Ca2+-dependent inactivation of CaV1.2 channels prevents Gd3+ block: does Ca2+ block the pore of inactivated channels?
Ca2+-dependent inactivation of CaV1.2 channels prevents Gd3+ block: does Ca2+ block the pore of inactivated channels?
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DOI：
10.1085/jgp.200709734
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发表时间：
2007-06
影响因子：
3.8
通讯作者：
Shirokov, Roman
中科院分区：
文献类型：
--
作者：
Babich, Olga;Matveev, Victor;Harris, Andrew L;Shirokov, Roman
镧系元素钆（Gd 3+）在选择性过滤器上阻断CaV 1.2通道。在这里，我们研究了Gd 3+块是否干扰钙依赖性失活，这需要通过相同的网站钙2+进入。使用短暂的脉冲到200 mV，缓解Gd 3+阻滞，但不失活，我们监测了开放和开放阻断通道的比例如何在失活过程中变化。我们发现，被阻断的通道失活程度要低得多。这是由于Gd 3+阻断Ca 2+内流而增强失活所预期的。然而，我们还发现，Gd 3+块的程度没有改变时，失活减少废除的Ca 2 +/钙调蛋白相互作用，表明Gd 3+不阻断失活的通道。因此，Gd 3+阻断和失活是相互排斥的，表明在共同位点的作用。这些观察结果表明，失活导致在选择性过滤器的变化，要么隐藏的Gd 3+网站或降低其亲和力，或Ca2+占据在失活通道的选择性过滤器的结合位点。后一种可能性得到先前发现的支持，即选择性EEEE基因座的EEQE突变没有Ca 2+依赖性失活（Zong Z.Q.，J.Y. Zhou和T.田边。1994.生物化学、生物物理通信资源201：1117 - 11123），并且当从细胞外培养基中除去Ca2+时，Ca2+失活的通道传导Na+（Babich O.，D. Isaev和R.希罗科夫2005. 565：709 - 717）。基于这些结果，我们提出，失活增加的选择性过滤器的钙离子的亲和力，使钙离子阻塞的孔。一个最小的模型，其中失活的“门”是渗透离子的选择性过滤器的亲和力的增加，成功地模拟了特征U形电压依赖性的失活在Ca2+。
Lanthanide gadolinium (Gd3+) blocks CaV1.2 channels at the selectivity filter. Here we investigated whether Gd3+ block interferes with Ca2+-dependent inactivation, which requires Ca2+ entry through the same site. Using brief pulses to 200 mV that relieve Gd3+ block but not inactivation, we monitored how the proportions of open and open-blocked channels change during inactivation. We found that blocked channels inactivate much less. This is expected for Gd3+ block of the Ca2+ influx that enhances inactivation. However, we also found that the extent of Gd3+ block did not change when inactivation was reduced by abolition of Ca2+/calmodulin interaction, showing that Gd3+ does not block the inactivated channel. Thus, Gd3+ block and inactivation are mutually exclusive, suggesting action at a common site. These observations suggest that inactivation causes a change at the selectivity filter that either hides the Gd3+ site or reduces its affinity, or that Ca2+ occupies the binding site at the selectivity filter in inactivated channels. The latter possibility is supported by previous findings that the EEQE mutation of the selectivity EEEE locus is void of Ca2+-dependent inactivation (Zong Z.Q., J.Y. Zhou, and T. Tanabe. 1994. Biochem. Biophys. Res. Commun. 201:1117–11123), and that Ca2+-inactivated channels conduct Na+ when Ca2+ is removed from the extracellular medium (Babich O., D. Isaev, and R. Shirokov. 2005. J. Physiol. 565:709–717). Based on these results, we propose that inactivation increases affinity of the selectivity filter for Ca2+ so that Ca2+ ion blocks the pore. A minimal model, in which the inactivation “gate” is an increase in affinity of the selectivity filter for permeating ions, successfully simulates the characteristic U-shaped voltage dependence of inactivation in Ca2+.