Department of Nanobio Medical Science, Dankook University College of Medicine, Cheonan, Korea.
Clin Exp Otorhinolaryngol. 2011 Mar;4(1):18-23. doi: 10.3342/ceo.2011.4.1.18. Epub 2011 Mar 17.
We tested the possibility of differential expression and function of the potassium-chloride (KCC2) and sodium-potassium-2 chloride (NKCC1) co-transporters in the lateral superior olive (LSO) of heterozygous (+/cir) or homozygous (cir/cir) mice.
Mice pups aged from postnatal (P) day 9 to 16 were used. Tails from mice were cut for DNA typing. For Immunohistochemical analysis, rabbit polyclonal anti-KCC2 or rabbit polyclonal anti-NKCC1 was used and the density of immunolabelings was evaluated using the NIH image program. For functional analysis, whole cell voltage clamp technique was used in brain stem slices and the changes of reversal potentials were evaluated at various membrane potentials.
Immunohistochemical analysis revealed both KCC2 and NKCC1 immunoreactivities were more prominent in heterozygous (+/cir) than homozygous (cir/cir) mice on P day 16. In P9-P12 heterozygous (+/cir) mice, the reversal potential (E(gly)) of glycine-induced currents was shifted to a more negative potential by 50 µM bumetanide, a known NKCC1 blocker, and the negatively shifted E(gly) was restored by additional application of 1 mM furosemide, a KCC2 blocker (-58.9±2.6 mV to -66.0±1.5 mV [bumetanide], -66.0±1.5 mV to -59.8±2.8 mV [furosemide+bumetanide], n=11). However, only bumetanide was weakly, but significantly effective (-60.1±2.9 mV to -62.7±2.6 mV [bumetanide], -62.7±2.6 mV to -62.1±2.5 mV [furosemide+bumetanide], n=7) in P9-P12 homozygous (cir/cir) mice.
The less prominent immunoreactivities and weak or absent responses to bumetanide or furosemide suggest impaired function or delayed development of both transporters in homozygous (cir/cir) mice.
我们检测钾氯（KCC2）和钠钾 2 氯（NKCC1）协同转运体在杂合子（+/cir）或纯合子（cir/cir）小鼠外侧上橄榄核（LSO）中的差异表达和功能的可能性。
使用出生后第 9 至 16 天的幼鼠。从幼鼠的尾巴上剪下一段用于 DNA 分型。对于免疫组织化学分析，使用兔多克隆抗 KCC2 或兔多克隆抗 NKCC1，并用 NIH 图像程序评估免疫标记的密度。对于功能分析，在脑切片中使用全细胞膜片钳技术，并在各种膜电位下评估反转电位的变化。
免疫组织化学分析显示，在 P 日 16 天，杂合子（+/cir）小鼠的 KCC2 和 NKCC1 免疫反应性均比纯合子（cir/cir）小鼠更为明显。在 P9-P12 杂合子（+/cir）小鼠中，甘氨酸诱导电流的反转电位（E(gly)）被 50µM 布美他尼（一种已知的 NKCC1 阻断剂）向更负的方向移动，并且通过额外应用 1mM 呋塞米（一种 KCC2 阻断剂）恢复负移的 E(gly)（-58.9±2.6mV 至-66.0±1.5mV[布美他尼]，-66.0±1.5mV 至-59.8±2.8mV[呋塞米+布美他尼]，n=11）。然而，只有布美他尼在 P9-P12 纯合子（cir/cir）小鼠中作用微弱，但有统计学意义（-60.1±2.9mV 至-62.7±2.6mV[布美他尼]，-62.7±2.6mV 至-62.1±2.5mV[呋塞米+布美他尼]，n=7）。
免疫反应性不明显以及对布美他尼或呋塞米的反应较弱或缺失提示两种转运体在纯合子（cir/cir）小鼠中的功能受损或发育迟缓。
