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

Intercellular channels formed by connexins (yap junctions) are sensitive to the application of transjunctional voltage (V-j), to which they gate by th e 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). Singl e channel studies of both intercellular and conductive hemichannels have de monstrated the existence of two separate gating mechanisms, termed "V-j-gat ing" 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 C x32 hemichannels, Vi-gating occurs at negative V-j (Oh, S., J. B. Rubin, M. V. L. Bennett, V. K. Verselis, and T. A. Bargiello. 1999. J. Con. Physiol. 114:339-364; Oh, S., C. K. Abrams, V. K. Verselis, and T. A. Bargiello. 20 00. J. Gen. Physiol. 116:13-31). A negative charge substitution at the seco nd amino acid position in the N-terminus reverses the polarity of V-j-gatin g 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). W e 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 conclu de that the ist through 10th amino acid residues lie within the transjuncti onal electric field and within the channel pore, as in this position they c ould sense changes in V-j and be largely insensitive to changes in absolute membrane potential (V-m). Conductive hemichannels formed by Cx32*Cx43E1 co ntaining a negatively charged residue at either the 8th or 10th position di splay bi-polar V-j-gating; that is, the open probability of hemichannels fo rmed by these connexins is reduced at both positive and negative potentials and is maximal at intermediate voltages. In contrast, Cx32*Cx43E1 hemichan nels 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 i s that the Cx32 hemichannel can adopt at least two different open conformat ions. The 1st-5th residues are located within the electric field in all ope n channel conformations, while the 8th and 10th residues lie within the ele ctric field in one conformation and outside the electric field in the other conformation.