Departamento de Farmacobiología, Cinvestav, Sede Sur., Mexico City, Mexico.
Unidad de Investigación Médica en Inmunología e Infectología, Centro Médico Nacional, La Raza, Instituto Mexicano del Seguro Social., Mexico City, Mexico.
J Neurochem. 2021 Jun;157(6):2039-2054. doi: 10.1111/jnc.15204. Epub 2020 Oct 19.
PKC and PKA phosphorylation inhibit TREK-1 channels downstream of G protein-coupled receptor activation in vitro. However, the role of phosphorylation of TREK-1 in neuropathic pain is unknown. The purpose of this study was to investigate whether altered TREK-1 channel function by PKA and PKC modulators contributes to antiallodynia in neuropathic rats. Furthermore, we investigated if the in vitro described sites for PKC and PKA phosphorylation (S300 and S333, respectively) participate in the modulation of TREK-1 in naïve and neuropathic rats. L5/L6 spinal nerve ligation (SNL) induced tactile allodynia. Intrathecal injection of BL-1249 (TREK-1 activator) reversed nerve injury-induced tactile allodynia, whereas spadin (TREK-1 blocker) produced tactile allodynia in naïve rats and reversed the antiallodynic effect induced by BL-1249 in neuropathic rats. Intrathecal administration of rottlerin or Rp-cAMPs (PKC and PKA inhibitors, respectively) enhanced the antiallodynia observed with BL-1249 in neuropathic rats. In contrast, pretreatment with PdBu or forskolin (PKC and PKA activators, respectively) reduced the BL-1249-induced antiallodynia. Intrathecal injection of two high-activity TREK-1 recombinant channels, using a in vivo transfection method with lipofectamine, with mutations at PKC/PKA phosphosites (S300A and S333A) reversed tactile allodynia in neuropathic rats, with no effect in naïve rats. In contrast, transfection of two low-activity TREK-1 recombinant channels with phosphomimetic mutations at those sites (S300D and S333D) produced tactile allodynia in naïve rats and interfered with antiallodynic effects of rottlerin/BL-1249 or Rp-cAMPs/BL-1249. Data suggest that TREK-1 channel activity can be dynamically tuned in vivo by PKC/PKA to provoke allodynia and modulate its antiallodynic role in neuropathic pain.
蛋白激酶 C（PKC）和蛋白激酶 A（PKA）磷酸化可抑制体外 G 蛋白偶联受体激活后 TREK-1 通道的开放。然而，TREK-1 磷酸化在神经病理性疼痛中的作用尚不清楚。本研究旨在探讨 PKA 和 PKC 调节剂改变 TREK-1 通道功能是否有助于神经病理性大鼠的抗痛觉过敏。此外，我们还研究了在体外描述的 PKC 和 PKA 磷酸化位点（分别为 S300 和 S333）是否参与调节在正常和神经病理性大鼠中的 TREK-1。L5/L6 脊神经结扎（SNL）诱导触觉痛觉过敏。鞘内注射 BL-1249（TREK-1 激活剂）逆转神经损伤引起的触觉痛觉过敏，而 spadin（TREK-1 阻断剂）在正常大鼠中引起触觉痛觉过敏，并逆转神经病理性大鼠中 BL-1249 引起的抗痛觉过敏作用。鞘内给予 rottlerin 或 Rp-cAMPs（分别为 PKC 和 PKA 抑制剂）增强了神经病理性大鼠中 BL-1249 引起的抗痛觉过敏。相反，用 PdBu 或 forskolin（分别为 PKC 和 PKA 激活剂）预处理可降低 BL-1249 诱导的抗痛觉过敏。采用脂质体体内转染方法，鞘内注射两种高活性 TREK-1 重组通道，这些通道具有 PKC/PKA 磷酸化位点（S300A 和 S333A）的突变，逆转了神经病理性大鼠的触觉痛觉过敏，而在正常大鼠中没有作用。相反，转染两种低活性 TREK-1 重组通道，这些通道在这些位点具有磷酸模拟突变（S300D 和 S333D），在正常大鼠中引起触觉痛觉过敏，并干扰了 rottlerin/BL-1249 或 Rp-cAMPs/BL-1249 的抗痛觉过敏作用。数据表明，TREK-1 通道活性可在体内被 PKC/PKA 动态调节，引发痛觉过敏，并调节其在神经病理性疼痛中的抗痛觉过敏作用。
