Zufall F, Stengl M, Franke C, Hildebrand J G, Hatt H
Physiologisches Institut der Technischen Universität München, Germany.
J Neurosci. 1991 Apr;11(4):956-65. doi: 10.1523/JNEUROSCI.11-04-00956.1991.
Whole-cell and single-channel voltage-clamp techniques were used to identify and characterize the ionic currents of insect olfactory receptor neurons (ORNs) in vitro. The cells were isolated from the antennae of male Manduca sexta pupae at stages 3-5 of adult development and maintained in primary cell culture. After 2-3 weeks in vitro, the presumptive ORNs had resting potentials of -62 +/- 12 mV (n = 18) and expressed at least 1 type of Na+ channel and at least 3 types of K+ channels. Na+ currents, recorded in the whole-cell mode, were reversibly blocked by 0.1 microM tetrodotoxin. The predominant type of K+ channel observed was a voltage-activated K+ channel (gamma = 30 pS) with characteristics similar to those of the delayed rectifier. The activity of the 30-pS K+ channel could be inhibited by the application of nucleotides to the cytoplasmic face of inside-out patches of membrane. The nucleotides had relative potencies as follows: ATP greater than cGMP greater than cAMP, with an inhibition constant for ATP of Ki = 0.18 mM. Raising the intracellular Ca2+ concentration from 0.1 to 5 microM induced the opening of a Ca2(+)-activated K+ channel (gamma = 66 pS at 0 mV) that had a low voltage sensitivity. A third, transient type of K+ channel (gamma = 12-18 pS) could be activated by depolarizing voltage steps from very negative resting potentials. Properties of this channel were similar to those of the "A-channel." These results support the conclusion that M. sexta ORNs differentiate in vitro and provide the basis for studying primary mechanisms of olfactory transduction.
采用全细胞和单通道电压钳技术在体外鉴定和表征昆虫嗅觉受体神经元（ORN）的离子电流。细胞取自成年发育3 - 5阶段雄性烟草天蛾蛹的触角，并维持在原代细胞培养中。在体外培养2 - 3周后，假定的ORN的静息电位为-62±12 mV（n = 18），并表达至少1种类型的Na⁺通道和至少3种类型的K⁺通道。在全细胞模式下记录的Na⁺电流可被0.1 μM河豚毒素可逆性阻断。观察到的主要K⁺通道类型是电压激活的K⁺通道（γ = 30 pS），其特性与延迟整流器相似。向膜的内向外膜片的胞质面施加核苷酸可抑制30 - pS K⁺通道的活性。核苷酸的相对效力如下：ATP＞cGMP＞cAMP，ATP的抑制常数Ki = 0.18 mM。将细胞内Ca²⁺浓度从0.1 μM提高到5 μM会诱导一个低电压敏感性的Ca²⁺激活K⁺通道（0 mV时γ = 66 pS）开放。第三种瞬时类型的K⁺通道（γ = 12 - 18 pS）可通过从非常负的静息电位进行去极化电压阶跃来激活。该通道的特性与“A通道”相似。这些结果支持了烟草天蛾ORN在体外分化的结论，并为研究嗅觉转导的主要机制提供了基础。
