2-aminoethoxydiphenyl borate directly inhibits channels composed of connexin26 and/or connexin32.
2-aminoethoxydiphenyl borate (2-APB), a commonly used blocker of IP3-induced calcium ion release and of store-operated channels, inhibits gap junction conductance when applied to cultured cells. The character and pharmacology of this inhibition was explored using 1) hemichannels composed of connexin32 (Cx32) and/or connexin26 (Cx26) purified from native sources and from transfected HeLa cells in which the connexin had a cleavable C-terminal epitope tag and 2) the corresponding junctional channels. Using reconstituted hemichannels in a liposome-based transport-specific fractionation assay (TSF), 2-APB reversibly inhibited homomeric Cx32 and heteromeric Cx26/Cx32 channels from native tissue and their tagged forms from HeLa cells. The IC50-TSF value of the inhibition was approximately 47 microM at pH 6.5. 2-APB did not inhibit tagged homomeric Cx26 channels even after tag cleavage (leaving several amino acids at the carboxyl terminus). Protonated 2-APB is the inhibitory agent, but channel sensitivity to 2-APB also increases as pH is lowered. To help define the chemical requirements for inhibition, the effects of four structural analogs of 2-APB were determined. The inhibitory action of 2-APB was shown to be distinct from that of aminosulfonates. 2-APB and its analogs, except phenytoin, inhibited dye-coupling through junctional channels formed by all the tagged channel forms except Cx26, consistent with the TSF studies. However 2-APB significantly inhibited dye coupling between cells expressing untagged Cx26, suggesting that an unmodified C terminus is required for action on Cx26 channels. These results show that protonated 2-APB directly and reversibly inhibits connexin channels composed of Cx26 and/or Cx32 and suggest involvement of the carboxyl-terminal domain.
Tao
Liang
L
Department of Pharmacology, Zhongsan College of Medicine, Sun Yet-San University, Guangzhou China 510080. taol@mail.sysu.edu.cn
Harris
Andrew L
AL
D001896:Boron Compounds / Q000737:chemistry / Q000494:pharmacology*; D000072259:Connexin 26; D017630:Connexins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020128:Inhibitory Concentration 50; D007365:Intercellular Junctions; D007473:Ion Channels / Q000037:antagonists & inhibitors*; D008081:Liposomes; D013329:Structure-Activity Relationship; D014162:Transfection; D000097002:Gap Junction beta-1 Protein
D001896:Boron Compounds; D000072259:Connexin 26; D017630:Connexins; D007473:Ion Channels; D008081:Liposomes; D000097002:Gap Junction beta-1 Protein; C000607042:GJB2 protein, human; C109986:2-aminoethoxydiphenyl borate