PKC modulation of calcium current and anesthetic action

描述（由申请人提供）： 电压门控性钙通道（Cav）和蛋白激酶C（PKC）都可以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。虽然潜在的PKC磷酸化位点存在于Cav通道的成孔α 1和辅助β和α 2/δ亚基中，但α 1亚基似乎在PKC诱导的这些通道调节中起主要作用。在本提案的目的I中，我们将通过直接（用佛波醇12-肉豆蔻酸酯13-乙酸酯; PMA）或受体刺激（用毒蕈碱M1）激活共表达这些受体和Cav通道的爪蟾卵母细胞中的PKC来鉴定Cav通道a1亚基中的磷酸化位点。将可能的PKC磷酸化位点突变为Ala或Glu，并使用电压钳测量研究响应于两种类型的PKC激活的全细胞Ba 2+（Ca 2+的替代物）电流。在目的II中，我们将确定参与直接或受体诱导的PKC敏感的Cav通道在爪蟾卵母细胞中表达的调制PKC同工酶。这将通过涉及PKC下调、激活诱导的易位、单个PKC同工酶的选择性丢失/抑制和“加回”研究的实验进行检查。在目的III中，我们将确定PKC同工酶的挥发性麻醉剂，氟烷或异氟烷调节爪蟾卵母细胞采用的方法在目的II。基于我们的研究结果，PKC的同工酶选择性激活剂或抑制剂可以与挥发性全身麻醉剂一起沿着使用，以i）增强麻醉剂的作用和ii）减少这些药剂由其非特异性作用引起的副作用。

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.