SLIP-MODE CONDUCTANCE IN HEART

PI在心脏细胞中发现了通过TTX敏感性Na+通道的Ca 2+进入[154]，称为“滑动模式电导”。通过蛋白激酶A（PKA）激活，Ca 2+相对于Na+的渗透性（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HEK 293细胞中证实了滑动模式电导，但β亚基必须共表达[38]，并且来自脑或骨骼肌的α亚基都不能取代心脏（见初步结果）。该建议将检查心脏Na+通道的滑动模式电导，量化其生理并确定其分子基础。计划的实验将测试的假设，即滑模式电导提供了显着的Ca 2+流入生理条件下。使用共聚焦Ca 2+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HEK 293细胞中检验假设。（一）.心脏中由滑移模式电导引起的[Ca 2 +]i瞬变的比例是多少？将对滑动模式电导进行定量检查，以确定Ca 2+内流，并确定其如何受到心脏中Ca 2+信号传导生理调节剂的影响（例如pH值、SR蛋白激酶C中的Ca 2+量）。将比较大鼠、小鼠和人心脏的心肌细胞。来自转基因和基因敲除小鼠的心脏细胞将使研究A-激酶锚定蛋白（AKAP）和β亚基在滑动模式电导。（二）、为什么心脏Na+通道是唯一滥用激活滑动模式电导？α亚基的心脏-骨骼肌嵌合体将用于确定心脏α亚基的哪些部分是滑移模式电导所必需的。影响通道动力学（例如快速失活）的突变将用于检查通道门控对滑动模式电导的影响。这些实验将拓宽我们对心脏中Ca 2+信号的理解。我们应该确定心脏Na+通道的Ca 2+渗透的分子基础，并表征其生理重要性。因此，计划的工作支持PI的长期计划，以扩大我们对心脏功能的理解。

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.