SLIP-MODE CONDUCTANCE IN HEART

PI在心肌细胞[154]中发现了通过TTX敏感的Na+通道的Ca~(2+)内流，被称为“滑模电导”。在蛋白激酶A(PKA)的激活下，Ca~(2+)相对于Na~+的通透性(P/Ca/P/Na)从接近零增加到大约1.0。虽然在表达心脏α亚基的HEK293细胞中证实了滑移模式电导，但β亚基必须共表达[38]，来自大脑和骨骼肌的α亚基都不能取代心脏(见初步结果)。这项建议将检测心脏Na+通道的滑模电导，量化其生理作用，并确定其分子基础。计划中的实验将检验滑移模电导在生理条件下提供显著的钙离子内流的假设。使用共聚焦钙成像和膜片钳方法，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细胞中验证这一假设。(1)。在心脏中，有多大比例的[Ca~(2+)]i瞬变是由滑模电导引起的？将定量检测滑模电导，以确定钙内流，并确定它如何受到心脏中钙信号的生理调节因素(例如，pH，SR蛋白激酶C中的钙含量)的影响。我们将对来自大鼠、小鼠和人类心脏的心肌细胞进行比较。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)和滑模电导中的β亚基成为可能。(2)。为什么心脏Na+通道独特地被滥用来激活滑模电导？心肌-骨骼肌α亚基的嵌合体将被用来确定心肌α亚基的哪一部分(S)是滑模电导所必需的。影响通道动力学的突变(例如，快速失活)将被用来检查通道选通对滑模电导的影响。拟议中的实验应该会拓宽我们对心脏中钙信号的理解。我们应该确定心肌Na+通道钙离子渗透的分子基础，并表征其生理意义。因此，计划中的工作支持了PI的长期计划，以扩大我们对心脏功能的了解。

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.