The two-pore-domain potassium channel (K_2P channel) THIK-1 has several predicted protein kinase A (PKA) phosphorylation sites. In trying to elucidate whether THIK-1 is regulated via PKA we expressed THIK-1 channels in a mammalian cell line (CHO cells) and used the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) as a pharmacological tool to induce activation of PKA. Using the whole-cell patch-clamp recording we found that THIK-1 currents were inhibited by application of IBMX with an IC₅₀ of 120 µM. Surprisingly, intracellular application of IBMX or of the second messenger cAMP via the patch pipette had no effect on THIK-1 currents. In contrast, extracellular application of IBMX produced a rapid and reversible inhibition of THIK-1. In patch-clamp experiments with outside-out patches, THIK‑1 currents were also inhibited by extracellular application of IBMX. Expression of THIK-1 channels in Xenopus oocytes was used to compare wild-type channels with mutated channels. Mutation of the putative PKA phosphorylation sites did not change the inhibitory effect of IBMX on THIK-1 currents. Mutational analysis of all residues of the (extracellular) helical cap of THIK-1 showed that mutation of the arginine residue at position 92, which is in the C2-P1 linker, markedly reduced the inhibitory effect of IBMX. This flexible linker region, which is unique for each K_2P channel subtype, may be a possible target of channel-specific blockers.

中文翻译：

---

磷酸二酯酶抑制剂IBMX从细胞外一侧阻断钾通道THIK-1。

两孔结构域钾通道（K _2P通道）THIK-1具有几个预测的蛋白激酶A（PKA）磷酸化位点。在试图阐明THIK-1是否通过PKA调控的过程中，我们在哺乳动物细胞系（CHO细胞）中表达了THIK-1通道，并使用了磷酸二酯酶抑制剂3-异丁基-1-甲基黄嘌呤（IBMX）作为药理学工具来诱导激活PKA。使用全细胞膜片钳记录，我们发现THIK-1电流被带有IC ₅₀的IBMX施加抑制120 µM。出人意料的是，通过贴片移液器在细胞内应用IBMX或第二信使cAMP对THIK-1电流没有影响。相反，在IBMX的细胞外应用产生THIK-1的快速和可逆的抑制作用。在用外而外的补丁进行补丁钳实验中，THKK-1电流也受到细胞外应用IBMX的抑制。THIK-1通道在非洲爪蟾中的表达卵母细胞用于比较野生型通道和突变通道。假定的PKA磷酸化位点的突变不会改变IBMX对THIK-1电流的抑制作用。对THIK-1（细胞外）螺旋帽所有残基的突变分析表明，在C2-P1接头中92位的精氨酸残基突变显着降低了IBMX的抑制作用。对于每个K _2P通道亚型而言，唯一的灵活链接区可能是特定于通道的阻断剂的目标。