Voltage Dependent Gating of Gap Junction Channels: Influence of the Second Extracellular Loop

Abstract

Patch clamp measurements with connexin transfected HeLa cells revealed two groups of connexin types which differ by their voltage dependent gating after changing the polarity of the transjunctional voltage. Positive and negative transjunctional voltage steps higher than the half maximum inactivation voltage closed individual channels, which was observed as an exponential decay of the initial to a steady state current. Repetitive pulses of the same polarity gave the same decay and when the polarity of the pulses was reversed, a similar decay was observed only for Cx37- and Cx43- but not for Cx32-, Cx40-, and Cx45-channels. The latter respond upon the first reversed pulse in a way which might be explained by a slow re-opening of closed channels and their subsequent closing. Further pulses of the same polarity resulted in the fast exponential decay as for Cx37- and Cx43- channels. The number and sequence of amino acids in the first extracellular loop is highly conserved for all investigated connexins, whereas a significant difference is obvious for the second extracellular loop, which is longer in Cx37 and Cx43. With the HeLa connexin transfectant Cx40*43E1,2 where both extracellular loops from Cx40 were replaced by Cx43, this slow re-opening was no longer observed. Since intracellular domains and membrane integrated helices of this transfectant were part of Cx40 and the first extracellular loop is highly conserved, we hypothesize that the second extracellular loop is involved in regulatory processes of gating.