鈉鉀幫浦次單元在斑馬魚離子調控機制中的角色

Abstract

魚類的鰓與皮膚上的離子細胞對於維持體內離子平衡與酸鹼平衡扮演著一個非常重要的角色。先前的研究中在斑馬魚(Danio rerio)上發現了三種離子細胞分別為富含鈉鉀幫浦細胞(NaR cells)、富含氫離子幫浦細胞 (HR cells)，以及鈉氯離子運輸細胞(NCC cells)。這三種離子細胞分別負責鈣離子吸收、鈉離子吸收及酸的排出，和氯離子吸收。研究報告指出鈉鉀幫浦其次單元 (NKA subunit) 有三種同功異構物atp1a1a.1, atp1a1a.2, 以及 atp1a1a.5 分別表現在富含鈉鉀幫浦細胞、鈉氯離子運輸細胞，以及富含氫離子幫浦細胞。本實驗的主要目的是測試上述的三種同功異構物是否對斑馬魚上的三種離子細胞扮演著重要的角色。 本實驗主要利用基因專一反股核酸(morpholino-modified antisense oligonucleotide) 抑制上述三種的同功異構物的蛋白質表現。實驗結果發現當atp1a1a.1被抑制後鈣離子的流入有減少的情況，表示atp1a1a.1對於魚體鈣離子吸收有著一定的程度的影響。抑制atp1a1a.2後會造成氯離子在魚的整體量減少，同時發現鈉氯離子運輸蛋白的基因表現量降低，推測atp1a1a.2在鈉氯離子運輸細胞的電化學平衡扮演著重要的角色，使得鈉氯離子運輸蛋白無法正常的表現造成整體的氯離子含量下降。在atp1a1a.5的抑制實驗中發現鈉離子的整體量會下降，顯示atp1a1a.5對於富含氫離子幫浦細胞在鈉離子吸收的機制上是十分重要的。 綜合以上結果，本實驗首次提供在生物體(in vivo)層次上的分子生理證據，證明了不同的鈉鉀幫浦次單元同功異構物在斑馬魚皮膚及鰓上離子細胞的離子調控機制中所扮演的角色。

Gill and skin ionocytes are the major cell types which play an essential role in the transepithelial transport of ions and maintenance of the acid-base balance in fish. Previous studies found three subtypes of ionocytes in zebrafish (Danio rerio), Na+-K+ ATPase-Rich Cells (NaR cells), H+-ATPase-Rich Cells (HR cells) and Na+-Cl- Co-transporter Cells (NCC cells). These subtypes are responsible for Ca2+ uptake, Na+ uptake/acid secretion and Cl- uptake, respectively. A recent study on zebrafish has reported three distinct Na+-K+ ATPase (NKA) α1-subunit genes, atp1a1a.1, atp1a1a.2, and atp1a1a.5, which are expressed in NaR cells, NCC cells and HR cells, respectively. The present study aims to test the hypothesis of whether the NKA α1 isoforms play distinct roles in the ion regulation pathways in the three types of ionocytes. Knockdown experiments with specific morpholinos against the three NKA α1 genes were conducted. The results showed that knockdown of atp1a1a.1 decreased the Ca2+ influx in the morphants. This indicates the role of atp1a1a.1 in Ca2+ uptake mechanism in NaR cells is important. Knockdown of atp1a1a.2 decreased the Cl- content and the mRNA expression of NCC in the morphants. These results suggest that loss-of-function of ATP1a1a.2 has may impact the intracellular electrochemistry and thus affect the transcription/translation of other transporters, consequently resulting in impairment to Cl- uptake function in NCC cells. Knockdown of atp1a1a.5 in zebrafish embryos showed a significant decrease of whole body Na+ content, implying an importance of ATP1a1a.5 in Na+ uptake mechanisms in HR cells. These results for the first time provide in vivo molecular physiological evidence for the different roles of NKA α1-subunit isoforms in zebrafish gill/skin ion regulation mechanisms.