Yu Haibo, Zou Beiyan, Wang Xiaoliang, Li Min
State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
The Solomon H. Snyder Department of Neuroscience, High Throughput Biology Center and Johns Hopkins Ion Channel Center, Johns Hopkins University, Baltimore, MD, USA.
Br J Pharmacol. 2015 Jul;172(13):3370-82. doi: 10.1111/bph.13127. Epub 2015 Apr 24.
A-type potassium channels (IA) are important proteins for modulating neuronal membrane excitability. The expression and activity of Kv 4.2 channels are critical for neurological functions and pharmacological inhibitors of Kv 4.2 channels may have therapeutic potential for Fragile X syndrome. While screening various compounds, we identified tyrphostin AG879, a tyrosine kinase inhibitor, as a Kv 4.2 inhibitor from. In the present study we characterized the effect of AG879 on cloned Kv 4.2/Kv channel-interacting protein 2 (KChIP2) channels.
To screen the library of pharmacologically active compounds, the thallium flux assay was performed on HEK-293 cells transiently-transfected with Kv 4.2 cDNA using the Maxcyte transfection system. The effects of AG879 were further examined on CHO-K1 cells expressing Kv 4.2/KChIP2 channels using a whole-cell patch-clamp technique.
Tyrphostin AG879 selectively and dose-dependently inhibited Kv 4.2 and Kv 4.3 channels. In Kv 4.2/KChIP2 channels, AG879 induced prominent acceleration of the inactivation rate, use-dependent block and slowed the recovery from inactivation. AG879 induced a hyperpolarizing shift in the voltage-dependence of the steady-state inactivation of Kv 4.2 channels without apparent effect on the V1/2 of the voltage-dependent activation. The blocking effect of AG879 was enhanced as channel inactivation increased. Furthermore, AG879 significantly inhibited the A-type potassium currents in the cultured hippocampus neurons.
AG879 was identified as a selective and potent inhibitor the Kv 4.2 channel. AG879 inhibited Kv 4.2 channels by preferentially interacting with the open state and further accelerating their inactivation.
A型钾通道（IA）是调节神经元膜兴奋性的重要蛋白质。Kv 4.2通道的表达和活性对神经功能至关重要，Kv 4.2通道的药理学抑制剂可能对脆性X综合征具有治疗潜力。在筛选各种化合物时，我们从酪氨酸激酶抑制剂中鉴定出 tyrphostin AG879作为Kv 4.2抑制剂。在本研究中，我们表征了AG879对克隆的Kv 4.2/钾通道相互作用蛋白2（KChIP2）通道的影响。
为了筛选药理活性化合物库，使用Maxcyte转染系统对瞬时转染Kv 4.2 cDNA的HEK-293细胞进行铊通量测定。使用全细胞膜片钳技术进一步检测AG879对表达Kv 4.2/KChIP2通道的CHO-K1细胞的影响。
Tyrphostin AG879选择性地且剂量依赖性地抑制Kv 4.2和Kv 4.3通道。在Kv 4.2/KChIP2通道中，AG879显著加速失活速率、使用依赖性阻断并减缓失活后的恢复。AG879在Kv 4.2通道稳态失活的电压依赖性上诱导超极化偏移，而对电压依赖性激活的V1/2没有明显影响。随着通道失活增加，AG879的阻断作用增强。此外，AG879显著抑制培养的海马神经元中的A型钾电流。
AG879被鉴定为Kv 4.2通道的选择性强效抑制剂。AG879通过优先与开放状态相互作用并进一步加速其失活来抑制Kv 4.2通道。
