Mitochondrial Connexins and Mitochondrial Contact Sites with Gap Junction Structure

Abstract

Mitochondria contain connexins, a family of proteins that is known to form gap junction channels. Connexins are synthesized in the endoplasmic reticulum and oligomerized in the Golgi to form hemichannels. Hemichannels from adjacent cells dock with one another to form gap junction channels that aggregate into plaques and allow cell-cell communication. Cell-cell communication was once thought to be the only function of connexins and their gap junction channels. In the mitochondria, however, connexins have been identified as monomers and assembled into hemichannels, thus questioning their role solely as cell-cell communication channels. Accordingly, mitochondrial connexins have been suggested to play critical roles in the regulation of mitochondrial functions, including potassium fluxes and respiration. However, while much is known about plasma membrane gap junction channel connexins, the presence and function of mitochondrial connexins remain poorly understood. In this review, the presence and role of mitochondrial connexins and mitochondrial/connexin-containing structure contact sites will be discussed. An understanding of the significance of mitochondrial connexins and their connexin contact sites is essential to our knowledge of connexins' functions in normal and pathological conditions, and this information may aid in the development of therapeutic interventions in diseases linked to mitochondria.

Keywords: annular gap junctions; connexin; contact sites; mitochondria.

Figure 1

Schematic diagram of connexin trafficking, gap junction plaque formation, internalization and proposed mechanisms for connexin delivery to the mitochondrial membrane. (1) Connexins are synthesized in the ER and (2) oligomerized into hexameric hemichannels in the Golgi. The hemichannels are then transported to plasma membrane in (3) secretory vesicles where they (4) dock with hemichannels on adjacent cells to form a gap junction channels. The channels cluster to form (5) functional gap junction plaques. Gap junction plaques are removed from the plasma membrane by an invagination process that eventually results in the release of (6) annular gap junction vesicles, which contain membranes from both the donor and host cell. (7) Annular gap junctions are (7) degraded by lysosomes. The proposed mechanisms for the delivery of connexins to the mitochondrial include the close physical associations with (8) annular gap junctions, (9) gap junction plaques and (10) secretory vesicles. Figure created with https://www.biorender.com, accessed on 27 April 2023.

Figure 2

Microscopy images demonstrating gap junction structures in association with mitochondria. (A) Immunofluorescent images showing colocalization (yellow in the merged image) of annular gap junctions (arrows) stained with anti-Cx43 antibody (green) and mitochondria stained with Mitotracker (red). (B) Superresolution-stimulated emission depletion (STED) microscopy of cells stained with anti-Cx43 antibody (green) and Mitotracker (red), which demonstrates annular gap junctions, cell surface puncta representative of gap junction formation (arrowheads), gap junction plaques and the close associations between mitochondria and annular gap junctions (inset on right (arrows). (C) Transmission electron microscopy image revealing the physical contact between an annular gap junction and mitochondria. Scale bars = (A): 10 nm, (B): 1000 nm, (C) 100 nm. Image from reference [6].

Figure 3

Microscopy images demonstrating gap junction structures in association with mitochondria. (A–E) Microscopy images demonstrate contact sites between mitochondria (M) and annular gap junctions (AGJ). Black boxes indicate contact between annular gap junctions and mitochondria. (C) An enlarged image of the area shown in (B) illustrating continuity between annular gap junctions and mitochondria. Scale bars: 500 nm. Image from reference [6].

Figure 4

The molecular architecture of Cx43 on the plasma membrane and putative orientation at the mitochondrial/connexin contact site. (A) Cx43 has four transmembrane domains (M1–M4). The amino-terminus, carboxyl-terminus and cytoplasmic loop (CL) extend into the cytoplasm, while the two extracellular loops face the extracellular space. In gap junctions, the extracellular loops (E1 and E2) of two apposing cells are in close contact. (B) The Cx43 configuration seen in A is depicted in the gap junction plaque shown between two cells (bracket). At the contact site between the mitochondria and gap junction plaque membranes two possible configurations have been suggested (dashed box in (B) and the entirety of (C,D)). Either (C) the hemichannels on the outer mitochondrial membranes oriented with the carboxyl terminus facing the cytoplasm and carboxyl terminal to carboxyl docking would be possible or (D) hemichannels or connexin monomers on the outer mitochondrial membrane have their extracellular loops oriented toward the cytoplasm, which would allow for interactions between the “extracellular loop” and the carboxyl terminus. IMM = inner mitochondrial membrane; OMM = outer mitochondrial membrane; C = carboxyl terminus; PM = plasma membrane; IMS = Intermembrane space; EC = extracellular space. Figure created with https://www.biorender.com. Given the orientation of the connexin in the plasma membrane with the extracellular loops in the extracellular space, it is unlikely that mitochondrial connexins or other possible proteins found on the outer membrane of the mitochondria could interact with the amino acids of the gap junction plaque or annular vesicle connexin extracellular loops. Instead, it has been suggested that binding may occur between the carboxyl-terminal tail segments of connexins, which extend into the cytoplasm (Figure 4C). Specifically, in this proposed model, binding would occur between the carboxyl-terminal regions of the mitochondria connexin and gap junction plaque or annular vesicle connexin molecules (Figure 4C). Support for binding at the carboxyl-terminal regions is provided by the findings that carboxyl-terminal regions can dimerize [77,78,79]. During the gap junction plaque internalization process, which results in the release of annular gap junction vesicles within the cytoplasm, the docked hemichannels remain bound at their extracellular loops [22,24,26,31,80,81]. Thus, the annular gap junction vesicle has a double membrane that is derived from the membrane of the donor cell and the membrane of the recipient cell, and the extracellular loops are located within the space between these two membranes. The carboxyl-terminal region of the connexin hemichannels is derived from the membrane of the host cell, and thus, they are located within the cytoplasm. The carboxyl-terminal region tail of annular gap junction vesicle channels would therefore potentially be available for direct or indirect interaction with the proteins (e.g., connexins) on the outer mitochondrial membrane. However, interactions between the carboxyl-terminal tail and the “external loops” (E1 and E2) (the regions of the connexin molecule that would be docked in the extracellular space, but depending on the orientation on outer mitochondrial membrane, could be projecting into the cytoplasm) (Figure 4D) cannot be ruled out and have been also suggested [9].

Figure 5

Connexin (Cx43) orientation in the inner mitochondrial membrane. (A) Connexins localize to the inner mitochondrial membrane as single channels and/or in hexameric hemichannel structures. (B) The enlarged illustration of mitochondrial cristae depicts inner mitochondrial membrane-localized connexin oriented, such that extracellular loops extend into the matrix while carboxyl-terminal and N-terminal regions extend into the intermembrane space. The figure was produced using Procreate software version 5.3.2.

Figure 6

Proposed model for mitochondrial connexin trafficking and possible role in ischemic preconditioning. Connexin trafficking from the gap junction plaques, annular gap junction vesicles or other locations to the inner mitochondrial membrane is facilitated by a Hsp90 and Tom 20 complex and TIM. Monomeric Cx43 or that in hemichannels would then interact with complex I of the electron transport chain to increase oxidative phosphorylation, which in turn inhibits apoptosis. The interaction of hemichannels with the K⁺ channel increases potassium production. Hemichannels can also block ROS production. The reduction in ROS and the blocking of apoptosis lead to ischemic preconditioning. IMM = inner mitochondrial membrane; IMS = intermembrane space; OMM = outer mitochondrial membrane; TIM = translocase of the inner membrane; TOM = translocase of the outer membrane; HSP90 = heat shock protein 90; ROS = reactive oxygen species; Cx43 = Connexin 43. Figure created using https://www.biorender.com.