Permeability of homotypic and heterotypic gap junction channels formed of cardiac connexins mCx30.2, Cx40, Cx43, and Cx45

Abstract

We examined the permeabilities of homotypic and heterotypic gap junction (GJ) channels formed of rodent connexins (Cx) 30.2, 40, 43, and 45, which are expressed in the heart and other tissues, using fluorescent dyes differing in net charge and molecular mass. Combining fluorescent imaging and electrophysiological recordings in the same cell pairs, we evaluated the single-channel permeability (P(gamma)). All homotypic channels were permeable to the anionic monovalent dye Alexa Fluor-350 (AF(350)), but mCx30.2 channels exhibited a significantly lower P(gamma) than the others. The anionic divalent dye Lucifer yellow (LY) remained permeant in Cx40, Cx43, and Cx45 channels, but transfer through mCx30.2 channels was not detected. Heterotypic channels generally exhibited P(gamma) values that were intermediate to the corresponding homotypic channels. P(gamma) values of mCx30.2/Cx40, mCx30.2/Cx43, or mCx30.2/Cx45 heterotypic channels for AF(350) were similar and approximately twofold higher than P(gamma) values of mCx30.2 homotypic channels. Permeabilities for cationic dyes were assessed only qualitatively because of their binding to nucleic acids. All homotypic and heterotypic channel configurations were permeable to ethidium bromide and 4,6-diamidino-2-phenylindole. Permeability for propidium iodide was limited only for GJ channels that contain at least one mCx30.2 hemichannel. In summary, we have demonstrated that Cx40, Cx43, and Cx45 are permeant to all examined cationic and anionic dyes, whereas mCx30.2 demonstrates permeation restrictions for molecules with molecular mass over approximately 400 Da. The ratio of single-channel conductance to permeability for AF(350) was approximately 40- to 170-fold higher for mCx30.2 than for Cx40, Cx43, and Cx45, suggesting that mCx30.2 GJs are notably more adapted to perform electrical rather than metabolic cell-cell communication.

Fig. 1

A: representative images demonstrating the presence or absence of dye transfer in homotypic junctions formed of different cardiac connexins (Cxs). Images were recorded ∼10 or more min after opening the patch in cell 1, the dye donor cell (indicated by 2 asterisks). Cell 2 is the dye acceptor cell (indicated by 1 asterisk). DAPI, 4,6-diamidino-2-phenylindole. B: examples of merged images showing that formation of heterotypic junctions is extensive (see arrows) between cells expressing mCx30.2-EGFP (in red) and Cx40-CFP (a; in green) and Cx43-CFP (b; in green). C: a representative image demonstrating propidium iodide transfer in a cell pair forming a Cx43-EGFP/Cx45-CFP heterotypic junction.

Fig. 2

Permeability of gap junction (GJ) channels. A: time course of the changes in Lucifer yellow (LY) fluorescence in a HeLaCx43-EGFP cell pair. FI, fluorescence intensity. B: plot of single-channel permeability (P_γ) vs. time, calculated from A. C: changes in Alexa Fluor-350 (AF³⁵⁰) fluorescence in cell 1 and cell 2 of a HeLaCx30.2-EGFP cell pair. D: plot of P_γ vs. time, calculated from C. Vertical arrows indicate the time of patch opening in cell 1. Linear regression lines of the first order are shown.

Fig. 3

Summary of data from single-channel permeability studies. Bars show averaged P_γ values of homotypic (A) and heterotypic (B) GJ channels for AF³⁵⁰ and LY.

Fig. 4

Representative examples showing the time course of AF³⁵⁰ leakage from individual cells expressing mCx30.2-EGFP and Cx43-EGFP and from HeLa parental cells. Solid lines are a fit of experimental points to a single exponential function.