2-Aminoethoxydiphenyl Borate Directly Inhibits Channels Composed of Connexin26 and/or Connexin32

Abstract

2-Aminoethoxydiphenyl borate (2-APB), a commonly used blocker of IP 3 -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 IC 50-TSF value of the inhibition was ϳ47M 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.

Fluxes theory in experiments with random distributed channels on vesicles

“…Working with transport-specific fractionation of liposomes, into which connexin channels were reconstituted, it was suggested that the channels are Poisson distributed among the liposomes. 2 This is in accordance to hypothetical modes of monomeric association to form channels on liposomes. 7 Based on channel Poisson distributions and using inside out vesicles, Alvarez et al developed a method to estimate the number of Ca 2+ -dependent K+ channels in the human red cell.…”

“…This distribution has been used in analyzing of several experiments. [2][3][4][5][6] Thus, the liposome population must be corrected in accordance to Poisson distribution for adequate interpretations of results. Working with transport-specific fractionation of liposomes, into which connexin channels were reconstituted, it was suggested that the channels are Poisson distributed among the liposomes.…”

The Effects of 2-Aminoethoxydiphenyl Borate and Diphenylboronic Anhydride on Gap Junctions Composed of Connexin43 in TM4 Sertoli Cells

“…For example, using reconstituted Cx hemichannels obtained from native tissues and stably-transfected cells, we characterized the direct inhibitions of Cx26 and/or Cx32 channels by 2-APB and DPBA. 26) Some studies also implicated a reduction in junctional channel open probability induced by 2-APB through inhibition of electrical communication 49) or through a direct extracellular action on Cxs. 50) Thus a comparative analysis among the tested compounds to find the pharmacophore for Cx43 channel inhibition is necessary.…”

“…26) In the present study, the effects of these compounds on the function of GJ composed of Cx43, an another important member of the Cx family, were explored. We found that 2-APB and DPBA could reversibly inhibit the function of Cx43 channel in a concentrationdependent manner in TM 4 Sertoli cells, whereas other analogues of 2-APB, DPDM, DPTTF and PHT, had no effect on the GJ channels.…”

“…We previously reported that 2-APB inhibited GJ channels composed of Cx26 and/or Cx32 by acting on Cx channel directly. 26) The direct and indirect evidences strongly support a potential role of 2-APB as a novel and efficient inhibitor of GJ channels. However, 2-APB was mostly used by electrical measurement of coupling with the treatment time scale within minutes, very little is known about its effect on permeability of Cx43 channel and the underlying mechanisms still remain to be elucidated.…”