Reversal of the gating polarity of gap junctions by negative charge substitutions in the N-terminus of connexin 32

Intercellular channels formed by connexins (gap junctions) are sensitive to the application of transjunctional voltage (V(j)), to which they gate by the separate actions of their serially arranged hemichannels (Harris, A. L., D. C. Spray, and M. V. L. Bennett. 1981. J. Gen. Physiol. 77:95-117). Single channel studies of both intercellular and conductive hemichannels have demonstrated the existence of two separate gating mechanisms, termed 'V(j)-gating' and 'loop gating' (Trexler, E. B., M. V. L. Bennett, T. A. Bargiello, and V. K. Verselis. 1996. Proc. Natl. Acad. Sci. U.S.A. 93:5836-5841). In Cx32 hemichannels, V(j)-gating occurs at negative V(j) (Oh, S., J. B. Rubin, M. V. L. Bennett, V. K. Verselis, and T. A. Bargiello. 1999. J. Gen. Physiol. 114:339-364; Oh, S., C. K. Abrams, V. K. Verselis, and T. A. Bargiello. 2000. J. Gen. Physiol. 116:13-31). A negative charge substitution at the second amino acid position in the N-terminus reverses the polarity of V(j)-gating of Cx32 hemichannels (Verselis, V. K., C. S. Ginter, and T. A. Bargiello. 1994. Nature. 368:348-351; Oh et al., 2000. J. Gen. Physiol. 116:13-31). We report that placement of a negative charge at the 5th, 8th, 9th, or 10th position can reverse the polarity of Cx32 hemichannel V(j)-gating. We conclude that the 1st through 10th amino acid residues lie within the transjunctional electric field and within the channel pore, as in this position they could sense changes in V(j) and be largely insensitive to changes in absolute membrane potential (V(m)). Conductive hemichannels formed by Cx32*Cx43E1 containing a negatively charged residue at either the 8th or 10th position display bi-polar V(j)-gating; that is, the open probability of hemichannels formed by these connexins is reduced at both positive and negative potentials and is maximal at intermediate voltages. In contrast, Cx32*Cx43E1 hemichannels with negative charges at either the 2nd or 5th positions are uni-polar, closing only at positive V(j). The simplest interpretation of these data is that the Cx32 hemichannel can adopt at least two different open conformations. The 1st-5th residues are located within the electric field in all open channel conformations, while the 8th and 10th residues lie within the electric field in one conformation and outside the electric field in the other conformation.