Intracellular Domains of Mouse Connexin26 and -30 Affect Diffusional and Electrical Properties of Gap Junction Channels

Abstract

To evaluate the influence of intracellular domains of connexin (Cx) on channel transfer properties, we analyzed mouse connexin (Cx) Cx26 and Cx30, which show the most similar amino acid sequence identities within the family of gap junction proteins. These connexin genes are tightly linked on mouse chromosome 14. Functional studies were performed on transfected HeLa cells stably expressing both mouse connexins. When we examined homotypic intercellular transfer of microinjected neurobiotin and Lucifer yellow, we found that gap junctions in Cx30-transfected cells, in contrast to Cx26 cells, were impermeable to Lucifer yellow. Furthermore, we observed heterotypic transfer of neurobiotin between Cx30-transfectants and HeLa cells expressing mouse Cx30.3, Cx40, Cx43 or Cx45, but not between Cx26 transfectants and HeLa cells of the latter group. The main differences in amino acid sequence between Cx26 and Cx30 are located in the presumptive cytoplasmic loop and C-terminal region of these integral membrane proteins. By exchanging one or both of these domains, using PCR-based mutagenesis, we constructed Cx26/30 chimeric cDNAs, which were also expressed in HeLa cells after transfection. Homotypic intercellular transfer of injected Lucifer yellow was observed exclusively with those chimeric constructs that coded for both cytoplasmic domains of Cx26 in the Cx30 backbone polypeptide chain. In contrast, cells transfected with a construct that coded for the Cx26 backbone with the Cx30 cytoplasmic loop and C-terminal region did not show transfer of Lucifer yellow. Thus, Lucifer yellow transfer can be conferred onto chimeric Cx30 channels by exchanging the cytoplasmic loop and the C-terminal region of these connexins. In turn, the cytoplasmic loop and C-terminal domain of Cx30 prevent Lucifer yellow transfer when swapped with the corresponding domains of Cx26. In chimeric Cx30/Cx26 channels where the cytoplasmic loop and C-terminal domains had been exchanged, the unitary channel conductance was intermediate between those of the parental channels. Moreover, the voltage sensitivity was slightly reduced. This suggests that these cytoplasmic domains interfere directly or indirectly with the diffusivity, the conductance and voltage gating of the channels.

Compartmentalized and Signal-Selective Gap Junctional Coupling in the Hearing Cochlea

“…We observed extensive transfer of LY at P0 and P8. Based on heterologous expression data, this can be interpreted as passage through gap junction channels comprising Cx26-only connexons (Manthey et al, 2001;Marziano et al, 2003). We observed junctional puncta predominantly labeled for Cx26 at these ages.…”

“…The molecular permeability of gap junctions consisting of Cx26 and Cx30 have been studied previously in vitro using dyes of different charge and molecular weight (MW), including Lucifer yellow (LY) (charge, -2; MW, 443 Da) and Neurobiotin (NBN) (charge, ϩ1; MW, 287 Da). In heterologous expression systems, gap junctions between cells expressing Cx26 alone are permeable to LY and NBN (Manthey et al, 2001;Marziano et al, 2003). Gap junctions between cells expressing Cx30 alone transfer NBN but are impermeable to LY.…”

Two Distinct Heterotypic Channels Mediate Gap Junction Coupling between Astrocyte and Oligodendrocyte Connexins

“…Using a Kruskal-Wallis test followed by Dunn's multiple comparison test, only Cx32/Cx30 and Cx47/Cx43 channels have G j that is significantly greater (*p Ͻ 0.001) than those of the corresponding control pairs (32/CTR and 30/CTR or 43/CTR and 47/CTR). studies have shown that homotypic channels containing only Cx32 (Elfgang et al, 1995), Cx43 (Elfgang et al, 1995), or Cx47 (Teubner et al, 2001;Orthmann-Murphy et al, 2007) are permeable to LY (MW, 443; valence, Ϫ2), but homotypic Cx30 channels are either minimally permeable (Beltramello et al, 2003) or impermeable (Manthey et al, 2001). As shown in Figure 5, when 0.1% LY was injected into one cell, we detected LY in the neighboring cell for pairs containing Cx47/Cx43 (Fig.…”

Connexin30 mediated intercellular communication plays an essential role in epithelial repair in the cochlea

“…Immunolabelling suggests that the small gap junction plaques that are present on the membranes of Deiters' cells in the Cx30 null mice are composed of Cx26, and there was no evidence for its upregulation, nor for the upregulation of some other connexin, with loss of Cx30 . The permeability properties of gap junction channels that contain Cx30, either alone or in heteromeric configuration with Cx26, are different from those formed of Cx26 only (Manthey et al, 2001;Marziano et al, 2003;Yum et al, 2007); Cx30 appears to play a role in restricting the passage of large anionic species. Thus, in the normal functionally mature organ of Corti anionic signalling molecules, such as IP3, c-AMP and ATP, may be unable to pass easily between Deiters' cells or between Hensen's and Deiters' cells.…”

“…There are 21 connexin genes in the human genome and 20 in that of the mouse (Bedner et al, 2012). There is some understanding of the varying biophysical properties of channels of different connexin composition (Kanaporis et al, 2011;Manthey et al, 2001;Yum et al, 2007) but the functional significance of expression of a particular connexin in a tissue is less clear.…”