Gap Junction Permeability: Transfer of Negative and Positive Charged Probes

Gap junction channels are composed of connexins, which exhibit specific permeability to the variety of larger solutes including second messengers, polypeptides and siRNAs.Here, we report the permeability of solutes with different size and net charge Lucifer Yellow (443 (no counterion), -2); Carboxyfluorescein (376, -2); AlexaFluor350 (326, -1); Ethidium bromide (314, +1) and NBD-m-TMA (280, +1) through gap junction channels in HeLa cells expressing Cx26, Cx40, Cx43 and Cx45.The channel permeability was determined using simultaneous measurements of junctional conductance and the cell-cell flux of a fluorescent probe.All four connexins transferred negative charged probes: LY, CF and AF350, however, Cx40 and Cx26 exhibited reduced permeability when compared to Cx43. These connexins revealed following permeability ratios for LY/ CF/ AF350, respectively, relative to the ubiquitous cation K+: 0.029/ 0.032/ 0.069 for Cx43; 0.014/ 0.021/ 0.049 for Cx45; 0.0044/ 0.014/ 0.0245 for Cx26 and 0.0026/ 0.0034/ 0.0206 for Cx40.The positive charged NBD and EthBr exhibited the following permeability relative to K+: 0.045 and 0.0125 for Cx43; 0.048 and 0.0036 for Cx45; 0.055 and 0.026 for Cx26; 0.040 and 0.009 for Cx40.In summary, all negative charged species showed a similar permeability order: Cx43>Cx45>Cx26>Cx40. For positively charged species the permeability orders were: Cx26≈Cx43≈Cx40≈Cx45 (NBD) and Cx26≥Cx43≈Cx40>Cx45 (EthBr). Reduced EthBr permeation through Cx45 channels in comparison to other connexins suggests a size-dependent discrimination of the solute. However, the reduced correlation between junctional conductance and positive charged probes flux suggests intracellular binding of the solute. Therefore quantitative comparison of positively charged solutes has to be taken cautiously.These results confirm that channels formed from individual connexins can discriminate for solutes based on size and charge suggesting that channel selectivity may be a key factor in cell signaling.Supported by AHA0335236N, NIHGM55263.