Cheng Lan, Sanguinetti Michael C
Nora Eccles Harrison Cardiovascular Research and Training Institute, Department of Physiology, University of Utah, Salt Lake City, UT 84112, USA.
Mol Pharmacol. 2009 May;75(5):1210-21. doi: 10.1124/mol.108.054437. Epub 2009 Feb 13.
Niflumic acid, 2-[[3-(trifluoromethyl)phenyl]amino]pyridine-3-carboxylic acid (NFA), is a nonsteroidal anti-inflammatory drug that also blocks or modifies the gating of many ion channels. Here, we investigated the effects of NFA on hyperpolarization-activated cyclic nucleotide-gated cation (HCN) pacemaker channels expressed in X. laevis oocytes using site-directed mutagenesis and the two-electrode voltage-clamp technique. Extracellular NFA acted rapidly and caused a slowing of activation and deactivation and a hyperpolarizing shift in the voltage dependence of HCN2 channel activation (-24.5 +/- 1.2 mV at 1 mM). Slowed channel gating and reduction of current magnitude was marked in oocytes treated with NFA, while clamped at 0 mV but minimal in oocytes clamped at -100 mV, indicating the drug preferentially interacts with channels in the closed state. NFA at 0.1 to 3 mM shifted the half-point for channel activation in a concentration-dependent manner, with an EC(50) of 0.54 +/- 0.068 mM and a predicted maximum shift of -38 mV. NFA at 1 mM also reduced maximum HCN2 conductance by approximately 20%, presumably by direct block of the pore. The rapid onset and state-dependence of NFA-induced changes in channel gating suggests an interaction with the extracellular region of the S4 transmembrane helix, the primary voltage-sensing domain of HCN2. Neutralization (by mutation to Gln) of any three of the outer four basic charged residues in S4, but not single mutations, abrogated the NFA-induced shift in channel activation. We conclude that NFA alters HCN2 gating by interacting with the extracellular end of the S4 voltage sensor domains.
尼氟灭酸，即2-[[3-(三氟甲基)苯基]氨基]吡啶-3-羧酸（NFA），是一种非甾体抗炎药，它还能阻断或改变许多离子通道的门控。在此，我们使用定点诱变和双电极电压钳技术，研究了NFA对非洲爪蟾卵母细胞中表达的超极化激活环核苷酸门控阳离子（HCN）起搏通道的影响。细胞外NFA作用迅速，导致HCN2通道激活和失活减慢，且通道激活的电压依赖性发生超极化偏移（1 mM时为-24.5±1.2 mV）。在用NFA处理的卵母细胞中，通道门控减慢和电流幅度降低在钳制于0 mV时很明显，但在钳制于-100 mV时最小，这表明该药物优先与处于关闭状态的通道相互作用。0.1至3 mM的NFA以浓度依赖性方式使通道激活的半值点发生偏移，EC(50)为0.54±0.068 mM，预测最大偏移为-38 mV。1 mM的NFA还使HCN2的最大电导降低了约20%，推测是通过直接阻断孔道实现的。NFA诱导的通道门控变化的快速起始和状态依赖性表明其与S4跨膜螺旋的细胞外区域相互作用，S4跨膜螺旋是HCN2的主要电压感应结构域。S4中外部四个带正电荷的碱性残基中的任意三个被中和（通过突变为Gln），而不是单个突变，消除了NFA诱导的通道激活偏移。我们得出结论，NFA通过与S4电压传感器结构域的细胞外末端相互作用来改变HCN(2)门控。
