N-terminal inactivation domains of beta subunits are protected from trypsin digestion by binding within the antechamber of BK channels.
N-terminal inactivation domains of beta subunits are protected from trypsin digestion by binding within the antechamber of BK channels.
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DOI：
10.1085/jgp.200810079
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发表时间：
2009-03
期刊：
影响因子：
--
通讯作者：
Lingle CJ
中科院分区：
文献类型：
--
作者：
Zhang Z;Zeng XH;Xia XM;Lingle CJ
辅助性β亚基的N端产生大电导Ca2+活化的K+ (BK)通道失活，首先通过侧门户进入分离BK孔模块和大c端细胞质结构域的前厅，从而到达它们的孔阻塞位置。先前的研究表明，β2亚基失活结构域在以失活构象结合时不受胰蛋白酶的消化。其他结果表明，即使当通道关闭时，失活结构域在前厅内结合时也可以保护胰蛋白酶不被消化。在这里，我们对该模型进行了额外的测试，并检验了其对其他β亚基N端的适用性。首先，我们发现β2失活片段的特定突变可以在封闭通道条件下加速胰蛋白酶的消化，这支持了β2 N末端通过前厅内的结合受到保护的观点。其次，我们发现细胞质通道阻断剂区分了失活介导的保护和封闭通道条件下的保护，暗示了两个不同的保护位点。总之，这些结果证实了β 2n末端可以通过在不同于BK中心腔的某些部位相互作用而占据BK通道前厅的想法。相比之下，β3a N端被消化的速度比β2 N端快10倍以上。对导致消化率差异的因素的分析表明，前厅内N端的结合限制了胰蛋白酶对可消化的碱性残基的可及性，即使这些残基位于前厅外。我们的分析表明，多达两个N端可能同时受到保护而不被消化。这些结果表明，除了直接参与失活的位点外，失活结构域还有结合位点。
N termini of auxiliary β subunits that produce inactivation of large-conductance Ca2+-activated K+ (BK) channels reach their pore-blocking position by first passing through side portals into an antechamber separating the BK pore module and the large C-terminal cytosolic domain. Previous work indicated that the β2 subunit inactivation domain is protected from digestion by trypsin when bound in the inactivated conformation. Other results suggest that, even when channels are closed, an inactivation domain can also be protected from digestion by trypsin when bound within the antechamber. Here, we provide additional tests of this model and examine its applicability to other β subunit N termini. First, we show that specific mutations in the β2 inactivation segment can speed up digestion by trypsin under closed-channel conditions, supporting the idea that the β2 N terminus is protected by binding within the antechamber. Second, we show that cytosolic channel blockers distinguish between protection mediated by inactivation and protection under closed-channel conditions, implicating two distinct sites of protection. Together, these results confirm the idea that β2 N termini can occupy the BK channel antechamber by interaction at some site distinct from the BK central cavity. In contrast, the β3a N terminus is digested over 10-fold more quickly than the β2 N terminus. Analysis of factors that contribute to differences in digestion rates suggests that binding of an N terminus within the antechamber constrains the trypsin accessibility of digestible basic residues, even when such residues are positioned outside the antechamber. Our analysis indicates that up to two N termini may simultaneously be protected from digestion. These results indicate that inactivation domains have sites of binding in addition to those directly involved in inactivation.
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