Permeability of R6G across Cx43 hemichannels through a novel combination of patch clamp and surface enhanced Raman spectroscopy.

Abstract

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.