We have measured the permeability of rhodamine-6G across Cx43 hemichannels reconstituted on a pipette tip. Cx43 hemichannels were overexpressed in Sf9 cells, and affinity-purified. The hemichannels were reconstituted in a lipid bilayer on a pipette tip by the tip-dip method. R6G in the pipette permeated across the channels into the bath. The permeability of R6G was quantified by measuring R6G concentration in the bath after several hours by surface enhanced Raman spectroscopy (SERS) with 100 nm silver colloid particles. The ratio of the permeability of dye to salt, as extracted by this combined electrical-SERS technique, is compatible with similar ratios for other dyes across whole gap junction channels. The results for the permeability ratio were further compared to fluorescence measurements. The novel combination of patch and SERS techniques can be extended to quantifying the transport of biologically significant non-fluorescent molecules, such as cAMP and IP3, across 1 nm sized pores, such as the gap junction channel.

中文翻译：

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通过膜片钳和表面增强拉曼光谱的新颖组合实现 R6G 跨 Cx43 半通道的渗透性


我们测量了罗丹明-6G 在移液器吸头上重建的 Cx43 半通道的渗透性。 Cx43 半通道在 Sf9 细胞中过表达，并进行亲和纯化。通过尖端浸入法在移液管尖端上的脂质双层中重建半通道。移液器中的 R6G 穿过通道渗透到浴中。 R6G 的渗透性通过使用 100 nm 银胶体颗粒的表面增强拉曼光谱 (SERS) 测量几个小时后浴中的 R6G 浓度来量化。通过这种组合电 SERS 技术提取的染料与盐的渗透率比率与整个间隙连接通道上其他染料的类似比率兼容。渗透率比的结果进一步与荧光测量结果进行比较。贴片和 SERS 技术的新颖组合可以扩展到量化具有生物学意义的非荧光分子（例如 cAMP 和 IP3）穿过 1 nm 大小的孔（例如间隙连接通道）的运输。