Functional analysis of hemichannels and gap-junctional channels formed by connexins 43 and 46.

PURPOSE

The gap junctions (GJs) mediating direct cell-cell interaction are formed by clusters of membrane-spanning proteins known as connexins (Cxs). These channels play a key role in signal transmission, and their permeability, time-, and voltage-dependence are governed by the properties of the specific Cxs forming the gap junctions. Retinal pigment epithelium (RPE) cells express Cx43 and Cx46. Here, we employed a heterologous expression system to explore the functional properties of the hemichannels and GJs that could be formed by different combinations of these Cxs. Specifically, we examined the response kinetics of GJs formed by pairing cells expressing Cx43 or Cx46, or those expressing both, i.e., designated as Cx43*Cx46.

METHODS

The Xenopus oocyte expression system and a two-electrode voltage clamp technique were used to study the properties of hemichannels and GJs formed in oocytes transfected with Cx43 and/or Cx46 mRNA.

RESULTS

Depolarizing voltages activated hemicurrents of similar amplitude from single oocytes transfected with Cx46 or Cx43Cx46, but not in oocytes expressing Cx43 alone. Incorporating Cx43 with Cx46 altered the gating charge, but not the voltage sensitivity of the hemichannels. In addition, Cx43Cx46 hemichannel currents exhibited faster activation kinetics than homomeric Cx46 hemichannels. Both homotypic GJs formed by Cx43 and Cx46, and heteromeric Cx43Cx46 GJs exhibited large junctional conductances with amplitudes of 6.5+/-3.0 microS (Cx43), 8.9+/-3.4 microS (Cx46), and 8.5+/-1.8 microS (Cx4346); a significantly lower conductance (1.8+/-0.7 microS) was observed for heterotypic GJs formed by Cx43 and Cx46. There were also differences in their gating kinetics. Whereas the kinetics of homotypic Cx46 could be described by a single exponential function (tau=0.91 s), double exponential functions were required for homotypic Cx43 (tau(1)=0.24, tau(2)=3.4 s), heterotypic Cx43/Cx46 (tau(1)=0.29, tau(2)=3.6 s), and heteromeric Cx43Cx46/Cx43Cx46 (tau(1)=1.2, tau(2)=8.1 s) junctions.

CONCLUSIONS

The failure of oocytes expressing Cx43 to exhibit hemichannel activity is an intrinsic membrane property of this Cx, and cannot be attributed to a lack of expression; western blot analysis showed clearly that Cx43 was expressed in oocytes in which it was injected. Our results provide further evidence that Cx43 and Cx46 form both heterotypic and heteromeric channels when co-expressed, an indication that various combinations of Cxs may participate in gap-junctional communication between RPE cells.