NO RELATED MODULATION OF CARDIAC CALCIUM CHANNELS

描述(改编自申请人的摘要)：一般假设 要测试的是：1)NO和RSNOS/OONO-(示例性蛋白质硫醇 没有供体和氧化剂)可以调节L型钙 通过两种不同的机制：i)间接机制 涉及可溶鸟酰环化酶(NO)的激活；以及ii)直接 S-亚硝化(RSNOS)或氧化的机理 肌膜和/或细胞外临界硫醇基团的(RSNOS/OONO-) 位于L通道亚单位复合体中的可接近的“氧化还原开关” 或相关的调控分子(S)；2)特定的细胞机制 这些影响的基础可能在不同的心脏之间存在显著差异 肌细胞类型(工作肌细胞与起搏肌细胞)；以及3)细胞氧化还原 肌膜/细胞外氧化还原过程中临界硫醇的状态 开关是心脏和心脏功能调节的重要决定因素。 回答是“不”。这些问题将通过以下组合来解决 膜片钳技术(全细胞和单通道)研究自然 在雪貂用酶法分离的心肌细胞(心室、SA结节)和 分子生物学技术(免疫定位、原位杂交) 以确定组织、细胞和亚细胞之间的相互关系 一氧化氮和氧化还原相关调控的功能效应。

DESCRIPTION (adapted from the applicant's abstract): The general hypotheses to be tested are: 1) NO and RSNOs/OONO- (exemplary protein thiol NO+donating and oxidizing agents, respectively) can modulate L-type calcium current (IcaL) by two distinct mechanisms: i) an indirect mechanism involving activation of soluble guanylyl cyclase (NO); and ii) a direct mechanism involving either S-nitrosylation (RSNOs) or oxidation (RSNOs/OONO-) of critical thiol groups in sarcolemmal and/or extracellularly accessible "redox switches" located either in the L-channel subunit complex or an associated regulatory molecule(s); 2) Specific cellular mechanisms underlying these effects may vary significantly among different cardiac myocyte types (working versus pacemaking myocytes); and 3) Cellular redox state of critical thiols located in the sarcolemmal/ extracellular redox switches is an important determinant in both modulation of cardiac ICaL and in the response "NO". These issues will be addressed using a combination of patch clamp techniques (both whole cell and single channel) to study native IcaL in ferret enzymatically isolated myocytes (ventricle, SA node) and molecular biological techniques (immunolocalization, in situ hybridization) to determine the tissue, cellular and subcellular correlates underlying the functional effects of NO- and redox-related modulation of IcaL.