PKC modulation of calcium current and anesthetic action

描述(由申请人提供)： 电压门控钙通道(Cav)和蛋白激酶C(PKC)均可被挥发性麻醉药调节，但其作用的性质一直存在争议。我们假设麻醉剂对CAV通道和突触传递的抑制是由特定的PKC同工酶介导的。尽管潜在的PKC磷酸化位点存在于Cav通道的成孔α1和辅助β和α2/Delta亚基中，但α1亚基似乎在PKC诱导的这些通道的调节中起主要作用。在这项建议的第一个目标中，我们将通过直接(用佛波醇12-肉豆蔻酸13-乙酸酯；PMA)或受体刺激(用毒鼠强M1)激活共同表达这些受体和Cav通道的非洲爪哇卵母细胞中的PKC来确定Cav通道A1亚基中的磷酸化位点。可能的PKC磷酸化位点将突变为Ala或Glu，并将使用电压钳测量来研究两种类型的PKC激活响应的全细胞Ba2-电流。在AIM II中，我们将鉴定参与非洲爪哇卵母细胞表达的PKC敏感的Cav通道的直接或受体诱导调节的PKC同工酶。这将通过涉及PKC下调、激活诱导的易位、个别PKC同工酶的选择性丢失/抑制和‘加回’研究的实验来检验。在AIM III中，我们将采用AIM II中提到的方法来鉴定非洲爪哇卵母细胞中受挥发性麻醉剂、氟烷或异氟醚调节的PKC同工酶。根据我们的研究结果，PKC同工酶的选择性激活剂或抑制剂可以与挥发性全麻药一起使用，以增强麻醉剂的作用，以及II)减少这些药物非特异性作用所引起的副作用。

DESCRIPTION (provided by applicant): Voltage-gated Ca2+-channels (Cav) and protein kinase C (PKC) can both be regulated by volatile anesthetics, but the nature of the effect has been controversial. We hypothesize that anesthetic inhibition of Cav channels and synaptic transmission are mediated by specific PKC isozymes. Although potential PKC phosphorylation sites are present in the pore-forming alpha1 and the auxiliary beta and alpha2/delta subunits of Cav channels; alpha1subunits seem to play the major role in PKC-induced regulation of these channels. In Aim I of this proposal, we will identify phosphorylation sites in the a1 subunits of Cav channels by direct (with phorbol 12-myristate 13-acetate; PMA) or receptor-stimulated (with muscarinic M1) activation of PKC in Xenopus oocytes coexpressing these receptors and Cav channels. Possible PKC phosphorylation sites will be mutated to Ala or Glu and whole-cell Ba2+ (a substitute for Ca2+)-current in response to two types of PKC activation will be investigated using voltage-clamp measurements. In Aim II we will identify PKC isozymes involved in the direct or receptor-induced modulation of PKC-sensitive Cav channels expressed in Xenopus oocytes. This will be examined using experiments involving down-regulation of PKC, activation-induced translocation, selective loss/inhibition of individual PKC isozymes and 'add-back' studies. In Aim III we will identify the PKC isozymes modulated by volatile anesthetics, halothane or isoflurane in Xenopus oocytes by employing the methods mentioned in Aim II. Based on the results of our studies, isozyme selective activators or inhibitors of PKC may be used along with the volatile general anesthetics to i) potentiate the action of anesthetics and ii) to reduce the side effects of these agents caused by their non-specific actions.