鈣離子結合蛋白5在興奮性細胞的生理功能研究

Abstract

鈣離子結合蛋白 (Ca2+-binding protein 1-5, CaBP1-5) 是一群與calmodulin有高度相似性結構的蛋白質，目前僅知CaBP1及CaBP4會調控鈣離子通道的活性，而其它蛋白的功能則尚不清楚。本篇報告中，我們以PCR技術從大鼠 (Rattus norvegicus) 胚胎的大腦組織中選殖出CaBP5基因，再予以表現於兩類型興奮性細胞中，來探討其對刺激-分泌耦合機轉的調控。細胞螢光染色結果顯示，CaBP5-EYFP主要分布於細胞質中，有些則會集中於細胞核。利用細胞電生理技術，我們發現 CaBP5的大量表現會使神經細胞的鈣離子通道的開啟降低至較負電位，失去第一個 EF-hand正常功能的突變型 (5EF1) 則沒有作用。但在牛腎上腺嗜鉻細胞 (bovine chromaffin cell) 中，CaBP5對其鈣離子通道沒有同樣影響，藉由測量細胞膜電容的變化發現CaBP5的表現，也不會影響胞吐和胞吞作用。在牛腎上腺嗜鉻細胞中，CaBP5對鈣離子通道沒有同樣的作用，也沒有對胞吐 (exocytosis) 和胞吞作用 (endocytosis) 造成明顯的影響；另外，CaBP5對鈉離子電流沒有影響，但5EF1的表現增強了鈉離子電流。以上觀察顯示CaBP5可能參與神經細胞膜的鈣離子電流調控，而對鈉離子電流的影響不明顯；但在牛腎上腺嗜鉻細胞，5EF1增強了鈉離子電流的大小。

Ca2+ -binding proteins 1-5 (CaBP1-5) are a group of proteins with structure similar to calmodulin. CaBP1 and CaBP4 have been reported to modulate the Ca2+ currents and synaptic plasticity of neurons; however, functions of other proteins have not been extensively investigated. In this report, functions of CaBP5 in excitable cells were studied. CaBP5 was cloned from rat (Rattus norvegicus) embryonic brain tissue and inserted into pEYFP-N1 for expression. Normally it was localized in the cytosol, but it may concentrat in the nucleus in some cells. We applied whole-cell patch clamping technique to monitor the electrophysiological properties of excitable cells. The results show that CaBP5 shifted the activation of Ca2+ channels to hyperpolarized voltages in primary neuron culture, but mutant CaBP5 with the first nonfunctional EF-hand didn’t have the same effect. When CaBP5 was expressed in cultured bovine adrenal chromaffin cells, neither the Ca2+ currents nor the exocytosis was affected, but the Na+ currents were enhanced. These observations indicate that CaBP5 may be involved in regulation of Ca2+ channels in neurons but enhanced Na+ currents in chromaffin cells.