Kronengold J, Trexler E B, Bukauskas F F, Bargiello T A, Verselis V K
Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, New York, NY 10461, USA.
J Gen Physiol. 2003 Oct;122(4):389-405. doi: 10.1085/jgp.200308861. Epub 2003 Sep 15.
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.
间隙连接（GJ）通道为信号分子的直接细胞间传递提供了一条重要途径。此前我们表明，第一细胞外环域（E1）中的固定负电荷强烈影响由Cx46形成的通道和半通道的电荷选择性、电导率和整流特性。在这里，我们使用含有Cx46半通道的切除膜片，在单通道水平上对E1中的残基应用半胱氨酸替代可及性方法（SCAM），以确定它们是否位于孔道内衬。我们证明，在开放的半通道中，E1氨基端的残基D51、G46和E43可被添加到细胞质或细胞外侧的带正电和负电的甲硫基磺酸盐（MTS）试剂修饰。沿孔道长度的修饰位置效应以及带相反电荷的修饰试剂对半通道电导率和整流的相反效应与位于通道孔道内一致，并表明可及残基侧链对离子通量有主要的静电影响。用MTS-EA+、MTS-ET+或MTS-ES-修饰的半通道对进一步修饰具有抗性，并且用带正电残基替代产生的效应与用带正电MTS试剂修饰产生的效应在静电上相似，这表明所有六个亚基可能都被修饰了。MTS-ET+引起的电导率大幅降低表现为单通道电流的逐步降低，这表明反应发生在单个亚基上。使用MTS-ET+将单通道SCAM扩展到第一个跨膜结构域TM1，发现在细胞外端的A39和L35处仍可及。TM3中的拓扑互补区域没有反应性的证据。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。如果这些数据代表开放通道和半通道，那么它们表明E1构成了这个内部孔道形成壁的很大一部分，而TM1在跨膜跨度的细胞外部分作为孔道内衬发挥作用。
