RECONSTITUTION OF EXPRESSED K-CA CHANNELS FROM XENOPUS-OOCYTES TO LIPID BILAYERS
RECONSTITUTION OF EXPRESSED K-CA CHANNELS FROM XENOPUS-OOCYTES TO LIPID BILAYERS
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DOI：
10.1016/s0006-3495(94)80883-5
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发表时间：
1994-04-01
影响因子：
3.4
通讯作者：
TORO, L
中科院分区：
文献类型：
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作者：
PEREZ, G;LAGRUTTA, A;TORO, L
大电导钙激活钾(K-Ca)通道从天然细胞膜到平面脂质双层的重构为研究单通道的性质，包括离子传导、药理学和门控提供了一个强有力的方法。最近，来源于果蝇Slowpoke(Slo)基因的钾-钙通道已被克隆并在非洲爪哇卵母细胞中异源表达。在这篇报道中，我们描述了克隆和表达的非洲爪哇卵母细胞膜上的Slo K-Ca通道在脂双层中的重组。重组通道具有天然钾-钙通道的功能特性。它们在对称的钾(250 Mm)中具有约260 ps的平均单位电导，并且对电压和钙敏感。在50µM钙离子下，它们的半激活电位接近-20 mV，对钙的亲和力在微摩尔范围内。重组的Slo钾钙通道对外源性赤潮毒素(40-500 nM)不敏感，对外源性四乙基铵(K-D=158mM，0 mV)和内Ba2+(40 mV，K-D=76mM)的微摩尔浓度敏感。此外，在40 mV时，它们可被内部施加的“球状”灭活肽(K-D=480µM)所阻断。这些结果表明，在非洲爪哇卵母细胞中表达的克隆K-Ca通道可以很容易地被结合到脂质双层中，在可控的内外实验条件下，可以进行详细的机制研究。
Reconstitution of large conductance calcium-activated potassium (K-Ca) channels from native cell membranes into planar lipid bilayers provides a powerful method to study single channel properties, including ion conduction, pharmacology, and gating. Recently, K-Ca channels derived from the Drosophila Slowpoke (Slo) gene have been cloned and heterologously expressed in Xenopus oocytes. In this report, we describe the reconstitution of cloned and expressed Slo K-Ca channels from Xenopus oocyte membranes into lipid bilayers. The reconstituted channels demonstrate functional properties characteristic of native K-Ca channels. They possess a mean unitary conductance of approximate to 260 pS in symmetrical potassium (250 mM), and they are voltage- and calcium-sensitive. At 50 mu M Ca2+, their half-activation potential was near -20 mV; and their affinity for calcium is in the micromolar range. Reconstituted Slo K-Ca channels were insensitive to external charybdotoxin (40-500 nM) and sensitive to micromolar concentrations of external tetraethylammonium (K-D = 158 mu M, at 0 mV) and internal Ba2+ (K-D = 76 mu M, at 40 mV). In addition, they were blocked by internally applied ''ball'' inactivating peptide (K-D = 480 mu M, at 40 mV). These results demonstrate that cloned K-Ca channels expressed in Xenopus oocytes can be readily incorporated into lipid bilayers where detailed mechanistic studies can be performed under controlled internal and external experimental conditions.