Biophys J, July 2001, p. 137-152, Vol. 81, No. 1

Gating Properties of Gap Junction Channels Assembled from Connexin43 and Connexin43 Fused with Green Fluorescent Protein

Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461 USA

We used cell lines expressing wild-type connexin43 (Cx43) and Cx43 fused with enhanced green fluorescent protein (Cx43-EGFP) to examine mechanisms of gap junction channel gating. Previously it was suggested that each hemichannel in a cell-cell channel possesses two gates, a fast gate that closes channels to a nonzero conductance or residual state via fast (<~2 ms) transitions and a slow gate that fully closes channels via slow transitions (>~10 ms). Here we demonstrate that transjunctional voltage (V_j) regulates both gates and that they are operating in series and in a contingent manner in which the state of one gate affects gating of the other. Cx43-EGFP channels lack fast V_j gating to a residual state but show slow V_j gating. Both Cx43 and Cx43-EGFP channels exhibit slow gating by chemical uncouplers such as CO₂ and alkanols. Chemical uncouplers do not induce obvious changes in Cx43-EGFP junctional plaques, indicating that uncoupling is not caused by dispersion or internalization of junctional plaques. Similarity of gating transitions during chemical gating and slow V_j gating suggests that both gating mechanisms share common structural elements. Cx43/Cx43-EGFP heterotypic channels showed asymmetrical V_j gating with fast transitions between open and residual states only when the Cx43 side was relatively negative. This result indicates that the fast V_j gate of Cx43 hemichannels closes for relative negativity at its cytoplasmic end.

Biophys J, July 2001, p. 137-152, Vol. 81, No. 1
© 2001 by the Biophysical Society   0006-3495/01/07/137/16  $2.00

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