Ma Zhongming, Wong Kin Yu, Horrigan Frank T
Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
J Gen Physiol. 2008 May;131(5):483-502. doi: 10.1085/jgp.200809980.
Copper is an essential trace element that may serve as a signaling molecule in the nervous system. Here we show that extracellular Cu2+ is a potent inhibitor of BK and Shaker K+ channels. At low micromolar concentrations, Cu2+ rapidly and reversibly reduces macrosocopic K+ conductance (G(K)) evoked from mSlo1 BK channels by membrane depolarization. GK is reduced in a dose-dependent manner with an IC50 and Hill coefficient of 2 microM and 1.0, respectively. Saturating 100 microM Cu2+ shifts the GK-V relation by +74 mV and reduces G(Kmax) by 27% without affecting single channel conductance. However, 100 microM Cu2+ fails to inhibit GK when applied during membrane depolarization, suggesting that Cu2+ interacts poorly with the activated channel. Of other transition metal ions tested, only Zn2+ and Cd2+ had significant effects at 100 microM with IC(50)s > 0.5 mM, suggesting the binding site is Cu2+ selective. Mutation of external Cys or His residues did not alter Cu2+ sensitivity. However, four putative Cu2+-coordinating residues were identified (D133, Q151, D153, and R207) in transmembrane segments S1, S2, and S4 of the mSlo1 voltage sensor, based on the ability of substitutions at these positions to alter Cu2+ and/or Cd2+ sensitivity. Consistent with the presence of acidic residues in the binding site, Cu2+ sensitivity was reduced at low extracellular pH. The three charged positions in S1, S2, and S4 are highly conserved among voltage-gated channels and could play a general role in metal sensitivity. We demonstrate that Shaker, like mSlo1, is much more sensitive to Cu2+ than Zn2+ and that sensitivity to these metals is altered by mutating the conserved positions in S1 or S4 or reducing pH. Our results suggest that the voltage sensor forms a state- and pH-dependent, metal-selective binding pocket that may be occupied by Cu2+ at physiologically relevant concentrations to inhibit activation of BK and other channels.
铜是一种必需的微量元素，可能在神经系统中作为信号分子发挥作用。在此我们表明，细胞外Cu2+是BK和Shaker钾通道的强效抑制剂。在低微摩尔浓度下，Cu2+能迅速且可逆地降低由膜去极化从mSlo1 BK通道诱发的宏观钾电导（G(K)）。GK以剂量依赖方式降低，IC50和希尔系数分别为2 microM和1.0。饱和的100 microM Cu2+使GK-V关系正向移动74 mV，并使G(Kmax)降低27%，而不影响单通道电导。然而，在膜去极化期间施加100 microM Cu2+时未能抑制GK，这表明Cu2+与激活通道的相互作用较差。在测试的其他过渡金属离子中，只有Zn2+和Cd2+在100 microM时具有显著作用，IC(50) > 0.5 mM，这表明结合位点对Cu2+具有选择性。外部半胱氨酸或组氨酸残基的突变不会改变Cu2+敏感性。然而，基于这些位置的取代改变Cu2+和/或Cd2+敏感性的能力，在mSlo1电压传感器的跨膜片段S1、S2和S4中鉴定出四个假定的Cu2+配位残基（D133、Q151、D153和R207）。与结合位点中存在酸性残基一致，在低细胞外pH值下Cu2+敏感性降低。S1、S2和S4中的三个带电位置在电压门控通道中高度保守，可能在金属敏感性方面发挥普遍作用。我们证明，与mSlo1一样，Shaker对Cu2+的敏感性远高于Zn2+，并且通过突变S1或S4中的保守位置或降低pH值，对这些金属的敏感性会发生改变。我们的结果表明，电压传感器形成了一个依赖状态和pH的金属选择性结合口袋，在生理相关浓度下可能被Cu2+占据，以抑制BK和其他通道的激活。
