Na-K-2Cl联合转运子-1和α_2 Na,K-ATP酶在耳蜗钾离子循环中的作用及与听觉功能的关系

Role of ion channel Na-K-2CI and α_2Na,K-ATPase in cochlear potassuim cycling and auditory function

目的应用 Na-K-2Cl 联合转运子-1(Na-K-2Cl Co-transporter 1,NKCC1)^(-/-)、NKCC1^(+/-)、α_2,Na,K-ATP 酶^(+/-)和 NKCC1^(+/-)/α_2Na,K-ATP 酶^(+/-)等不同基因型小鼠作为实验平台,对耳蜗钾离子循环模式与听觉功能进行研究,检验 NKCC1 和α_2Na,K-ATPase 在耳蜗钾循环中的地位和作用。方法应用 NKCC1^(-/-)、NKCC1^(+/-)和α_2Na,K-ATPase^(+/-)等基因敲除和转基因小鼠,同时培育 NKCC1^(+/-)/α_2Na,K-ATPase^(+/-)双半拷贝基因小鼠,采用听性脑干反应和耳蜗内电位检测技术对小鼠的听觉功能进行检测。应用 NKCC1 通道阻断剂速尿和 Na,K-ATP 酶通道阻断剂哇巴因,对 Castel鼠系进行通道阻断-听觉功能实验,进一步在小鼠体内检测耳蜗钾循环模式,对内耳钾循环与听觉生理之间的关系进行印证。结果 NKCC1^(+/-)小鼠和α_2Na,K-ATP 酶^(+/-)小鼠的听性脑干反应短声阈值(声压级)分别为(38.49±12.29)dB 和(53.32±7.62)dB,与野生型小鼠的(23.13±3.78)dB 比较均有提高,差异有统计学意义(t 值分别为3.559和10.267,P<0.05)。NKCC1^(+/-)小鼠和α_2Na,K-ATP 酶^(+/-)小鼠的耳蜗内电位值分别(78±7)mV 和(71±14)mV,分别低于野生型小鼠的(98±16)mV。NKCC1^(+/-)/α_2Na,K-ATP 酶^(+/-)小鼠听性脑干反应短声阈值为(24.14±8.83)dB,低于α_2Na,K-ATPase^(+/-)小鼠和 NKCC1^(+/-)小鼠,差异有统计学意义(t 值分别为6.128和2.255,P<0.05)。注射速尿后的 Castel 小鼠听性脑于反应听阈明显升高,与注射前比较其差异有统计学意义(t=12.162,P<0.05);注射哇巴因后的 Castel 小鼠也出现听阈升高(t=7.644,P<0.05)。而次序注射哇巴因和速尿后,Castel 小鼠听性脑干反应听阈明显低于单独注射速尿(t=4.515,P<0.01)。结论 NKCC1和α_2,Na,K-ATP 酶是耳蜗钾循环中的关键通道,任何一通道蛋白出现功能失衡均可影响内淋巴钾离子浓度及耳蜗内电位的维持,进而影响听觉功能。NKCC1 和α_2Na,K-ATP 酶在耳蜗钾循环诸通道中处于一种限制性动态平衡关系,在内耳钾代谢和耳蜗听觉功能的发挥中具有重要意义。本实验在小鼠实体中验证了 NKCC1和α_2Na,K-ATP 酶在内耳钾循环径路中的重要作用,同时也模拟了内耳钾循环模式。

Objective To investigate the auditory function and the role of NKCCl and α2Na,K-ATPase in the potassium recycling of cochlea. Methods NKCCl ＋/-/α2Na,K-ATPase ＋/- mice model was established from NKCCl ＋/- and α2Na,K-ATPase ＋/- mice. The auditory function of all strain mice were detected by auditory brainstem response（ABR） and endocochlear potential（EP） to investigate the role of NKCCl and α2Na,K-ATPase in the potassium recycling of cochlea. Furosemide and ouabain were applied to block the two channels in Castel mice line which can long-time maintain normal auditory function and then their auditory function was detected by ABR to authenticate the mode of potassium recycling in vivo and the relationship between cochlear potassium recycling and NKCCl ＋/- and α2Na,K-ATPase. Results The mean value for ABR thresholds in response to stimulus was elevated in NKCCl ＋/- and α2Na, K-ATPase ＋/- mice [ （38.49 ±12. 29） dB and （53.32 ±7.62 ） dB ） ] respectively, which was significantly increased compared with that observed in wild type mice [（23.13 ±3.78）dB ,P 〈0.05）]; The EP value of NKCCl ＋/- [ （78 ±7） mV] and α2Na, K-ATPase＋/- mice[ （71 ± 14） mV] was decreased compared with that of NKCCl ＋/-/α2Na, K-ATPase ＋/- mice [ （86 ± 11 ） mV ]. The auditory function of NKCCl ＋/-/α2 Na,K-ATPase＋/- mice could simulate the model of cochlear potassium recycling well. NKCCl and Na,K- ATPase were great of importance in the potassium recycling, while the two ion channels were in restrict dynamic equilibrium. Castel mice line after administration with furosemide developed significant ABR threshold shifts （ P 〈 0. 05 ） compared with control group. Castel mice line after administration with ouabain also developed greatly significant ABR threshold shifts （ P 〈 0. 05 ） compared with control group. ABR threshold shifts in mice after administration both furosemide and ouabain was attenuated compared with only administration with furosemide（P 〈0. 01 ）. Conclusions Ion channel NKCCl and α2Na,K-ATPase played important roles in the inner ear potassium recycling. Dysfunction of either of them could influence potassium concentration in the endolymph and lead to hearing loss subsequently. The role of NKCCl and α2Na, K- ATPase in cochlear potassium recycling was authenticated in vivo. The two ion channels contribute the key role for dynamic equilibrium in cochlear potassium recycling and are of great importance for the metabolism of potassium in the inner ear to maintain the normal auditory function.