Connexin diversity and gap junction regulation by pHi

Abstract

The molecular mechanisms controlling pH‐sensitivity of gap junctions formed of two different connexins are yet to be determined. We used a proton‐sensitive fluorophore and electrophysiological techniques to correlate changes in intracellular pH (pH_i) with electrical coupling between connexin‐expressing Xenopus oocytes. The pH sensitivities of α₃ (connexin46), α₂ (connexin38), and α₁ (connexin43) were studied when these proteins were expressed as: 1) nonjunctional hemichannels (for α₃ and α₂), 2) homotypic gap junctions, and 3) heterotypic gap junctions. We found that α₃ hemichannels are sensitive to changes in pH_i within a physiological range (pKa = 7.13 ± 0.03; Hill coefficient = 3.25 ± 1.73; n = 8; mean ± SEM); an even more alkaline pKa was obtained for α₂ hemichannels (pKa = 7.50 ± 0.03; Hill coefficient = 3.22 ± 0.66;n = 13). The pH sensitivity curves of α₂ and α₃ homotypic junctions were indistinguishable from those recorded from hemichannels of the same connexin. Based on a comparison of pKa values, both α₃ and α₂ gap junctions were more pH_i‐dependent than α₁. The pH sensitivity of α₂‐containing heterotypic junctions could not be predicted from the behavior of the two connexons in the pair. When α₂ was paired with α₃, the pH sensitivity curve was similar to that obtained from α₂ homotypic pairs. Yet, pairing α₂ with α₁ shifted the curve similar to homotypic α₁ channels. Pairing α₂ with a less pH sensitive mutant of α₁ (M257) yielded the same curve as when α₁ was used. However, the pH sensitivity curve of α₃/α₁channels was similar to α₃/α₃, while α₃/M257 was indistinguishable from α₃/α₁. Our results could not be consistently predicted by a probabilistic model of two independent gates in series. The data show that dissimilarities in the pH regulation of gap junctions are due to differences in the primary sequence of connexins. Moreover, we found that pH regulation is an intrinsic property of the hemichannels, but pH sensitivity is modified by the interactions between connexons. These interactions should provide a higher level of functional diversity to gap junctions that are formed by more than one connexin. Dev. Genet. 24:123–136, 1999.