@article{3b0698f4d18b44888acc6c5ab2d9ae15,
title = "Gating properties of gap junction channels assembled from connexin43 and connexin43 fused with green fluorescent protein",
abstract = "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 (Vj) 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 Vj gating to a residual state but show slow Vj 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 Vj gating suggests that both gating mechanisms share common structural elements. Cx43/Cx43-EGFP heterotypic channels showed asymmetrical Vj gating with fast transitions between open and residual states only when the Cx43 side was relatively negative. This result indicates that the fast Vj gate of Cx43 hemichannels closes for relative negativity at its cytoplasmic end.",
author = "Bukauskas, {Feliksas F.} and Angele Bukauskiene and Bennett, {Michael V.L.} and Verselis, {Vytas K.}",
note = "Funding Information: Attachment of EGFP to the CT of Cx43 selectively disrupts fast V j gating to γ res but preserves both slow V j gating and gating by chemical agents. Closure of channels by chemical agents, particularly fatty acids and alkanols, has been suggested to occur by perturbation of the channel at the lipid-channel interface ( Burt et al., 1991 ). Previously we showed that clusters or plaques of GJ channels can be resolved in living cells at locations of cell-cell contact using Cx43-EGFP and that plaques are required for electrical coupling ( Bukauskas et al., 2000 ). Here we monitored fluorescence of plaques in electrically coupled cell pairs and showed that exposure to the uncoupling agent heptanol caused complete uncoupling but no measurable change in fluorescence intensity or size and shape of the plaque. Thus, the uncoupling effect of alkanols is not related to dispersion of junctional plaques. Similarly, plaques did not show evidence of dispersing when exposed to CO 2 . However, fluorescence of GFP and other color variants are known to be sensitive to pH (reviewed by Tsien, 1998 ), and by lowering cytoplasmic pH with CO 2 we observed that EGFP retained its pH sensitivity when attached to Cx43 ( Fig. 10 C ). Thus, connexins fused to EGFP, or more appropriately with mutants of GFP selected for their pH-sensitive properties ( Miesenbock et al., 1998 ), can be exploited to measure intracellular pH in very close proximity to the junctions and better quantitate conductance changes with pH. In summary, Cx43 GJ channels contain two types of gate sensitive to V j : one characterized by fast gating transitions to the residual state and the other by slow gating transitions to the fully closed state. These two mechanisms interact in a manner consistent with contingent gating and placement of the gating elements in series in the pore. Fast V j gating is selectively lost in Cx43-EGFP channels and this loss is confined to the hemichannel composed of Cx43-EGFP. Gating asymmetry in Cx43/Cx43-EGFP heterotypic channels shows that the fast V j gate has a negative gating polarity; i.e., the hemichannel closes when its cytoplasmic face is made relatively negative. We suggest that all connexins that form GJ channels contain two V j gates and that relative differences in sensitivity and kinetics of V j dependence of these gates provide the complex array of gating properties observed among GJ channels. Indeed, both types of gating transitions have been reported in channels formed of Cx26, Cx32, Cx37, and Cx40 ( Bukauskas et al., 1995a,b; Oh et al., 1999; Ramanan et al., 1999 ). All GJ channels also display chemical gating, and this form of gating shares several features in common with slow V j gating. Both mechanisms close channels completely, and closures are characterized by slow transitions to a fully closed state ( Bukauskas and Peracchia, 1997 ). Also, chemically induced uncoupling by alkanols, Ca 2+ , and H + was shown to be reversible by voltage ( Obaid et al., 1983; Weingart and Bukauskas, 1998; Peracchia et al., 1999 ). Taken together, these data support the possibility that chemical gating and slow V j gating share common structural elements. We thank Dr. Laird for kindly providing us with the Cx43-EGFP construct and Dr. Klaus Willecke and Dr. Ross Johnson for kindly providing us with the HeLaCx43 and Novikoff cell lines. We thank Dr. Seunghoon Oh and Dr. Brady Trexler for valuable technical help. This study was supported by National Institutes of Health grants NS36706 to F.F.B. and GM54179 to V.K.V. ",
year = "2001",
doi = "10.1016/S0006-3495(01)75687-1",
language = "English (US)",
volume = "81",
pages = "137--152",
journal = "Biophysical journal",
issn = "0006-3495",
publisher = "Biophysical Society",
number = "1",
}