Roe M W, Worley J F, Mittal A A, Kuznetsov A, DasGupta S, Mertz R J, Witherspoon S M, Blair N, Lancaster M E, McIntyre M S, Shehee W R, Dukes I D, Philipson L H
Department of Medicine, University of Chicago, Chicago, Illinois 60637, USA.
J Biol Chem. 1996 Dec 13;271(50):32241-6. doi: 10.1074/jbc.271.50.32241.
Voltage-dependent delayed rectifier K+ channels regulate aspects of both stimulus-secretion and excitation-contraction coupling, but assigning specific roles to these channels has proved problematic. Using transgenically derived insulinoma cells (betaTC3-neo) and beta-cells purified from rodent pancreatic islets of Langerhans, we studied the expression and role of delayed rectifiers in glucose-stimulated insulin secretion. Using reverse-transcription polymerase chain reaction methods to amplify all known candidate delayed rectifier transcripts, the expression of the K+ channel gene Kv2.1 in betaTC3-neo insulinoma cells and purified rodent pancreatic beta-cells was detected and confirmed by immunoblotting in the insulinoma cells. betaTC3-neo cells were also found to express a related K+ channel, Kv3.2. Whole-cell patch clamp demonstrated the presence of delayed rectifier K+ currents inhibited by tetraethylammonium (TEA) and 4-aminopyridine, with similar Kd values to that of Kv2.1, correlating delayed rectifier gene expression with the K+ currents. The effect of these blockers on intracellular Ca2+ concentration ([Ca2+]i) was studied with fura-2 microspectrofluorimetry and imaging techniques. In the absence of glucose, exposure to TEA (1-20 mM) had minimal effects on betaTC3-neo or rodent islet [Ca2+]i, but in the presence of glucose, TEA activated large amplitude [Ca2+]i oscillations. In the insulinoma cells the TEA-induced [Ca2+]i oscillations were driven by synchronous oscillations in membrane potential, resulting in a 4-fold potentiation of insulin secretion. Activation of specific delayed rectifier K+ channels can therefore suppress stimulus-secretion coupling by damping oscillations in membrane potential and [Ca2+]i and thereby regulate secretion. These studies implicate previously uncharacterized beta-cell delayed rectifier K+ channels in the regulation of membrane repolarization, [Ca2+]i, and insulin secretion.
电压依赖性延迟整流钾通道调节刺激-分泌偶联和兴奋-收缩偶联的多个方面，但确定这些通道的具体作用已被证明存在问题。利用转基因衍生的胰岛素瘤细胞（βTC3-neo）和从啮齿动物胰岛中纯化的β细胞，我们研究了延迟整流钾通道在葡萄糖刺激的胰岛素分泌中的表达和作用。使用逆转录聚合酶链反应方法扩增所有已知的候选延迟整流钾通道转录本，通过免疫印迹在胰岛素瘤细胞中检测并证实了钾通道基因Kv2.1在βTC3-neo胰岛素瘤细胞和纯化的啮齿动物胰腺β细胞中的表达。还发现βTC3-neo细胞表达相关的钾通道Kv3.2。全细胞膜片钳显示存在被四乙铵（TEA）和4-氨基吡啶抑制的延迟整流钾电流，其解离常数（Kd）值与Kv2.1相似，将延迟整流钾通道基因表达与钾电流相关联。用fura-2显微荧光分光光度法和成像技术研究了这些阻滞剂对细胞内钙离子浓度（[Ca2+]i）的影响。在无葡萄糖的情况下，暴露于TEA（1-20 mM）对βTC3-neo或啮齿动物胰岛的[Ca2+]i影响最小，但在有葡萄糖的情况下，TEA激活了大幅度的[Ca2+]i振荡。在胰岛素瘤细胞中，TEA诱导的[Ca2+]i振荡由膜电位的同步振荡驱动，导致胰岛素分泌增强4倍。因此，激活特定的延迟整流钾通道可通过抑制膜电位和[Ca2+]i的振荡来抑制刺激-分泌偶联，从而调节分泌。这些研究表明，以前未被表征的β细胞延迟整流钾通道参与膜复极化、[Ca2+]i和胰岛素分泌的调节。
