Connexin diversity and gap junction regulation by pHi

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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 (pHi) 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 pHi 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 pHi-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.