Departamento de Farmacobiología, Centro de Investigación y de Estudios Avanzados del IPN (Cinvestav-IPN), Mexico Distrito Federal, Mexico.
Biochem Biophys Res Commun. 2012 Feb 10;418(2):420-5. doi: 10.1016/j.bbrc.2012.01.048. Epub 2012 Jan 18.
Hyperglycemia alters the tight control of intracellular calcium dynamics in retinal cells and may lead to the development of diabetic retinopathy. The potassium channel interacting protein 3 (KChIP3) also known as DREAM (Downstream Regulatory Element Antagonist Modulator) or calsenilin (KChIP3/DREAM/calsenilin), a member of the neuronal calcium sensor protein family, is expressed in Müller glial cells and upregulated under high glucose experimental culture conditions. Here, we analyzed the expression and function of KChIP3 in the retina of streptozotocin induced diabetic Long Evans rats by immunofluorescence confocal microscopy, western blot, co-immunoprecipitation, whole cell patch clamp recording on isolated cells and KChIP3 gene silencing by RNA interference. Three weeks after streptozotocin application, KChIP3 was increased throughout the different retinal layers and this process was not linked to augmented apoptosis. KChIP3 co-immunoprecipitated with voltage gated K(+) channels of the K(V)4.2-4.3 subtype in retinal extracts from control and hyperglycemic rats. Electrophysiological analysis showed that control cells did not express A type (K(V)4-mediated) K(+) currents but most of the cells from streptozotocin treated retinas displayed macroscopic currents with an inactivating component sensitive to 4-AP, suggesting the persistence of the A type currents at early times after treatment. siRNA analysis in Müller cells cultures grown under high glucose experimental conditions corroborated that, when the expression of KChIP3 is 50% reduced, the number of cells expressing A type currents decreases significantly. Together these data suggest an altered expression and function of KChIP3 after streptozotocin induced hyperglycemia that might help explain some pathological alterations in early diabetic retinopathy.
高血糖会改变视网膜细胞内钙离子动力学的紧密控制，可能导致糖尿病视网膜病变的发展。钾通道相互作用蛋白 3（KChIP3）也称为 DREAM（下游调节元件拮抗剂调节剂）或钙敏蛋白（KChIP3/DREAM/calsenilin），是神经元钙传感器蛋白家族的一员，在 Muller 胶质细胞中表达，并在高糖实验培养条件下上调。在这里，我们通过免疫荧光共聚焦显微镜、western blot、共免疫沉淀、分离细胞的全细胞膜片钳记录和 RNA 干扰沉默 KChIP3 基因，分析了链脲佐菌素诱导的糖尿病 Long Evans 大鼠视网膜中 KChIP3 的表达和功能。链脲佐菌素给药 3 周后，KChIP3 在不同的视网膜层中增加，这个过程与细胞凋亡增加无关。KChIP3 与控制和高血糖大鼠视网膜提取物中的电压门控 K(+)通道的 K(V)4.2-4.3 亚基共免疫沉淀。电生理分析表明，对照细胞不表达 A 型（K(V)4 介导）K(+)电流，但大多数来自链脲佐菌素处理视网膜的细胞显示出具有失活成分的宏观电流，对 4-AP 敏感，提示在治疗后早期 A 型电流持续存在。在高糖实验条件下培养的 Muller 细胞培养物中的 siRNA 分析证实，当 KChIP3 的表达减少 50%时，表达 A 型电流的细胞数量显著减少。这些数据表明，链脲佐菌素诱导高血糖后 KChIP3 的表达和功能发生改变，这可能有助于解释早期糖尿病视网膜病变中的一些病理改变。
