Mix and match: investigating heteromeric and heterotypic gap junction channels in model systems and native tissues

Abstract

This review is based in part on a roundtable discussion session: "Physiological roles for heterotypic/heteromeric channels" at the 2013 International Gap Junction Conference (IGJC 2013) in Charleston, South Carolina. It is well recognized that multiple connexins can specifically co-assemble to form mixed gap junction channels with unique properties as a means to regulate intercellular communication. Compatibility determinants for both heteromeric and heterotypic gap junction channel formation have been identified and associated with specific connexin amino acid motifs. Hetero-oligomerization is also a regulated process; differences in connexin quality control and monomer stability are likely to play integral roles to control interactions between compatible connexins. Gap junctions in oligodendrocyte:astrocyte communication and in the cardiovascular system have emerged as key systems where heterotypic and heteromeric channels have unique physiologic roles. There are several methodologies to study heteromeric and heterotypic channels that are best applied to either heterologous expression systems, native tissues or both. There remains a need to use and develop different experimental approaches in order to understand the prevalence and roles for mixed gap junction channels in human physiology.

Keywords: Connexin; Gap junction; Membrane transport; Quality control.

Figure 1. Structure and interactions between human connexins

A. Shown is a dendrogram arranged using amino acid homology [5], where human connexin protein names were used (connexin; Cx) and gene names are in italics. B. Line diagram of a generic connexin, showing both the N-terminus (NH₂) and C-terminus (CT) oriented towards the cytoplasm. Other protein elements include the two Extracellular Loop (EL) domains, four Transmembrane (TM ) domains, Cytoplasmic Loop (CL) domain. C. Diagram of different classes of channels, including homomeric, heterotypic and heteromeric.

Figure 2. Heteromeric and heterotypic compatibility.

A. Location of heteromeric and heterotypic compatibility motifs. Shown are selected connexins, gene homology group, heteromeric specificity motif in the transition between the cytoplasmic loop (CL) and third transmembrane (TM3) domains and heterotypic specificity motif in the second extracellular loop (EL2) domain. B. Heteromeric interactions indicated by the wheel diagram where connexins known to form heteromeric channels are connected by dashed lines. The solid dividing line separates beta connexins (white) from other connexins to indicate a lack of heteromeric compatibility. C. Heterotypic interactions indicated by the wheel diagram where connexins known to form heteromeric channels are connected by dashed lines. There are conflicting data regarding heterotypic Cx30 and Cx31 containing channels, this is indicated by the gray dashed lines (see text). The solid dividing lines show three putative compatibility groups which do not align with overall amino acid homology groups. Based on [1, 18, 20, 34, 35, 38, 39, 65-67, 89, 90, 92, 97, 101, 106-112, 113].

Figure 3. Connexin oligomerization pathways.

Connexins are co-translationally inserted into the ER membrane. Depending on the connexin subtype, oligomerization can occur either in the ERGIC (W type, Cx32, white) or the TGN (R type, Cx43, shaded). The potential for connexin oligomerization in the ER, driven by high levels of connexin expression, is also shown for Cx32. Hemichannels are subsequently transported to the plasma membrane, where they can function as channels or pair with hemichannels on adjacent cells to form complete intercellular channels. Channels at the plasma membrane further assemble into semi-crystalline arrays known as gap junction plaques, which can contain from tens to thousands of channels. Homogenous plaques are composed of either a single connexin or heteromeric connexins (not shown). Heterogeneous plaques contain regions enriched for different connexins. Adapted from [2], with permission.

Figure 4. Hypothetical monomer stability model for regulated hetero-oligomerization.

Different connexins are depicted by different colored ovals, yellow represents quality control chaperones which stabilize monomeric connexins (e.g. ERp29 [13]) A. A cell expressing connexins with matched monomer stability would have oligomerization occur in the same intracellular compartment and be expected to form fully heteromeric channels. A pair of connexins with matched monomer stability are Cx40 (light) and Cx43 (dark) [7]. B. A cell expressing connexins where one connexin has greater monomer stability than another would have restricted hetero-oligomerization, leading to monomeric channels. A pair of connexins with this type of mismatched monomer stability are Cx46 (light) and Cx43 (dark) [15]. C. A cell expressing connexins where one connexin has less monomer stability than another would have reduced hetero-oligomerization as a result of prior hexamer formation. A pair of connexins with this type of mismatched monomer stability are Cx37 (light) and Cx43 (dark) [7]. In either case B or C, expression and function of the connexin quality control pathway can potentially act as a “rheostat” to regulate hetero-oligomerization by a direct effect on relative monomer stability.

Figure 5. Structural homology model of a heterotypic Cx26/Cx32 gap junction channel.

One pair of end-to-end docked Cx26 and Cx32 subunits is highlighted as ribbon and line diagrams overlaid on the structural model. Six hydrogen bonds at the EL2-EL2 docking interface are predicted by the homology model (inset). Residues involved in the spatial positioning of the interface are labeled and the central carbon atoms of the residues involved in hydrogen bonding are represented by a dark oval. Hydrogen-bonds shown are between Cx32 N175 and Cx26 K168, T177 and D179 and between Cx26 N176 and Cx32 K167, T176 and D178. Adapted from [28] with permission.