阳离子对豚鼠Ⅱ型前庭毛细胞ACh-敏感性电流的调制

Cation ions modulate the ACh-sensitive current in type II vestibular hair cells of guinea pigs

本文旨在探讨哺乳动物前庭胆碱能传出神经系统的作用机制,应用全细胞膜片钳技术研究新鲜分离的豚鼠Ⅱ型前庭毛细胞ACh-敏感性电流的特性以及细胞内外的阳离子对ACh-敏感性电流的调制作用。结果显示,Ⅱ型前庭毛细胞对细胞外ACh敏感,ACh激活缓慢持久的外向性电流,室温下此电流的再次完全激活时间约为(60±10)s。ACh-敏感性电流的反转电位为(-66±8)mV,提示此电流主要由K+参与形成,其直接作用是使毛细胞超极化。ACh-敏感性电流对较高浓度的四乙胺(tetraethylammonium chloride,TEA)敏感,提示细胞外ACh激活钙依赖性钾电流。进一步检测细胞内外阳离子对 ACh-敏感性电流的调制作用发现,细胞外Na+和细胞内Ca2+释放不参与此电流的激活过程,而细胞外K+、细胞外Ca2+和细胞内Mg2+对ACh-敏感性电流具有重要的调制作用。进一步提示,ACh是哺乳动物前庭传出神经系统重要的神经递质。 Na+不参与ACh-敏感性电流的激活过程提示,ACh-敏感性电流可能由非α9-N型胆碱能受体(α9-nAChR)介导。ACh诱导的Ⅱ型前庭毛细胞超极化作用受细胞外Ca2+浓度和细胞内Mg2+浓度调制。

Molecular biological studies and electrophysiological data have demonstrated that acetylcholine （ACh） is the principal cochlear and vestibular efferent neurotransmitter among mammalians. However, the functional roles of ACh in type Ⅱ vestibular hair cells among mammalians are still unclear, with the exception of the well-known α9-containing nicotinic ACh receptor （α9-nAChR） in cochlear hair cells and frog saccular hair cells. In this study, the properties of the ACh-sensitive current were investigated by whole-cell patch clamp technique in isolated type Ⅱ vestibular hair cells of guinea pigs. The direct effect of extracellular ACh was to induce a hyperpolarization effect in type Ⅱ vestibular hair cells. Type Ⅱ vestibular hair cells displayed a sustained outward current in response to the perfusion of ACh. It took about 60 s for the ACh-sensitive current to get a complete re-activation. The reversal potential of the ACh-sensitive current was （-66±8） mV, which indicated that potassium ion was the main carrier of this current. The blocking effect by the submillimolar concentration of tetraethylammonium （TEA） further indicated that extracellular ACh stimulated the calcium-dependent potassium current. Following replacement of the compartment of NaCl in the normal external solution with TrisCl, LiCl or saccharose respectively, the amplitude of the ACh-sensitive current was not affected. Blocking of the release of intracellular Ca^2＋ stores by intracellular application of heparin failed to inhibit the ACh-sensitive current. Therefore, extracellular Na^＋ and the inositol 1,4,5-trisphosphate （IP3）- dependent intracellular Ca^2＋ release were not involved in the activation of the ACh-sensitive current. However, the ACh-sensitive current was strongly affected by the concentration of the extracellular K^＋, extracellular Ca^2＋ and intracellular Mg^2＋. The amplitude of the ACh-sensitive current was strongly inhibited by high concentration of extracellular K^＋. In the Ca^2＋ -free external solution, ACh only activated a very small current; however, the ACh-sensitive current demonstrated a Ca^2＋ -dependent inhibition effect in high concentration of Ca^2＋ solution. In addition, the ACh-sensitive current was inhibited by increasing of the concentration of intracellular Mg^2＋. In conclusion, the present results demonstrate that ACh plays an important role in the vestibular efferent system. The fact that Na^＋ is not involved in the ACh-sensitive current will not favor the well-known profile of α9-nAChR, which is reported to display a small but important permeability to Na^＋. It is also suggested that, in vivo, the amplitude of the ACh-induced hyperpolarization may strongly depend on the concentration of extracellular Ca^2＋ and intracellular Mg^2＋.