MECHANISM OF REGULATION OF A GLOMERULAR K CHANNEL

肾小球系膜细胞（MC）是调节滤过率的收缩细胞 (GFR)通过调节肾小球毛细血管表面积。 比如血管 平滑肌、MC对体积和压力调节激素作出反应， 如血管紧张素II、心钠素（ANP）和一氧化氮 （否）。 因此，MC在血液调节中具有重要的生理作用 压力和液体和容量的动态平衡。 我们的实验室最近发现， BK通道在NO和ANP调节的舒张中起主要作用 肾小球系膜细胞 BK对血管舒张剂的反应是 双相，激活期约为10至30秒， 一个灭活阶段，可以持续60秒，然后返回到 基线水平。 在激活期，NO和ANP，通过刺激 鸟苷酸环化酶和产生cGMP，导致特定的cGMP激活 失活阶段可以通过以下方式抑制： 冈田酸（okadaicacid）或岩藻酸，蛋白磷酸酶1的抑制剂， 2A. 控制BK的调节和信号通路 活化/失活循环将是本提案的主要焦点。 膜片钳方法揭示了两个群体的BK：一个， 对磷酸酶和cGMP激活的蛋白激酶（BK 1）有反应， 对这些药物无反应（BK 2）。 这些结果表明 BK 2不具有与BK 1相同的磷酸化序列位点。 是 假设这些BK群体具有不同的信使RNA， 可以被转录调控。 初步电生理检查 研究表明，当MC暴露时，BK 2优先表达 24小时后再注射DB-cGMP 初步的分子研究表明， 至少有两种变体的信息被转录为hkD， 编码人类BK通道。 一种变体不包含替代品 外显子插入物（hmc 2），另一个包含29个AA插入物， 共有PKG磷酸化位点（hmc 1）。 我们建议使用分子 方法来确定哪一种变体的hkD是BK 1的等价物， PKG磷酸化位点，并定量差异表达。 hmc 1和hmc 2转录本在长期条件下的表达 MC暴露于cGMP。 cGMP在转录水平的调节 将提供额外的反馈机制，以适应慢性 暴露于一氧化氮或心房利钠肽。 这些结果 这将有助于更好地了解系膜K 通道和GFR在细胞和分子水平。

Mesangial cells (MC) are contractile cells that modulate filtration rate (GFR) by regulating glomerular capillary surface area. Like vascular smooth muscle, MC respond to volume and pressure regulatory hormones, such as angiotensin II, atrial natriuretic peptide (ANP) and nitric oxide (NO). Thus, MC have a critical physiological role in regulation of blood pressure and fluid and volume homeostasis. Our laboratory has recently discovered that large, calcium-activated K channels (BK) play a major role in both NO and ANP regulated relaxation of mesangial cells. The response of BK to vasorelaxing agents is biphasic, with an activation phase of approximately 10 to 30 seconds and an inactivation phase which can last for 60 seconds before returning to baseline levels. In the activation phase, NO and ANP, by stimulating guanylyl cyclase and generating cGMP, lead to specific cGMP-activated kinase stimulation of BK. The inactivation phase can be inhibited by okadaic acid or cantharidic acid, inhibitors of protein phosphatase 1 and 2A. The regulatory and signaling pathways controlling the BK activation/inactivation cycle will be a major focus of this proposal. The patch clamp method has revealed two populations of BK: one which responds to phosphatases and cGMP-activated protein kinase (BK1) and one that is unresponsive (BK2) to these agents. These results suggest that BK2 do not have the same phosphorylation sequence sites as BK1. It is postulated that these populations of BK have distinct messenger RNA which can be transcriptionally regulated. Preliminary electrophysiological studies show that BK2 are preferentially expressed when MC are exposed for 24 hours to DB-cGMP. Preliminary molecular studies show that there are at least two variants of message transcribed for hslo, the gene encoding human BK channels. One variant does not contain alternative exon inserts (hmc2) and another contains a 29 AA insert with two consensus PKG phosphorylation sites (hmc1). We propose to use molecular methods to establish which variant of hslo is the equivalent of BK1, the PKG phosphorylation site on BK1, and quantitate the differential expression of hmc1 and hmc2 transcripts under conditions of longterm exposure of MC to cGMP. Regulation by cGMP at the transcriptional level would provide an additional feedback mechanism to adapt to chronic exposure to nitric oxide or atrial natriuretic peptide. These results should lead to a better understanding of the regulation of mesangial K channels and GFR at the cellular and molecular levels.