Abstract
Most vertebrate gap junction channels are gated by voltage, they close with a potential difference between the cells (Vj). However, closure is only partial, even with a large potential difference the junctional conductance decreases slowly to about one third to one half of the maximal conductance. This incomplete closure of the channels would have a minimal effect on the electrical continuity of the cells and the function of this voltage gate is undetermined.Like other membrane channels, gap junction channels exhibit subconductance states. In some connexin channels, a voltage gradient increases the probability of the channels to reside at subconductance states. It is tempting to assume that the subconductance state has a different selective permeability than the full conductance state, i.e. that the cut-off limit for larger molecules such as second messengers is reduced.In order to avoid the technical problems associated with studies on complete gap junction channels we decided to address the question of a functional role of voltage gate initially in a more amenable model system. Cx46 forms regular gap junction channels; however, when expressed in Xenopus oocytes also forms open hemichannels in the membrane without another membrane in apposition. Cx46 hemichannels exhibit permeability and gating properties indistinguishable from those of complete gap junction channels. Thus cx46 hemichannels are the suitable objects to study voltage effects on channel permeability.Here we show that the voltage gate (Vj gate) changes the permeation filter of gap junctions. Following the transit of fluorescent test molecules and of cAMP as assayed with CFTR as a reporter through cx46 hemichannels at different potentials, the influx of fluorescent dye is diminished at positive holding potential compared to that at negative holding potential. Similarly, in paired oocytes the flux of these tracer molecules is found to be affected by Vj. Further sugar exclusion study at single channel level on cx46 single hemichannel also proves the voltage gate can slow down the entry of molecules of the size of sucrose or larger at positive potential.