Therapeutic strategies targeting connexins

Abstract

The connexin family of channel-forming proteins is present in every tissue type in the human anatomy. Connexins are best known for forming clustered intercellular channels, structurally known as gap junctions, where they serve to exchange members of the metabolome between adjacent cells. In their single-membrane hemichannel form, connexins can act as conduits for the passage of small molecules in autocrine and paracrine signalling. Here, we review the roles of connexins in health and disease, focusing on the potential of connexins as therapeutic targets in acquired and inherited diseases as well as wound repair, while highlighting the associated clinical challenges.

Figure 1:. Connexin topology and the connexin family.

A: Connexin topology within the lipid bilayer illustrating the main functional domains and the connexin gene subclasses. The connexin polypeptide passes through the membrane 4 times (gray), resulting in both the amino and carboxyl terminal domains being exposed to the cytoplasm and connected by two extracellular and one cytoplasmic-exposed loops . The highly conserved amino terminal domain (red) plays a role in regulating the channel pore ^, while the two conserved disulfide-linked extracellular loops (blue) govern hemichannel docking between adjacent cells . The cytoplasmic loop (purple) size varies extensively amongst connexin subtypes and it has been reported to participate in pH gating of the channel for at least one connexin . The cytoplasmic tail (yellow) length and amino acid sequence is the most varied region amongst the connexin family members. This domain is frequently phosphorylated in many connexins and responsible for binding the vast majority of the connexin interactome ^,. B: The connexin family is divided into 5 subclasses (α, β, γ, δ, ε) based on homology. Connexins may oligomerize into homomeric or heteromeric hexamers with other connexins within the same subclass and, on occasion, with connexins from other subclasses leading to highly diversified channel arrangements . While most connexins are known to form functional channels, it remains unclear if this is the case for Cx23 .

Figure 2:. Canonical life cycle of connexins.

Two connexins (pink, blue) are shown to co-translationally insert into the endoplasmic reticulum where they appear as monomers or proceed to co-oligomerize into homomeric or heteromeric single membrane channels, while being resident in the endoplasmic reticulum or after transport to the Golgi apparatus. After constitutive or microtubule-facilitated delivery to the cell surface, connexins may function as hemichannels or proceed to form homotypic or heterotypic gap junction channels that cluster into fully assembled gap junctions. Typically, after a short residency at the cell surface, gap junctions, or fragments of gap junctions, are internalized as unique double-membraned connexosomes before their ultimate degradation in lysosomes. Throughout the typical short life expectancy of connexins, they interact with a large interactome that facilitates their assembly, turnover and function.

Figure 3:. Links between connexins, the connexin interactome and early stage tumourigenesis.

A: Cx43 interacts with many proteins including some (noted here) that have been reported to be involved in tumourigenesis. Cx43 binding proteins are indicated as enhancing (red type), reducing (green type), or playing unclear roles (blue) in tumourigenesis but their mode of action may, in part, be due to their interactions with Cx43. Calmodulin interacts with the intracellular loop, while the others interact with motifs encoded within the C-terminal tail. The names of the Cx43 interactors are positioned near the site of the C-terminal tail where they bind, except TSG101, NOV, β-catenin, BAX, p62, EPS15 and PKC as their specific binding sites within the C-terminal domain are less well defined. A more complete list of Cx43 interactors can be found in other publications ^,,. B: Connexins present in gap junctions (primarily Cx26, Cx32, Cx43) have been widely reported to inhibit the promotion, growth and invasion stages of tumourigenesis, but may have more complex roles in later stages of disease progression that is tumour type dependent .

Figure 4:. Current and potential connexin modulating therapeutic strategies.

Given the short half-life of connexins, therapeutic strategies can be designed to modulate their expression and/or function at multiple points throughout their life cycle. The purple boxes and numbers represent strategies that have entered therapeutic testing or under development. These include; targeting connexin mRNA levels by using antisense technology to reduce connexin protein levels (A), ultimately leading to a reduction in connexin hemichannels, which can also be blocked by select inhibitors (B), regulating GJ size by promoting hemichannel recruitment into GJ (C), and increasing GJIC (D). Blue boxes and numbers denote potential future therapeutic strategies. It is possible to imagine and design drugs that will drive the expression of connexin encoding genes (a) in cases where the expression of a second connexin family member may rescue the pathology invoked by a disease-linked connexin gene mutation. In the age of CRISPR/Cas9 technology, it is also possible to consider gene editing to repair disease-linked connexin gene mutations (b), or possibly rescue trafficking defective connexin mutants so they make it to the cell surface where they may still retain sufficient channel function to prevent disease (c). Novel antibody targeting or other strategies that block leaky mutant or dysregulated connexin hemichannels may have therapeutic value (d), as would regulating the overall stability of gap junctions that have assembled at sites of cell-cell contact (e).