霍乱毒素对三氟氯氰菊酯抗性及敏感棉铃虫神经细胞L型钙通道的调节作用

霍乱毒素（CTX）可激活兴奋性异三聚体G蛋白（Gα_s）的α-亚基和刺激电压门控L-型钙通道，而昆虫的L-型钙通道可能是拟除虫菊酯类杀虫剂的作用靶点。为进一步探讨农业害虫对拟除虫菊酯类杀虫剂产生抗药性的作用机理，我们检测了CTX对三氟氯氰菊酯抗性及敏感棉铃虫Helicoverpa armigera中枢神经细胞电压门控L-型钙通道的调节作用。分别急性分离三氟氯氰菊酯抗性及敏感的3～4龄棉铃虫幼虫胸腹神经节细胞，并在改良的L15培养基（加入或未加入700 ng/mL的CTX）中培养12～16 h。钡离子为载流子，应用全细胞膜片钳技术记录电压门控L_型钙通道电流。结果显示，CTX可使敏感组棉铃虫神经细胞L-型钙通道的峰值电流密度增大36％、峰值电压左移5 mV，但对抗性组棉铃虫神经细胞L-型钙通道无上述作用。并且，CTX对敏感组及抗性组棉铃虫神经细胞L_型钙通道的激活电位、翻转电位、激活曲线和失活曲线等其他一些参数的影响也不明显。在无CTX作用时，所检测到的抗性组与敏感组棉铃虫神经细胞L_型钙通道的上述参数值间差异不显著。结果提示，棉铃虫神经细胞内存在Gs腺苷酸环化酶(AC)-cAMP-蛋白激酶A (PKA)-L-型钙通道信号调节系统；与敏感棉铃虫神经细胞L-型钙通道相比，三氟氯氰菊酯抗性棉铃虫神经细胞L-型钙通道的活性相对不易受到CTX调节，这可能与昆虫对拟除虫菊酯产生抗药性的机理有关。

Modulations by CTX of the L-type Ca2+ channels in the central neurons of the cyhalothrin-resistant and cyhalothrin-susceptible cotton bollworm, Helicoverpa armigera

Cholera toxin (CTX) activates the α-subunit of stimulatory heterotrimeric G-proteins (Gαs) and stimulates voltage-gated L-type (Ca2+(L)) channels, which may be primary targets of pyrethroids. To investigate the potential mechanisms underlying the resistance to pyrethroids in agriculturally important insect pests, we examined the modulations by CTX of Ca2+(L) channels in the central neurons of the cyhalothrin-resistant (Cy-R) and cyhalothrin-susceptible (Cy-S) cotton bollworms (Helicoverpa armigera). Neurons were isolated from the 3rd-4th instar larvae of the Cy-R and Cy-S cotton bollworms, respectively. The isolated neurons from each group were cultured for 12-16 h in an improved L15 insect culture medium with or without CTX (700 ng/mL). Barium currents (IBa) through Ca2+(L)channels were recorded by the whole-cell patch-clamp technique. The results showed that CTX increased the IBa peak current density by 36.1 % and caused a hyperpolarizing shift by 5 mV in the I-V curve in the Cy-S neurons, but had no such effect in the Cy-R neurons. Moreover, CTX exerted little effects on other parameters such as the activation potential, reverse potential, activation and inactivation curves in either Cy-S or Cy-R neurons. No significant differences of the parameters mentioned above in the Ca2+(L) channels were detected between the Cy-S and Cy-R neurons cultured without CTX. The results suggest that the Gs-adenylyl cyclase (AC)-cAMP-protein kinase A (PKA)-Ca2+(L) channel signal transduction pathway may exist in cotton bollworm neurons, and the reduced sensitivity of Ca2+(L)channels to the CTX modulation in the Cy-R neurons (but not in the Cy-S neurons) may account for the reduced nerve sensitivity in the pyrethroids-resistant insects.