Yamazaki Muneharu, Kim Kyunghee X, Marcus Daniel C
Cellular Biophysics Laboratory, Department of Anatomy & Physiology, Kansas State University, Manhattan, KS 66506, USA.
BMC Physiol. 2011 Feb 1;11:4. doi: 10.1186/1472-6793-11-4.
Sodium absorption by Reissner's membrane is thought to contribute to the homeostasis of the volume of cochlear endolymph. It was previously shown that the absorptive transepithelial current was blocked by amiloride and benzamil. The most commonly-observed target of these drugs is the epithelial sodium channel (ENaC), which is composed of the three subunits α-,β- and γ-ENaC. However, other less-selective cation channels have also been observed to be sensitive to benzamil and amiloride. The aim of this study was to determine whether Reissner's membrane epithelial cells could support parasensory K+ absorption via amiloride- and benzamil-sensitive electrogenic pathways.
We determined the molecular and functional expression of candidate cation channels with gene array (GEO GSE6196), RT-PCR, and whole-cell patch clamp. Transcript expression analysis of Reissner's membrane detected no amiloride-sensitive acid-sensing ion channels (ASIC1a, ASIC2a, ASIC2b) nor amiloride-sensitive cyclic-nucleotide gated channels (CNGA1, CNGA2, CNGA4, CNGB3). By contrast, α-,β- and γ-ENaC were all previously reported as present in Reissner's membrane. The selectivity of the benzamil-sensitive cation currents was observed in whole-cell patch clamp recordings under Cl--free conditions where cations were the only permeant species. The currents were carried by Na+ but not K+, and the permeability of Li+ was greater than that of Na+ in Reissner's membrane. Complete replacement of bath Na+ with the inpermeable cation NMDG+ led to the same inward current as with benzamil in a Na+ bath.
These results are consistent with the amiloride/benzamil-sensitive absorptive flux of Reissner's membrane mediated by a highly Na+-selective channel that has several key characteristics in common with αβγ-ENaC. The amiloride-sensitive pathway therefore absorbs only Na+ in this epithelium and does not provide a parasensory K+ efflux route from scala media.
人们认为内淋巴囊膜对钠的吸收有助于维持耳蜗内淋巴液体积的稳态。先前的研究表明，阿米洛利和苯扎米能阻断经上皮吸收电流。这些药物最常见的作用靶点是上皮钠通道（ENaC），它由α-、β-和γ-ENaC三个亚基组成。然而，也观察到其他选择性较低的阳离子通道对苯扎米和阿米洛利敏感。本研究的目的是确定内淋巴囊膜上皮细胞是否能通过对阿米洛利和苯扎米敏感的生电途径支持副感觉性钾吸收。
我们通过基因芯片（GEO GSE6196）、逆转录聚合酶链反应（RT-PCR）和全细胞膜片钳技术确定了候选阳离子通道的分子和功能表达。对内淋巴囊膜的转录本表达分析未检测到对阿米洛利敏感的酸敏感离子通道（ASIC1a、ASIC2a、ASIC2b），也未检测到对阿米洛利敏感的环核苷酸门控通道（CNGA1、CNGA2、CNGA4、CNGB3）。相比之下，α-、β-和γ-ENaC此前均被报道存在于内淋巴囊膜中。在无氯离子条件下进行全细胞膜片钳记录时，观察到苯扎米敏感的阳离子电流具有选择性，此时阳离子是唯一的通透离子。这些电流由钠离子携带而非钾离子，在内淋巴囊膜中锂离子的通透性大于钠离子。用不可通透的阳离子NMDG+完全替代浴液中的钠离子，产生的内向电流与在钠离子浴液中加入苯扎米时相同。
这些结果与内淋巴囊膜中由高度钠选择性通道介导的对阿米洛利/苯扎米敏感的吸收通量一致，该通道与αβγ-ENaC具有几个关键的共同特征。因此，阿米洛利敏感途径在该上皮细胞中仅吸收钠离子，而不能提供从中阶流出副感觉性钾离子的途径。
