Functionally active t1-t1 interfaces revealed by the accessibility of intracellular thiolate groups in kv4 channels.
Functionally active t1-t1 interfaces revealed by the accessibility of intracellular thiolate groups in kv4 channels.
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DOI：
10.1085/jgp.200509288
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发表时间：
2005-07
期刊：
影响因子：
--
通讯作者：
Covarrubias M
中科院分区：
文献类型：
--
作者：
Wang G;Shahidullah M;Rocha CA;Strang C;Pfaffinger PJ;Covarrubias M
电压依赖性K+通道的门控涉及控制孔开放的跨膜区域的运动。然而，关于大的细胞内通道结构域对门控基础的构象变化的贡献知之甚少。在这里，我们研究了Kv 4通道的细胞内区域的功能作用，通过探测相关的半胱氨酸与巯基特异性试剂。我们发现，试剂应用到细胞内侧的由内而外补丁的结果在时间依赖性的不可逆抑制Kv4.1和Kv4.3电流。在Kv 4特异性辅助亚基存在或不存在的情况下，突变和电生理分析表明，14个细胞内半胱氨酸中没有一个是通道门控所必需的。C110，C131，和C132在亚基间的四聚化结构域（T1）的界面是目标负责的不可逆抑制甲硫基磺酸盐衍生物（MTSET）。该结果是令人惊讶的，因为Kv 4-T1晶体的结构研究预测了通过组成性高亲和力Zn 2+配位来保护目标硫醇盐基团。此外，添加的Zn 2+或有效的Zn 2+螯合剂（TPEN）不会显着调节MTSET对C110、C131或C132的可及性;此外，当三个关键半胱氨酸仍然作为可能的靶点时，激活状态的MTSET修饰速率比静息状态快200倍。生物化学实验证实了MTS试剂对完整的α-亚基和纯化的四聚体T1结构域的化学修饰。这些结果最终表明，Kv 4通道的T1-T1界面是功能活性和动态的，并且该界面中的关键反应性硫醇基团可能不受Zn 2+结合的保护。
Gating of voltage-dependent K+ channels involves movements of membrane-spanning regions that control the opening of the pore. Much less is known, however, about the contributions of large intracellular channel domains to the conformational changes that underlie gating. Here, we investigated the functional role of intracellular regions in Kv4 channels by probing relevant cysteines with thiol-specific reagents. We find that reagent application to the intracellular side of inside-out patches results in time-dependent irreversible inhibition of Kv4.1 and Kv4.3 currents. In the absence or presence of Kv4-specific auxiliary subunits, mutational and electrophysiological analyses showed that none of the 14 intracellular cysteines is essential for channel gating. C110, C131, and C132 in the intersubunit interface of the tetramerization domain (T1) are targets responsible for the irreversible inhibition by a methanethiosulfonate derivative (MTSET). This result is surprising because structural studies of Kv4-T1 crystals predicted protection of the targeted thiolate groups by constitutive high-affinity Zn2+ coordination. Also, added Zn2+ or a potent Zn2+ chelator (TPEN) does not significantly modulate the accessibility of MTSET to C110, C131, or C132; and furthermore, when the three critical cysteines remained as possible targets, the MTSET modification rate of the activated state is ∼200-fold faster than that of the resting state. Biochemical experiments confirmed the chemical modification of the intact α-subunit and the purified tetrameric T1 domain by MTS reagents. These results conclusively demonstrate that the T1–T1 interface of Kv4 channels is functionally active and dynamic, and that critical reactive thiolate groups in this interface may not be protected by Zn2+ binding.
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