NO RELATED MODULATION OF CARDIAC CALCIUM CHANNELS

描述（改编自申请人的摘要）：一般假设 待测试的是：1) NO 和 RSNOs/OONO-（示例性蛋白质硫醇 分别提供 NO 和氧化剂）可以调节 L 型钙 电流 (IcaL) 通过两种不同的机制： i) 间接机制 涉及可溶性鸟苷酸环化酶（NO）的激活； ii) 直接 涉及 S-亚硝基化 (RSNO) 或氧化的机制 肌膜和/或细胞外关键硫醇基团的 (RSNOs/OONO-) 可访问的“氧化还原开关”位于 L 通道亚基复合体中 或相关的调节分子； 2）特定的细胞机制 这些影响的背后可能在不同的心脏之间存在显着差异 肌细胞类型（工作肌细胞与起搏肌细胞）； 3) 细胞氧化还原 位于肌膜/细胞外氧化还原的关键硫醇状态 开关是心脏 ICaL 调节和调节的重要决定因素 在回答“否”中。 这些问题将通过以下措施的组合来解决： 膜片钳技术（全细胞和单通道）来研究天然 雪貂酶法分离的肌细胞（心室、SA 结）中的 IcaL 和 分子生物学技术（免疫定位、原位杂交） 确定潜在的组织、细胞和亚细胞相关性 IcaL 的 NO 和氧化还原相关调节的功能效应。

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.