Rapid and direct effects of pH on connexins revealed by the connexin46 hemichannel preparation

PH is a potent modulator of gap junction (GJ) mediated cell-cell communicat ion. Mechanisms proposed for closure of GJ channels by acidification includ e direct actions of H+ on GJ proteins and indirect actions mediated by solu ble inter mediates. Here we report on the effects of acidification on conne xin (Cx;)46 cell-cell channels expressed in Neuro-2a cells and Cx46 hemicha nnels expressed in Xenopus oocytes. Effects of acidification on hemichannel s were examined macroscopically and in excised patches that permitted rapid (<1 ms) and uniform pH changes at the er;posed hemichannel face. Both type s of Cx46 channel were found to be sensitive to cytoplasmic pH, and two eff ects were evident. A rapid and reversible closure,vas reproducibly elicited with short exposures to low pH, and a poorly reversible or irreversible lo ss occurred with longer exposures. We attribute the former to pH gating and the latter to pH inactivation. Half-maximal reduction of open probability for pH gating in hemichannels occurs at PII 6.4. Hemichannels remained sens itive to cytoplasmic pH when excised and when cytoplasmic [Ca2+] was mainta ined near resting (similar to 10(-7);M) levels. Thus, Cx46 hemichannel pH g ating does not depend on cytoplasmic intermediates ol a rise in [Ca2+]. Rap id application of low pH to the cytoplasmic face of open hemichannels resul ted in a minimum latency to closure near zero, indicating that Cx46 hemicha nnels directly sense pH. Application to closed hemichannels extended their closed time, suggesting that the pH sensor is accessible fr om the cytoplas mic side of a closed hemichannel. Rapid closure with significantly reduced sensitivity was observed with low pH application to the extracellular face, but could be explained by H+ permeation through the pore to reach an inter nal site. Closure by pH is voltage dependent and has the same polarity with low pH applied to either side. These data suggest that the pH sensor is lo cated directly on Cx46 near the pc:,re entrance on the cytoplasmic side.