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Received for publication June 6, 2006.
Revised November 7, 2006.
Accepted for publication November 9, 2006.
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 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, in addition to 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, as well as their tagged forms from HeLa cells. The IC50-TSF of the inhibition was 
47 µM at pH 6.5. 2-APB did not inhibit homomeric tagged Cx26 channels, even after tag cleavage (leaving several amino acids at the carboxy-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 carboxy-terminal domain.
Key words:
Ion channel regulation, Liposome, Structure/function/mechanism
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