Qu Chunyan, Liang Fenghe, Smythe Nancy M, Schulte Bradley A
Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC 29425, USA.
J Assoc Res Otolaryngol. 2007 Jun;8(2):205-19. doi: 10.1007/s10162-007-0072-0. Epub 2007 Mar 2.
Voltage-gated chloride channels (ClCs) are important mediators of cellular ion homeostasis and volume regulation. In an earlier study, we used immunohistochemical, Western blot, and reverse transcriptase PCR (RT-PCR) approaches to identify ClC-K variants in types II, IV, and V fibrocytes of the rodent spiral ligament. We have now confirmed the expression of ClC-K2 in these cells by in situ hybridization. All three of these fibrocyte subtypes are thought to be involved in cochlear K(+) recycling; thus, it is important to understand the precise mechanisms regulating their membrane conductance and the role played by ClCs in this process. In this study, we report the characterization of a secondary cell line derived from explants from the region of the rat spiral ligament underlying and inferior to the spiral prominence. The cultured cells were immunopositive for vimentin, Na,K/ATPase, Na,K,Cl-cotransporter, carbonic anhydrase isozyme II, and creatine kinase isozyme BB, but not for cytokeratins or Ca/ATPase, an immunostaining profile indicative of the type IV subtype. Evaluation of the cultures by RT-PCR and Western blot analysis confirmed the presence of both ClC-2 and -K2. Whole-cell patch clamp recordings identified two biophysically distinct Cl(-) currents in the cultured cells. One, an inwardly rectifying Cl(-) current activated by hyperpolarization or decreasing extracellular pH corresponded with the properties of ClC-2. The other, a weak outwardly rectifying Cl(-) current regulated by extracellular pH, Cl(-), and Ca(2+) resembled the channel characteristics of ClC-K2 when expressed in Xenopus oocytes. These findings suggest that at least two functionally different chloride channels are involved in regulating membrane anion conductance in cultured type IV spiral ligament fibrocytes.
电压门控氯离子通道（ClCs）是细胞离子稳态和体积调节的重要介质。在早期研究中，我们使用免疫组织化学、蛋白质印迹和逆转录聚合酶链反应（RT-PCR）方法，在啮齿动物螺旋韧带的II型、IV型和V型纤维细胞中鉴定ClC-K变体。我们现在通过原位杂交证实了ClC-K2在这些细胞中的表达。这三种纤维细胞亚型都被认为参与了耳蜗K(+)循环；因此，了解调节其膜电导的精确机制以及ClCs在此过程中所起的作用非常重要。在本研究中，我们报告了一种源自大鼠螺旋韧带螺旋隆起下方和下方区域外植体的二级细胞系的特征。培养的细胞对波形蛋白、Na,K/ATP酶、Na,K,Cl-协同转运蛋白、碳酸酐酶同工酶II和肌酸激酶同工酶BB呈免疫阳性，但对细胞角蛋白或Ca/ATP酶呈免疫阴性，这种免疫染色特征表明其为IV型亚型。通过RT-PCR和蛋白质印迹分析对培养物进行评估，证实了ClC-2和-K2的存在。全细胞膜片钳记录在培养细胞中鉴定出两种生物物理特性不同的Cl(-)电流。一种是由超极化或降低细胞外pH激活的内向整流Cl(-)电流，与ClC-2的特性相符。另一种是由细胞外pH、Cl(-)和Ca(2+)调节的弱外向整流Cl(-)电流，类似于在非洲爪蟾卵母细胞中表达时ClC-K2的通道特征。这些发现表明，至少两种功能不同的氯离子通道参与调节培养的IV型螺旋韧带纤维细胞膜阴离子电导。
