Chang Qing, Tang Wenxue, Ahmad Shoeb, Zhou Binfei, Lin Xi
Department of Otolaryngology, Emory University School of Medicine, Atlanta, GA, USA.
PLoS One. 2008;3(12):e4088. doi: 10.1371/journal.pone.0004088. Epub 2008 Dec 31.
Connexin26 (Cx26) and connexin30 (Cx30) are two major protein subunits that co-assemble to form gap junctions (GJs) in the cochlea. Mutations in either one of them are the major cause of non-syndromic prelingual deafness in humans. Because the mechanisms of cochlear pathogenesis caused by Cx mutations are unclear, we investigated effects of Cx30 null mutation on GJ-mediated ionic and metabolic coupling in the cochlea of mice. A novel flattened cochlear preparation was used to directly assess intercellular coupling in the sensory epithelium of the cochlea. Double-electrode patch clamp recordings revealed that the absence of Cx30 did not significantly change GJ conductance among the cochlear supporting cells. The preserved electrical coupling is consistent with immunolabeling data showing extensive Cx26 GJs in the cochlea of the mutant mice. In contrast, dye diffusion assays showed that the rate and extent of intercellular transfer of multiple fluorescent dyes (including a non-metabolizable D-glucose analogue, 2-NBDG) among cochlear supporting cells were severely reduced in Cx30 null mice. Since the sensory epithelium in the cochlea is an avascular organ, GJ-facilitated intercellular transfer of nutrient and signaling molecules may play essential roles in cellular homeostasis. To test this possibility, NBDG was used as a tracer to study the contribution of GJs in transporting glucose into the cochlear sensory epithelium when delivered systemically. NBDG uptake in cochlear supporting cells was significantly reduced in Cx30 null mice. The decrease was also observed with GJ blockers or glucose competition, supporting the specificity of our tests. These data indicate that GJs facilitate efficient uptake of glucose in the supporting cells. This study provides the first direct experimental evidence showing that the transfer of metabolically-important molecules in cochlear supporting cells is dependent on the normal function of GJs, thereby suggesting a novel pathogenesis process in the cochlea for Cx-mutation-linked deafness.
连接蛋白26（Cx26）和连接蛋白30（Cx30）是两个主要的蛋白质亚基，它们共同组装形成耳蜗中的缝隙连接（GJ）。其中任何一个发生突变都是人类非综合征性语前聋的主要原因。由于Cx突变导致耳蜗发病机制尚不清楚，我们研究了Cx30基因敲除突变对小鼠耳蜗中GJ介导的离子和代谢偶联的影响。一种新型的扁平耳蜗标本被用于直接评估耳蜗感觉上皮中的细胞间偶联。双电极膜片钳记录显示，Cx30的缺失并没有显著改变耳蜗支持细胞之间的GJ电导。保留的电偶联与免疫标记数据一致，该数据显示突变小鼠耳蜗中存在广泛的Cx26 GJ。相比之下，染料扩散试验表明，在Cx30基因敲除小鼠中，多种荧光染料（包括一种不可代谢的D-葡萄糖类似物2-NBDG）在耳蜗支持细胞间的转移速率和范围严重降低。由于耳蜗中的感觉上皮是一个无血管的器官，GJ促进的营养和信号分子细胞间转移可能在细胞内稳态中起重要作用。为了验证这种可能性，NBDG被用作示踪剂，以研究全身给药时GJ在将葡萄糖转运到耳蜗感觉上皮中的作用。Cx30基因敲除小鼠耳蜗支持细胞中NBDG的摄取显著减少。在使用GJ阻滞剂或葡萄糖竞争时也观察到了这种减少，这支持了我们测试的特异性。这些数据表明，GJ促进了支持细胞中葡萄糖的有效摄取。本研究提供了首个直接实验证据，表明耳蜗支持细胞中代谢重要分子的转移依赖于GJ的正常功能，从而提示了耳蜗中Cx突变相关耳聋的一种新发病机制。
