Single-channel SCAM identifies pore-lining residues in the first extracellular loop and first transmembrane domains of Cx46 hemichannels.
Single-channel SCAM identifies pore-lining residues in the first extracellular loop and first transmembrane domains of Cx46 hemichannels.
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DOI：
10.1085/jgp.200308861
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发表时间：
2003-10
期刊：
影响因子：
--
通讯作者：
Verselis VK
中科院分区：
文献类型：
--
作者：
Kronengold J;Trexler EB;Bukauskas FF;Bargiello TA;Verselis VK
间隙连接（GJ）通道为信号分子的直接细胞间传递提供了重要途径。之前我们表明，第一个细胞外环结构域 (E1) 中的固定负电荷强烈影响 Cx46 形成的通道和半通道的电荷选择性、电导和整流。在这里，使用含有 Cx46 半通道的切除斑块，我们在单通道水平上对 E1 中的残基应用取代半胱氨酸可及性方法 (SCAM)，以确定它们是否是孔衬里。我们证明，E1 氨基端的残基 D51、G46 和 E43 可以在开放半通道中修饰，以添加到细胞质或细胞外侧面的带正电荷和负电荷的甲硫磺酸盐 (MTS) 试剂。沿孔长度的修饰的位置效应以及带相反电荷的修饰试剂对半通道电导和整流的相反作用与通道孔中的放置一致，并且表明可接近残基的侧链对离子通量的主要静电影响。 MTS-EA+、MTS-ET+ 或 MTS-ES− 修饰的半通道难以进一步修饰，并且静电模拟带正电 MTS 试剂修饰引起的带正电残基的取代效果相似，表明所有六个亚基可能都被修饰。 MTS-ET+ 引起的电导大幅降低，表现为单通道电流的逐步降低，表明各个亚基发生了反应。使用 MTS-ET+ 将单通道 SCAM 延伸到第一个跨膜结构域 TM1，揭示了 A39 和 L35 细胞外端的持续可访问性。 TM3 中的拓扑互补区域没有显示出反应性的证据。结构模型显示细胞外间隙中的 GJ 通道具有连续的蛋白质内壁和外壁。如果代表开放通道和半通道，这些数据表明 E1 构成了内部成孔壁的重要部分，而 TM1 则在跨膜跨度的细胞外部分中充当孔衬里。
Gap junction (GJ) channels provide an important pathway for direct intercellular transmission of signaling molecules. Previously we showed that fixed negative charges in the first extracellular loop domain (E1) strongly influence charge selectivity, conductance, and rectification of channels and hemichannels formed of Cx46. Here, using excised patches containing Cx46 hemichannels, we applied the substituted cysteine accessibility method (SCAM) at the single channel level to residues in E1 to determine if they are pore-lining. We demonstrate residues D51, G46, and E43 at the amino end of E1 are accessible to modification in open hemichannels to positively and negatively charged methanethiosulfonate (MTS) reagents added to cytoplasmic or extracellular sides. Positional effects of modification along the length of the pore and opposing effects of oppositely charged modifying reagents on hemichannel conductance and rectification are consistent with placement in the channel pore and indicate a dominant electrostatic influence of the side chains of accessible residues on ion fluxes. Hemichannels modified by MTS-EA+, MTS-ET+, or MTS-ES− were refractory to further modification and effects of substitutions with positively charged residues that electrostatically mimicked those caused by modification with the positively charged MTS reagents were similar, indicating all six subunits were likely modified. The large reductions in conductance caused by MTS-ET+ were visible as stepwise reductions in single-channel current, indicative of reactions occurring at individual subunits. Extension of single-channel SCAM using MTS-ET+ into the first transmembrane domain, TM1, revealed continued accessibility at the extracellular end at A39 and L35. The topologically complementary region in TM3 showed no evidence of reactivity. Structural models show GJ channels in the extracellular gap to have continuous inner and outer walls of protein. If representative of open channels and hemichannels, these data indicate E1 as constituting a significant portion of this inner, pore-forming wall, and TM1 contributing as pore-lining in the extracellular portion of transmembrane span.
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DOI：
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发表时间：
1999-09
期刊：
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影响因子：
--
作者：
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