Supplementary material for Revilla et al. (2000) Proc. Natl. Acad. Sci. USA 97 (26), 14760–14765. (10.1073/pnas.250475897)

Asymmetry of G_jss/V_j relations is a form of V_m dependence. However, the asymmetry may result from a degree of sensitivity of the V_j gating process to V_m rather than from the existence of a separate V_m gating mechanism. It is not unreasonable that a gate sensitive to V_j also exhibits a degree of sensitivity to V_m, and it is somewhat surprising that there are any V_j sensitive junctions that are V_m insensitive, given the probable structure of the channels. For the RCx43 G_jss/V_j relation, the apparent V₀ and G_jmin were smaller for V_j generated by positive V_m in cell 1. The asymmetry in the G_jss/V_j relation is consistent with the V_m dependence, which increases g_j for increase in inside negativity. Furthermore, the duration of the V_j pulses was long enough to permit some gating by the V_m sensitive mechanism. However, the asymmetry was less for the 257 truncation mutant in which fast V_j gating was abolished. Because V_m gating was unaffected in the mutant, this result suggests that in the wild-type the asymmetry in V_j gating reflects a direct action on the V_j gates rather than modulation by the V_m gate. In Cx45 junctions, the kinetics of V_j sensitivity is faster, and there is less asymmetry at different V_m’s (1), indicating that the V_j gating in this system is quite insensitive to V_m (and that V_m gating transitions are infrequent relative to V_j gating events).

Banach and Weingart reported (2) a small asymmetry of the G_jss/V_j relation of junctions of RCx43 expressed in a cell line, but stated that they did not see changes in G_j when equal voltages were applied in the two cells. This result suggests that the asymmetry is due to V_m sensitivity of the V_j gating mechanism. We do not understand their failure to see V_m dependence of the kind described here. It may be that junctions formed from recombinant connexins expressed in transfected cell lines differ in this respect. Junctions formed of other connexins also exhibit V_m sensitivity (3), and G_j can be increased by depolarization, as in Cx45 expressed in Xenopus oocytes (1) or in Cx57 junctions expressed in a mammalian cell line (4), rather than decreased, as reported here for Cx43 junctions.

1. Barrio, L. C., Capel, J., Jarillo, J. A., Castro, C. & Revilla, A. (1997) Biophys. J. 73, 757–769.

2. Banach, K. & Weingart, R. (1996) Pflügers Arch. Eur. J. Physiol. 431, 775–785.

3. Barrio, L. C., Revilla, A., Gómez-Hernández, J. M., de Miguel, M. & González, D. (2000) in Gap Junctions: Molecular Basis of Cell Communication in Health and Disease, ed. Peracchia, C. (Academic, San Diego), pp. 175–188.

4. Manthey, D., Bukauskas, F., Lee, C. G., Kozak, C. A. & Willecke, K. (1999) J. Biol. Chem. 274, 14716–14723.