AKAP150-BK通道复合体对糖尿病冠状动脉血管功能调节的机制研究

Diabetes has become a disease of epidemic proportions and cardiovascular diseases are the most important causes of morbidity and mortality in diabetic patients. The large conductance calcium-activated potassium(BK) channels are proteins that regulate vessel tone and are critical determinants of blood flow in vital organs. Dysfunctional mechanisms of BK channel in diabetic coronary arties are yet to be completely described. The A-kinase anchor proteins (AKAPs) are a group of structurally diverse proteins, which have the common function of binding to the regulatory subunit of protein kinase (PKA) and confining the holoenzyme to discrete locations within the cell, as well as binding specific targeting of substrates to be regulated by phosphodiesterases(PDE) which break down cAMP, phosphatases which dephosphorylate downsteam PKA targets, and PKC. AKAP150 not only act as a signaling hub that assembles signaling proteins on a scaffold, but also bind ion channels. We have confirmed a physiological interaction between AKAP150 and BK channel in membrane. BK channels in coronary arterial smooth muscle cells from diabetic animals have a reduced sensitivity to calcium- and voltage-dependent activation. Several abnormal expression of protein kinase regulated by AKAP150 exacerbate the BK channel function. The hypotheses to be tested are abnormal vascular BK channel function in diabetes due to AKAP150 modulation. These studies will be performed using in vitro and in vivo models of diabetes. Whole cell and single channel patch clamp techniques, molecular approaches, and calcium imagine will be employed to determine the regulation of AKAP150 in diabetes on BK channel function and degradation. The goal of this project is to further delineate the molecular mechanisms through which AKAP150 modulate BK channels in diabetes. BK channels regulated by AKAP 150 suggest a possible target for management of occluded coronary artery diseases. The results may provide important novel insights into the mechanisms through which diabetes leads to abnormal function in the cardiovascular system.

目前糖尿病已成为心血管疾病的最主要的危险因素之一，可能与信号转导介导离子通道异常有关,但其机制尚不清楚。大电导钙离子激活的钾通道（BK通道）是冠状动脉平滑肌细胞的复极主要影响因素之一。A型激酶锚定蛋白150（AKAP150）在靶向定位和调节PKA介导的磷酸化,调控细胞多种功能,和信号传递中起着重要作用。当AKAP150表达异常或BK通道表达下降，都会导致冠状动脉收缩增强，故AKAP150和BK 通道信号转导异常可能是心肌梗死发生的重要原因。本研究在申请者前期工作基础上，采用膜片钳实验、分子生物学实验和细胞内钙离子浓度荧光测定等技术，研究KAP150信号功能及其介导的信号转导通路对糖尿病冠状动脉血管张力的调节作用，旨在阐明AKAP150对BK通道功能调节及糖尿病相关血管功能受损的分子机制，研究结果对指导筛选糖尿病冠状动脉阻塞性病变治疗的新靶点具有重要的理论意义和实用价值。

糖尿病是严重危害人类健康的慢性疾病，其中成人糖尿病发病率高达8%，已成为我国急性心肌梗死最主要的危险因素之一。糖尿病冠脉血管功能恶化所致心肌缺血，引起心肌梗死等严重心血管事件，作为血管平滑肌细胞上主要钾通道是BK通道,当细胞内钙离子浓度升高时，BK通道激活，钾离子外流，产生超极化，抑制钙离子通道开放，使钙离子内流减少，从而使血管扩张。因此，研究糖尿病冠状动脉BK通道病变发生机制并作为治疗靶点具有重要意义。A型激酶锚定蛋白150(AKAP150)在靶向定位和调节PKA介导的磷酸化,调控细胞多种功能，和信号传递中起着重要作用。BK通道表达下降，导致冠状动脉收缩增强，故AKAP150和BK通道信号转导异常是心肌梗死发生的重要原因。本研究采用膜片钳实验、分子生物学和细胞内钙离子浓度荧光测定等技术，研究KAP150-BK通道信号功能及其对糖尿病冠状动脉血管张力的调节作用。BK通道由a和beta亚基组成，beta亚基发挥调节功能。正常组BK通道电流密度为262.3±20.6pA/pF，糖尿病状态下，BK通道电流密度下降为67.2±13.1pA/pF，BK通道电流下降有显著差异；单通道记录发现BK通道开放概率，正常状态下，特异性beta激活剂可明显增加BK通道开放概率（由0.14±0.11增加到0.43±0.15），而糖尿病，beta亚基激活剂未见明显增加BK通道激活（由0.13±0.09增加到0.23±0.12），且BK-beta亚基对血管活性药物反应下降。我们进一步发现糖尿病状态下，BK通道beta表达下降，而BK通道a蛋白表达未见明显改变，且糖尿病时，mRNA未见明显下降，提示BK通道beta下降，为蛋白降解增加。基于免疫共沉淀显示AKAP与BK通道由功能结构基础的关联，在AKAP基因敲除动物模型，糖尿病状态下，BK通道未见明显损伤。综上，糖尿病血管BK通道功能受损，依赖于AKAP介导BK-beta亚基下调所致。研究结果对指导筛选糖尿病冠状动脉病变治疗的新靶点具有重要的理论意义和实用价值。