Horwitz Geoffrey C, Risner-Janiczek Jessica R, Holt Jeffrey R
Department of Otolaryngology and 2 F.M. Kirby Neurobiology Center, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115.
J Gen Physiol. 2014 Apr;143(4):481-97. doi: 10.1085/jgp.201311126. Epub 2014 Mar 17.
The hyperpolarization-activated, cyclic nucleotide-sensitive current, Ih, is present in vestibular hair cells and vestibular ganglion neurons, and is required for normal balance function. We sought to identify the molecular correlates and functional relevance of Ih in vestibular ganglion neurons. Ih is carried by channels consisting of homo- or heteromeric assemblies of four protein subunits from the Hcn gene family. The relative expression of Hcn1-4 mRNA was examined using a quantitative reverse transcription PCR (RT-PCR) screen. Hcn2 was the most highly expressed subunit in vestibular neuron cell bodies. Immunolocalization of HCN2 revealed robust expression in cell bodies of all vestibular ganglion neurons. To characterize Ih in vestibular neuron cell bodies and at hair cell-afferent synapses, we developed an intact, ex vivo preparation. We found robust physiological expression of Ih in 89% of cell bodies and 100% of calyx terminals. Ih was significantly larger in calyx terminals than in cell bodies; however, other biophysical characteristics were similar. Ih was absent in calyces lacking Hcn1 and Hcn2, but small Ih was still present in cell bodies, which suggests expression of an additional subunit, perhaps Hcn4. To determine the contributions of hair cell mechanotransduction and Ih to the firing patterns of calyx terminals, we recorded action potentials in current-clamp mode. Mechanotransduction currents were modulated by hair bundle defection and application of calcium chelators to disrupt tip links. Ih activity was modulated using ZD7288 and cAMP. We found that both hair cell transduction and Ih contribute to the rate and regularity of spontaneous action potentials in the vestibular afferent neurons. We propose that modulation of Ih in vestibular ganglion neurons may provide a mechanism for modulation of spontaneous activity in the vestibular periphery.
超极化激活的环核苷酸敏感电流（Ih）存在于前庭毛细胞和前庭神经节神经元中，是正常平衡功能所必需的。我们试图确定Ih在前庭神经节神经元中的分子相关性和功能意义。Ih由Hcn基因家族的四个蛋白质亚基的同源或异源组装体组成的通道携带。使用定量逆转录PCR（RT-PCR）筛选检测Hcn1-4 mRNA的相对表达。Hcn2是前庭神经元细胞体中表达最高的亚基。HCN2的免疫定位显示在前庭神经节所有神经元的细胞体中均有强烈表达。为了表征前庭神经元细胞体和毛细胞传入突触处的Ih，我们开发了一种完整的离体标本。我们发现在89%的细胞体和100%的花萼终末中有强烈的Ih生理表达。花萼终末中的Ih明显大于细胞体中的Ih；然而，其他生物物理特性相似。缺乏Hcn1和Hcn2的花萼中不存在Ih，但细胞体中仍存在少量Ih，这表明可能有另一个亚基，也许是Hcn4的表达。为了确定毛细胞机械转导和Ih对花萼终末放电模式的贡献，我们在电流钳模式下记录动作电位。机械转导电流通过毛束偏转和应用钙螯合剂破坏纤毛连接进行调节。Ih活性使用ZD7288和cAMP进行调节。我们发现毛细胞转导和Ih都有助于前庭传入神经元自发动作电位的频率和规律性。我们提出，前庭神经节神经元中Ih的调节可能为调节前庭外周的自发活动提供一种机制。
