The two-pore domain potassium channel (K2P-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 linker between cap helix 2 and pore helix 1, markedly reduced the inhibitory effect of IBMX. This flexible linker region, which is unique for each K2P-channel subtype, may be a possible target of channel-specific blockers. SIGNIFICANCE STATEMENT: The potassium channel THIK-1 is strongly expressed in the central nervous system. We studied the effect of 3-isobutyl-1-methyl-xanthine (IBMX) on THIK-1 currents. IBMX inhibits breakdown of cAMP and thus activates protein kinase A (PKA). Surprisingly, THIK-1 current was inhibited when IBMX was applied from the extracellular side of the membrane, but not from the intracellular side. Our results suggest that IBMX binds directly to the channel and that the inhibition of THIK-1 current was not related to activation of PKA.

中文翻译：

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磷酸二酯酶抑制剂 IBMX 从细胞外侧阻断钾通道 THIK-1。


双孔结构域钾通道（K2P 通道）THIK-1 具有多个预测的蛋白激酶 A (PKA) 磷酸化位点。为了阐明 THIK-1 是否通过 PKA 调节，我们在哺乳动物细胞系（CHO 细胞）中表达 THIK-1 通道，并使用磷酸二酯酶抑制剂 3-异丁基-1-甲基-黄嘌呤 (IBMX) 作为药理学工具诱导PKA的激活。使用全细胞膜片钳记录，我们发现 THIK-1 电流被 IBMX 的应用所抑制，IC50 为 120 µM。令人惊讶的是，通过贴片移液管在细胞内应用IBMX或第二信使cAMP对THIK-1电流没有影响。相比之下，IBMX 的细胞外应用对 THIK-1 产生快速且可逆的抑制。在使用外侧膜片的膜片钳实验中，IBMX 的细胞外应用也抑制了 THIK-1 电流。使用爪蟾卵母细胞中 THIK-1 通道的表达来比较野生型通道与突变通道。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，位于帽螺旋2和孔螺旋1之间的连接子中的第92位精氨酸残基的突变显着降低了IBMX的抑制作用。这种灵活的连接子区域对于每个 K2P 通道子类型都是唯一的，可能是通道特异性阻断剂的可能目标。意义陈述：钾通道 THIK-1 在中枢神经系统中强烈表达。我们研究了 3-异丁基-1-甲基-黄嘌呤 (IBMX) 对 THIK-1 电流的影响。 IBMX 抑制 cAMP 分解，从而激活蛋白激酶 A (PKA)。 令人惊讶的是，当从膜的细胞外侧施加 IBMX 时，THIK-1 电流受到抑制，但从细胞内侧施加则不受抑制。我们的结果表明 IBMX 直接与通道结合，并且 THIK-1 电流的抑制与 PKA 的激活无关。