斑馬魚仔魚側線毛細胞上的TRP通道之研究
Abstract
魚類的側線系統是由毛細胞(hair cells)和支持細胞組成的機械性感覺器官，負責感覺外在水體的流動。毛細胞的纖毛束在受到機械性刺激時會開啟非選擇性的陽離子通道，對於鈣離子有較高的選擇性。流入細胞的鈣離子會引發胞內的訊息傳導，使毛細胞傳出感覺訊號。
然而脊椎動物內耳與魚類側線毛細胞上的機械性感覺通道的分子組成仍具爭議性，目前主要認為transient receptor potential (TRP) channels家族的蛋白較可能是參與的分子，但證據仍有所欠缺。本研究利用斑馬魚胚胎為模式動物，進一步研究側線毛細胞上參與機械性感覺的分子。我們利用掃描式離子選擇性電極技術scanning ion selective electrode technique (SIET)在活體胚胎毛細胞上測得鈣離子流入，並證實這個通道會被neomycin、gentamicin、La3+及BAPTA所抑制。利用morphlino gene knockdown技術將TRPA1、TRPN1及TRPV4表現弱化，發現TRPA1及TRPN1弱化後毛細胞上鈣離子流入顯著降低。免疫染色也顯示這三個蛋白質在纖毛上表現，並且在基因弱化後，表現量明顯下降。本研究結果顯示，斑馬魚側線毛細胞上的TRPA1與TRPN1通道扮演著機械性感覺的角色。
The lateral-line, a mechanosensory organ of fish, is composed of hair cells and supporting cells. The mechanosensory channel on the hair bundles of hair cells is a non-selective cation channel with higher Ca2+ permeability. Mechanical deflection of the sensory hair bundles causes Ca2+ influx via the channel and further induces sensory transduction. Recently, a group of Ca2+ channels, the TRP (transient receptor potential) channels, was suggested to be the mechanosensory channel on hair cells. However, the molecular identity of the channel is still controversial. In this study, we used zebrafish as an in vivo model to investigate the mechanosensory channels on the lateral-line hair cell of newly hatch larvae. We used a non-invasive Scanning Ion-Selective Electrode Technique (SIET) to measure the Ca2+ influx atthe hair bundles as the hair were depressed by the Ca2+-sensitive microprobe. We successfully detect Ca2+ influx with this new method and found that the Ca2+ influx was blocked by neomycin, gentamicin, La3+ and BAPTA which were shown to block the mechanosensory channel in other studies. Isoform- specific antibody were generated to localize 3 zebrafish TRP proteins (TRPA1a, TRPN1 and TRPV4) on the lateral line hair bundles. Using morpholino-gene knockdown technique, the protein expression of the TRP channels were blocked and leaded to a remarkable decline of Ca2+ influx, suggesting that the TRP channels are involved in the mechanosensory of zebrafish lateral-line hair cells.
The lateral-line, a mechanosensory organ of fish, is composed of hair cells and supporting cells. The mechanosensory channel on the hair bundles of hair cells is a non-selective cation channel with higher Ca2+ permeability. Mechanical deflection of the sensory hair bundles causes Ca2+ influx via the channel and further induces sensory transduction. Recently, a group of Ca2+ channels, the TRP (transient receptor potential) channels, was suggested to be the mechanosensory channel on hair cells. However, the molecular identity of the channel is still controversial. In this study, we used zebrafish as an in vivo model to investigate the mechanosensory channels on the lateral-line hair cell of newly hatch larvae. We used a non-invasive Scanning Ion-Selective Electrode Technique (SIET) to measure the Ca2+ influx atthe hair bundles as the hair were depressed by the Ca2+-sensitive microprobe. We successfully detect Ca2+ influx with this new method and found that the Ca2+ influx was blocked by neomycin, gentamicin, La3+ and BAPTA which were shown to block the mechanosensory channel in other studies. Isoform- specific antibody were generated to localize 3 zebrafish TRP proteins (TRPA1a, TRPN1 and TRPV4) on the lateral line hair bundles. Using morpholino-gene knockdown technique, the protein expression of the TRP channels were blocked and leaded to a remarkable decline of Ca2+ influx, suggesting that the TRP channels are involved in the mechanosensory of zebrafish lateral-line hair cells.
Description
Keywords
斑馬魚胚胎, 毛細胞, TRP通道, 機械性感覺, zebrafish larva, hair cells, TRP channels, mechanosensory