Mechanism of Regulation of a Glomerular K Channel

心钠素(ANP)通过增加肾小球滤过率和钠排泄来响应血管容量的增加。肾小球血流动力学改变的部分机制是，在肾小球系膜细胞(MC)中，ANP刺激cGMP-PKG通路，激活大的钙激活的K通道(BK)，使膜电位超极化，使MC对收缩激动剂反应减弱。PKG-1似乎特异性地(不同于PKA和PKC)和直接激活MC和血管平滑肌中的BK。然而，在大脑和其他组织中，BK受到PKA的调节，或间接受到PKG的调节，PKG激活一种蛋白磷酸酶。BK由一个类似于其他电压门控K通道的保守跨膜特性的Alpha亚基(由Slopoke基因编码；hSLO-Alpha)和一个附属的Beta亚基组成，其结构因起源的组织而有很大不同。有证据表明，β亚基可以解释许多组织依赖的钙敏感性差异以及BK的其他生物物理和调节特性。最近开发的Mbeta1基因敲除小鼠的出现使我们能够确定BK-β是否对BK对PKG的敏感性起重要作用，并在调节系膜张力和GFR方面发挥重要作用。这些小鼠有明显的高血压，这与钙和血管平滑肌和肾小球系膜细胞ANP/cGMP/PKG调节BK功能障碍的观点一致。在功能和结构上，小鼠BK-β(Mbeta1)与人类Bkbeta(Hbeta2)非常相似。我们的初步数据表明：1.Hbeta2和Mbeta1在HEK293细胞中与hSlo-α共表达时，使BK对钙敏感，并通过cGMP途径激活。2.β-反义寡核苷酸使系膜BK对HEK293中hSlo-α的钙敏感性增强。3.Western印迹分析表明，ANPO可导致PKG1转位到质膜上。4.扩容Mbeta1基因敲除小鼠的GFR和Na排泄率均低于野生型对照小鼠的40%。因此，尽管hSlo-α亚基被PKG磷酸化，但β亚基是ANP和cGMP激活BK所必需的。这一途径在一定程度上导致了对容量负荷的反应，导致GFR升高。以下具体目标将利用体外(细胞培养)和体内-(β基因敲除)实验模型；#1.确定Hbeta2是否通过cGMP激活的激酶1(PGK1)对BK进行调节。#2.确定PKG磷酸化BK的机制。#3.确定BK-β亚基在ANP-和cGMP-激活肾小球系膜细胞BK中的生理意义。通过依赖cGMP的途径调节BK可能是维持血管张力和肾脏容量平衡的关键。最终，在分子水平上深入了解BK的调节机制，可以更好地管理某些形式的高血压。

Atrial natriuretic peptide (ANP) responds to an elevated vascular volume by signaling an increase in glomerular filtration rate and Na excretion. The mechanism for the altered glomerular hemodynamics is partly due to the fact that, in glomerular mesangial cells (MC), ANP stimulates the cGMP-PKG pathway which activates large, Ca-activated K channels (BK) and hyperpolarizes the membrane potential rendering MC hyporesponsive to contractile agonists. PKG-1 appears to specifically (distinct from PKA and PKC) and directly activate BK in MC and vascular smooth muscle. However, in brain and other tissues, BK are regulated by PKA or indirectly by PKG which activates a protein phosphatase. BK are comprised of an alpha subunit (which is encoded by the slopoke gene; hSLO-alpha) with conserved membrane spanning properties similar to other voltage-gated K channels and an accessory beta sub7unit with a structure that differs substantially depending on the tissue of origin. Evidence shows that the beta subunit can account for many of the tissue-dependent differences in Ca sensitivity and other biophysical and regulatory properties of BK. The advent of a recently developed Mbeta1 knockout mouse permits us to determine if BK-beta is important for the sensitivity of BK to PKG with an important role to modulate mesangial tone and GFR.. These mice are significantly hypertensive, consistent with the view of dysfunctional regulation of BK by Ca and ANP/cGMP/PKG in vascular smooth muscle and glomerular mesangial cells. Functionally and structurally, the mouse BK-beta (Mbeta1) is very similar to the human Bkbeta (Hbeta2). Our preliminary data shows: 1. Hbeta2 and Mbeta1, when co-expressed with hSlo-alpha in HEK293 cells, confers the sensitivity of BK to Ca and activation by the cGMP pathway. 2. Beta-antisense oligonucleotides render the Ca sensitivity of mesangial BK to that found for the hSlo-alpha expressed in HEK293. 3. Western blot analysis shows that ANPO causes the translocation of PKG1 to the plasma membrane. 4. The GFR and Na excretion rates of volume expanded Mbeta1 knockout mice are less than 40% of the values for wild type controls. Therefore, it is hypothesized that, although the hSlo-alpha subunit is phosphorylated by PKG, the beta subunit is necessary for the ANP- and cGMP-evoked activation of BK. This pathway partly contributes to an elevated GFR in response to a volume load. The following Specific Aims will utilize both in vitro (cell cultures) and in vivo -(beta knockout) experimental models; #1. Determine if Hbeta2 confers regulation of BK by cGMP activated kinase 1 (PGK1). #2. Determine the mechanism of phosphorylating BK by PKG. #3. Determine the physiological significance of the BK-beta subunit in ANP-and cGMP-evoke activation of BK in glomerular mesangial cells. Regulation of BK via the cGMP-dependent pathway may be critical for vascular tone and renal maintenance of volume balance. Ultimately, better management of some forms of hypertension could develop from an in-depth understanding of BK regulatory mechanism at the molecular level.