Talavera Karel, Staes Mik, Janssens Annelies, Droogmans Guy, Nilius Bernd
Laboratorium voor Fysiologie, KU Leuven, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium.
J Gen Physiol. 2004 Sep;124(3):225-38. doi: 10.1085/jgp.200409050. Epub 2004 Aug 16.
Arachidonic acid (AA) modulates T-type Ca(2+) channels and is therefore a potential regulator of diverse cell functions, including neuronal and cardiac excitability. The underlying mechanism of modulation is unknown. Here we analyze the effects of AA on the T-type Ca(2+) channel alpha(1G) heterologously expressed in HEK-293 cells. AA inhibited alpha(1G) currents within a few minutes, regardless of preceding exposure to inhibitors of AA metabolism (ETYA and 17-ODYA). Current inhibition was also observed in cell-free inside-out patches, indicating a membrane-delimited interaction of AA with the channel. AA action was consistent with a decrease of the open probability without changes in the size of unitary currents. AA shifted the inactivation curve to more negative potentials, increased the speed of macroscopic inactivation, and decreased the extent of recovery from inactivation at -80 mV but not at -110 mV. AA induced a slight increase of activation near the threshold and did not significantly change the deactivation kinetics or the rectification pattern. We observed a tonic current inhibition, regardless of whether the channels were held in resting or inactivated states during AA perfusion, suggesting a state-independent interaction with the channel. Model simulations indicate that AA inhibits T-type currents by switching the channels into a nonavailable conformation and by affecting transitions between inactivated states, which results in the negative shift of the inactivation curve. Slow-inactivating alpha(1G) mutants showed an increased affinity for AA with respect to the wild type, indicating that the structural determinants of fast inactivation are involved in the AA-channel interaction.
花生四烯酸（AA）可调节T型Ca(2+)通道，因此是包括神经元和心脏兴奋性在内的多种细胞功能的潜在调节因子。其调节的潜在机制尚不清楚。在此，我们分析了AA对在HEK-293细胞中异源表达的T型Ca(2+)通道α(1G)的影响。无论之前是否暴露于AA代谢抑制剂（ETYA和17-ODYA），AA在几分钟内即可抑制α(1G)电流。在无细胞的内向外膜片中也观察到电流抑制，这表明AA与通道存在膜限定的相互作用。AA的作用与开放概率降低一致，而单位电流大小不变。AA使失活曲线向更负的电位移动，增加了宏观失活的速度，并降低了在-80 mV而非-110 mV时从失活状态恢复的程度。AA在阈值附近诱导激活略有增加，且未显著改变失活动力学或整流模式。无论在AA灌注期间通道是处于静息状态还是失活状态，我们都观察到了持续性电流抑制，这表明AA与通道的相互作用不依赖于状态。模型模拟表明，AA通过将通道转换为不可用的构象并影响失活状态之间的转换来抑制T型电流，这导致失活曲线负移。慢失活α(1G)突变体相对于野生型对AA的亲和力增加，表明快速失活的结构决定因素参与了AA与通道的相互作用。
