LENS CELL-TO-CELL CHANNEL PROTEIN .1. SELF-ASSEMBLY INTO LIPOSOMES AND PERMEABILITY REGULATION BY CALMODULIN
LENS CELL-TO-CELL CHANNEL PROTEIN .1. SELF-ASSEMBLY INTO LIPOSOMES AND PERMEABILITY REGULATION BY CALMODULIN
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DOI：
10.1007/bf01868696
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发表时间：
1985-01-01
影响因子：
2.4
通讯作者：
PERACCHIA, C
中科院分区：
文献类型：
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作者：
GIRSCH, SJ;PERACCHIA, C
透镜纤维通过含有28-kdalton蛋白（MIP 26）的通信连接点偶联，所述28-kdalton蛋白被认为是细胞-细胞通道的主要组分。为了研究这些通道的渗透性和调节，开发了一种体外系统，其中将从小牛透镜分离的MIP 26掺入脂质体中，并通过溶胀测定法对所得通道进行渗透性研究。使用超声处理/重悬方法制备脂质体囊泡。通过冷冻断裂监测掺入效率。将囊泡重悬于6%葡聚糖T-10中。试验缓冲液是相同的，除了蔗糖等渗取代T-10。掺入MIP 26的囊泡在等渗条件下溶胀，表明蔗糖进入（通过通道），随后是水以维持渗透平衡。在缺乏钙调素的情况下，钙离子对通道通透性没有影响。相反，用等摩尔量的MIP 26和CaM制备的囊泡在Ca离子的存在下不溶胀，表明通道可以关闭。加入EGTA [乙二醇双（β-氨基乙基醚）N，N，N "，N"-四乙酸]对这些囊泡重新启动溶胀-证明通道门控机制是可逆的。Mg~（2+）对两种囊泡均无影响。
Lens fibers are coupled by communicating junctions which contain a 28-kdalton protein (MIP26) believed to be the main component of the cell-to-cell channel. To study the permeability properties and regulation of these channels, an in vitro system was developed in which MIP26 isolated from calf lens is incorporated into liposomes and the resulting channels are studied spectrophotometrically by a swelling assay. Liposome vesicles were prepared using a sonication/resuspension method. Incorporation efficiency was monitored by freeze-fracture. Vesicles were resuspended in 6% Dextran T-10. Assay buffer was identical, except for isotonic substitution of sucrose for T-10. MIP26-incorporated vesicles swell under isotonic conditions indicating sucrose entry (via channels) followed by water to maintain osmotic balance. In the absence of calmodulin Ca ion has no effect on channel permeability. On the contrary, vesicles prepared with equimolar amounts of MIP26 and CaM do not swell in the presence of Ca ion, indicating that the channels can be closed. Addition of EGTA [ethylene glycol bis(.beta.-aminoethyl ether)N,N,N'',N''-tetraacetic acid] to these vesicles reinitiates swelling-evidence that the channel gating mechanism is reversible. Mg2+ has no effect on either type of vesicle.