Chew Shit F, Hiong Kum C, Lam Sock P, Chen Xiu L, Ching Biyun, Ip Yuen K
Natural Sciences and Science Education, National Institute of Education, Nanyang Technological University, 1 Nanyang Walk, Singapore, 637616, Republic of Singapore,
J Comp Physiol B. 2015 Jan;185(1):57-72. doi: 10.1007/s00360-014-0867-3. Epub 2014 Oct 28.
The giant mudskipper, Periophthalmodon schlosseri, is an obligate air-breathing teleost that can actively excrete ammonia against high concentrations of environmental ammonia. This study aimed to clone and sequence the Na (+) :K (+) :2Cl (-) cotransporter 1 (nkcc1) from the gills of P. schlosseri, and to determine the effects of ammonia exposure on its mRNA expression and protein abundance after pre-acclimation to slightly brackish water (salinity 3; SBW) for 2 weeks. The complete coding cDNA sequences of nkcc1a consisted of 3453 bp, coding for 1151 amino acid with an estimated molecular mass of 125.4 kDa. Exposure to 75 mmol l(-1) NH4Cl in SBW had no effect on the mRNA expression of nkcc1a. However, western blotting revealed a significant increase in the protein abundance of multiple T4-immunoreactive bands of molecular mass 170-250 kDa in the gills of P. schlosseri exposed to ammonia. Furthermore, immunofluorescence microscopy demonstrated the colocalization of the increased T4-immunoreactive protein with Na(+)/K(+)-ATPase (Nka) α-subunit to the basolateral membrane of certain ionocytes in the gills of the ammonia-exposed fish. As Nkcc1 is known to have a basolateral localization, it can be concluded that ammonia exposure led to an increase in the expression of glycosylated Nkcc1, the molecular masses of which were reduced upon enzymatic deglycosylation, in the gills of P. schlosseri. The dependency on post-transcriptional and post-translational regulation of branchial Nkcc1 in P. schlosseri would facilitate prompt responses to changes in environmental condition. As NH4 (+) can replace K(+), NH4 (+) could probably enter ionocytes through the basolateral Nkcc1a during active ammonia excretion, but increased influx of Na(+), NH4 (+) and 2Cl(-) would alter the transmembrane Na(+) gradient. Consequently, exposure of P. schlosseri to ammonia would also result in an increase in branchial activity of Nka with decreased NH4 (+) affinity so as to maintain intracellular Na(+) and K(+) homeostasis as reported elsewhere.
弹涂鱼（Periophthalmodon schlosseri）是一种专性呼吸空气的硬骨鱼，能够在高浓度环境氨存在的情况下主动排泄氨。本研究旨在克隆和测序弹涂鱼鳃中的钠钾氯共转运体1（nkcc1），并确定在预先适应微咸水（盐度3；SBW）2周后，氨暴露对其mRNA表达和蛋白质丰度的影响。nkcc1a的完整编码cDNA序列由3453个碱基对组成，编码1151个氨基酸，估计分子量为125.4 kDa。在SBW中暴露于75 mmol l(-1) NH4Cl对nkcc1a的mRNA表达没有影响。然而，蛋白质印迹法显示，暴露于氨的弹涂鱼鳃中，分子量为170 - 250 kDa的多个T4免疫反应条带的蛋白质丰度显著增加。此外，免疫荧光显微镜显示，暴露于氨的鱼鳃中，增加的T4免疫反应蛋白与钠钾ATP酶（Nka）α亚基共定位于某些离子细胞的基底外侧膜。由于已知Nkcc1定位于基底外侧，因此可以得出结论，氨暴露导致弹涂鱼鳃中糖基化Nkcc1的表达增加，其分子量在酶促去糖基化后降低。弹涂鱼鳃中Nkcc1对转录后和翻译后调控的依赖性将有助于对环境条件变化做出快速反应。由于NH4(+)可以替代K(+)，在主动排泄氨的过程中，NH4(+)可能通过基底外侧的Nkcc1a进入离子细胞，但Na(+)、NH4(+)和2Cl(-)流入量的增加会改变跨膜Na(+)梯度。因此，弹涂鱼暴露于氨也会导致鳃中Nka活性增加，NH4(+)亲和力降低，从而如其他地方所报道的那样维持细胞内Na(+)和K(+)的稳态。
