Homotypic, Heterotypic and Heteromeric Gap Junctional Channels Formed by Connexin Proteins Expressed in Retinal Pigment Epithelial Cells

Abstract

Purpose: : Gap junctional channels (GJ) play a key role in signal transmission. Homotypic GJs are composed of two identical connexons. Heterotypic GJs are composed of two different connexons. In heteromeric GJs, each connexon is composed of a mixture of different connexins (Cx). The Cxs mediating GJ communication between retinal pigment epithelial (RPE) cells are Cx43 and Cx46. Here, we employ a heterologous expression system to explore the properties of hemichannels and GJs formed by the different combinations of these Cxs.

Methods: : Using the Xenopus oocyte expression system and two-electrode voltage clamp technique, we studied the properties of hemichannels and GJs formed in oocytes transfected with Cx43 and/or Cx46 mRNA.

Results: : Depolarizing voltage activated large hemicurrents in single oocytes transfected with Cx46, or Cx46/Cx43 (but not Cx43 alone). The hemicurrents recorded from Cx46 and Cx46/Cx43 oocytes exhibited similar activation threshold (-10 mV) and I-V relationships. With a +60 mV voltage pulse, hemicurrents had magnitudes of 14.1 +/- 1.2 µA (mean +/- SEM, Cx46, n = 10) and 13.4 +/- 1.3 µA (Cx46/Cx43, n = 13) and activation time constants (τ) of 2.4 +/- 0.3 s and 2.7 +/- 0.3 s, respectively. In paired oocytes, homotypic GJs formed by Cx43 or Cx46 had similar junctional conductance (G_j): 7 +/- 4 µS for Cx43 (n = 4) and 9 +/- 3 µS for Cx46 (n = 3). Preliminary data shows gating charges (n) at - and + junctional voltages (V_j) of 1.3 and 2.3 in Cx43, and 2.1 and 2.1 in Cx46 GJs. In contrast, heterotypic GJs showed smaller G_j (2 +/- 1 µS, n = 6) with gating asymmetry, as previously reported. The gating kinetics to a -150 mV voltage pulse can fit with a first-order exponential equation, and was different between Cx43 and Cx46 homotypic GJs (τ = 1.90 +/- 0.46 s and 0.92 +/- 0.03 s, respectively). In paired oocytes expressing both Cx43 and Cx46, GJs formed with G_j similar to that of the homotypic GJs (8 +/- 2 µS, n = 10), and with n of 1.7 and 1.8 at - and +V_j. The kinetics of these channels at -150 mV were also fit with a single term (τ = 1.73 +/- 0.29 s, correlation coefficient 0.950).

Conclusions: : Co-expressing Cx43 and Cx46 (Cxs expressed in RPE cells) forms heteromeric connexons, rather than a mixture of homotypic GJs, with characteristics that differ from their homotypic counterparts. On the other hand, co-expressing Cx43 with Cx46 does not alter the property of hemichannels in individual cells.

Keywords: gap junctions/coupling • retinal pigment epithelium • electrophysiology: non-clinical