Modulation of Calcium-Dependent Inactivation of L-Type Ca²⁺ Channels via β-Adrenergic Signaling in Thalamocortical Relay Neurons

Neuronal high-voltage-activated (HVA) Ca2+ channels are rapidly inactivated by a mechanism that is termed Ca2+-dependent inactivation (CDI). In this study we have shown that β-adrenergic receptor (βAR) stimulation inhibits CDI in rat thalamocortical (TC) relay neurons. This effect can be blocked by inhibition of cAMP-dependent protein kinase (PKA) with a cell-permeable inhibitor (myristoylated protein kinase inhibitor-(14–22)-amide) or A-kinase anchor protein (AKAP) St-Ht31 inhibitory peptide, suggesting a critical role of these molecules downstream of the receptor. Moreover, inhibition of protein phosphatases (PP) with okadaic acid revealed the involvement of phosphorylation events in modulation of CDI after βAR stimulation. Double fluorescence immunocytochemistry and pull down experiments further support the idea that modulation of CDI in TC neurons via βAR stimulation requires a protein complex consisting of CaV1.2, PKA and proteins from the AKAP family. All together our data suggest that AKAPs mediate targeting of PKA to L-type Ca2+ channels allowing their phosphorylation and thereby modulation of CDI.神经元高电压激活型（HVA）钙离子通道可通过被称为钙离子依赖性失活（CDI）的机制快速失活。本研究证实，β肾上腺素能受体（βAR）激活可抑制大鼠丘脑皮层（TC）中继神经元中的CDI。该效应可被细胞通透性抑制剂——肉豆蔻酰化蛋白激酶抑制剂-(14–22)-酰胺，或A激酶锚定蛋白（AKAP）St-Ht31抑制肽对环磷酸腺苷依赖的蛋白激酶（PKA）的阻断所抵消，提示上述分子在受体下游发挥关键作用。此外，使用冈田酸抑制蛋白磷酸酶（PP）的实验揭示，βAR激活后，CDI的调控依赖于磷酸化事件。双重荧光免疫细胞化学技术与下拉实验进一步证实，TC神经元中βAR激活对CDI的调控需要由CaV1.2、PKA以及AKAP家族蛋白组成的蛋白复合物参与。综合所有实验数据，本研究表明AKAP介导PKA靶向结合至L型钙离子通道，使其发生磷酸化，进而实现对CDI的调控。