of Ca2+ signaling in heart (e.g. pH, the amount of Ca2+ in the SR protein kinase C). Cardiac myocytes from rat, mouse and human hearts will be compared. Heart cells from transgenic and knockout mice will enable the investigation of A-kinase anchoring proteins (AKAPs) and the beta subunits in slip-mode conductance. (2). Why is the cardiac Na+ channel uniquely abuse to activate slip-mode conductance? Cardiac-skeletal muscle chimeras of the alpha subunit will be used to determine what part(s) of the cardiac alpha subunit is(are) necessary for slip-mode conductance. Mutations that affect channel kinetics (e.g. fast inactivation) will be used to examine the effects of channel gating on slip-mode conductance. The proposed experiments should broaden our understanding of Ca2+ signaling in heart. We should identify the molecular basis of Ca2+ permeation of cardiac Na+ channels and characterize its physiological importance. The planned work thus supports the PI's long-term plan to broaden our understanding of heart function.