Chemical Gating Mechanism Of Connexin26-containing Channels By Aminosulfonate

Abstract

Protonated taurine directly and reversibly inhibits homomeric and heteromeric Cx26-containing hemichannels but not homomeric Cx32 hemichannels. It is unknown if taurine interacts with Cx26 and/or Cx32 in heteromeric channels, which domains are involved, or if junctional channels are taurine-sensitive. These issues were addressed with channels composed of Cx26 and/or Cx32 with/without cleavable 3KDa carboxyl-terminal (CT) tags (T). Hemichannel activity was assessed in liposomes, and by extracellular dye-uptake in cells. In contrast to untagged hemichannels, Cx26T/Cx32 and Cx26T hemichannels were not taurine-sensitive, but Cx26/Cx32T hemichannels were. Tag cleavage (Tc, leaving 4aa at the carboxyl-terminus) restored taurine-sensitivity of Cx26Tc/Cx32 hemichannels, but taurine surprisingly narrowed rather than closed Cx26Tc hemichannels. Thus, the 3KDa CT tag blocks taurine-sensitivity, unless hemichannels also contain Cx32, and the short 4aa CT extension affects Cx26Tc channel open state. Taurine effects on junctional channels were assessed by intercellular dye-coupling. Taurine substantially reduced dye-coupling by Cx26 and Cx26/Cx32T channels, but not by Cx26T, Cx26T/Cx32 or Cx32T channels. Junctional channels therefore have identical taurine-sensitivity as their component hemichannels. An intracellular site for taurine action was shown by a membrane-impermeable blocker of taurine uptake. Thus, all data indicate taurine-induced pore closure utilizes the Cx26 CT. Taurine binding to Cx26-CT was assessed by natural-abundance 13C-HMQC-NMR. Overlapping resonances of Cx26-CT peptide in the presence and absence of taurine indicate no direct taurine binding to Cx26-CT. Peptide ‘elisa’ showed a pH dependent interaction occurs between Cx26-CT and the carboxyl-terminal 20aa of the Cx26 cytoplasmic loop (Cx26-CL). Acidification increases the binding affinity of Cx26-CL and Cx26-CT peptides, and only the protonated form of taurine negatively affects this interaction, suggesting that its disruption leads to channel closure. Structural analysis of Cx26-CT and Cx26-CL peptides in the presence and absence of taurine are ongoing. Supported by GM36044, DC7470.