Szamosfalvi Balázs, Cortes Pedro, Alviani Rebecca, Asano Kenichiro, Riser Bruce L, Zasuwa Gary, Yee Jerry
Department of Medicine, Division of Nephrology and Hypertension, Henry Ford Hospital, Detroit, Michigan 48202, USA.
Kidney Int. 2002 May;61(5):1739-49. doi: 10.1046/j.1523-1755.2002.00302.x.
Sulfonylurea agents exert their physiological effects in many cell types via binding to specific sulfonylurea receptors (SUR). SUR couple to inwardly-rectifying K+ channel (Kir6.x) to form tetradimeric ATP-sensitive K+ channels (KATP). The SUR subunits confer ATP-sensitivity on KATP and also provide the binding sites for sulfonylureas and other pharmacological agents. Our previous work demonstrated that the exposure of mesangial cells (MC) to sulfonylureas generated profound effects on MC glucose uptake and matrix metabolism and induced heightened cell contractility in association with Ca2+ transients. Because these responses likely resulted from the binding of sulfonylurea to a mesangial SUR2, we subsequently documented [3H]-glibenclamide binding to MC and the gene expression of several mesangial SUR2 transcripts. From these data, we inferred that MC expressed the components of a mesangial KATP and sought to establish their presence in primary MC.
To obtain mesangial SUR2 cDNA sequences, rapid amplification of cDNA ends (RACE) was utilized. DNA sequences were established by the fluorescent dye termination method. Gene expression of mesangial SUR2 and Kir6.1/2 was examined by reverse transcription polymerase chain reaction (RT-PCR) and Northern analysis. SUR2 proteins were identified by immunoblotting of mesangial proteins from membrane-enriched fractions with polyclonal antiserum directed against SUR2.
RACE cloning yielded two mesangial SUR2 cDNAs of 4.8 and 6.7 kbp whose open reading frames translated proteins of 964 and 1535 aa, respectively. Using probes specific to each cDNA, the presence of a unique, 5.5 kbp serum-regulated mesangial SUR2 splice variant was established. The sequence of this mesangial SUR2 (mcSUR2B) shares identity with the recently cloned rat SUR2B (rSUR2B), but, in comparison to rSUR2B, is truncated by 12 exons at the N-terminus where it contains a unique insert of 16 aa. Immunoblotting studies with anti-SUR2 antiserum demonstrated SUR2 proteins of 108 and 170 kD in membrane-enriched fractions of MC protein extracts. Complementary studies showed abundant gene expression of Kir6.1, thereby establishing gene expression of both components of KATP.
Based upon analogy to vascular smooth muscle cells (VSMC), there are at least two putative mesangial KATP that most likely represent hetero-octamers, comprised of either rSUR2B or mcSUR2 in complex with Kir6.1. Our results define the mesangial SUR2B as the possible first link in a chain of cellular events that culminates in MC contraction and altered extracellular matrix metabolism following exposure to sulfonylureas. In addition, our results serve as the basis for the future elucidation of the electrophysiologic characteristics of the mesangial KATP and the study of endogenous regulators of mesangial cell contractility.
磺脲类药物通过与特定的磺脲类受体（SUR）结合，在多种细胞类型中发挥其生理作用。SUR与内向整流钾通道（Kir6.x）偶联，形成四聚体ATP敏感性钾通道（KATP）。SUR亚基赋予KATP对ATP的敏感性，同时也为磺脲类药物和其他药理剂提供结合位点。我们之前的研究表明，将系膜细胞（MC）暴露于磺脲类药物会对MC葡萄糖摄取和基质代谢产生深远影响，并与Ca2+瞬变相关地诱导细胞收缩增强。由于这些反应可能是磺脲类药物与系膜SUR2结合的结果，我们随后记录了[3H] - 格列本脲与MC的结合以及几种系膜SUR2转录本的基因表达。从这些数据中，我们推断MC表达了系膜KATP的组成成分，并试图在原代MC中确定它们的存在。
为了获得系膜SUR2 cDNA序列，采用了cDNA末端快速扩增（RACE）技术。通过荧光染料终止法确定DNA序列。通过逆转录聚合酶链反应（RT-PCR）和Northern分析检测系膜SUR2和Kir6.1/2的基因表达。用针对SUR2的多克隆抗血清对富含膜的系膜蛋白提取物进行免疫印迹，鉴定SUR2蛋白。
RACE克隆产生了两个系膜SUR2 cDNA，大小分别为4.8和6.7 kbp，其开放阅读框分别编码964和1535个氨基酸的蛋白质。使用针对每个cDNA的特异性探针，确定了一种独特的、5.5 kbp血清调节的系膜SUR2剪接变体的存在。这种系膜SUR2（mcSUR2B）的序列与最近克隆的大鼠SUR2B（rSUR2B）具有同源性，但与rSUR2B相比，在N端被截断了12个外显子，其中包含一个16个氨基酸的独特插入序列。用抗SUR2抗血清进行的免疫印迹研究表明，在MC蛋白提取物的富含膜的部分中存在108和170 kD的SUR2蛋白。补充研究显示Kir6.1基因表达丰富，从而确定了KATP两个组成成分的基因表达。
基于与血管平滑肌细胞（VSMC）的类比，至少有两种假定的系膜KATP，最有可能代表由rSUR2B或mcSUR2与Kir6.1形成复合物组成的异源八聚体。我们的结果将系膜SUR2B定义为细胞事件链中可能的第一个环节，该事件链在暴露于磺脲类药物后最终导致MC收缩和细胞外基质代谢改变。此外，我们的结果为未来阐明系膜KATP的电生理特性以及研究系膜细胞收缩的内源性调节因子奠定了基础。
