Channel structure of a connexin 43 gap junction in a putative closed state (2023)

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1. 

   eLife

   22. May 2023 r

   eLife Rating

   Gap junctions formed by connexins are important for cellular communication and allow the direct movement of ions and small molecules between cells. By determining the cryo-EM structure of connexin 43 in a putative closed state with lipids, the study makes an important contribution to the development of a mechanistic model of connexin activation. The structure of connexin 43 is robust and its presentation will appeal to the channel and membrane protein communities.

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2. 

   eLife

   22. May 2023 r

   Reviewer #1 (public review):

   Gap junctions formed by connexins are important for cellular communication and allow the direct movement of ions and small molecules between cells. Although the structures of connexins have been reported previously, the structure of connexin 43, the most commonly expressed connexin and important in many physiological processes, was not known. Qi et al. used cryo-EM to elucidate the structure of connexin 43. They then compared this structure with the structure of other connexins. Connexin-gap junctions consist of two "semi-channels" made up of connexin hexamers. Half channels of two opposite cells join to form a full channel that allows molecules to move between cells. N-terminal helices of each of the 6 subunits of each half-channel allow control of whether the channels are open or closed. ...

   Reviewer #1 (public review):

   Gap junctions formed by connexins are important for cellular communication and allow the direct movement of ions and small molecules between cells. Although the structures of connexins have been reported previously, the structure of connexin 43, the most commonly expressed connexin and important in many physiological processes, was not known. Qi et al. used cryo-EM to elucidate the structure of connexin 43. They then compared this structure with the structure of other connexins. Connexin-gap junctions consist of two "semi-channels" made up of connexin hexamers. Half channels of two opposite cells join to form a full channel that allows molecules to move between cells. N-terminal helices of each of the 6 subunits of each half-channel allow control of whether the channels are open or closed. The N-termini of the previously resolved Cx26 and Cx46/50 structures lie downstream of the pore of the protein, leaving a central pore, so these channels were assumed to be open. The Qi et al. has the N-terminus at an elevated position with a narrower pore in the middle. This led them to speculate as to whether it was a 'locked' form of the protein. They also noticed that when looking at only proteins, there were gaps between the N-terminal helices, but these gaps were filled with lipid-like molecules. Hence, they speculated that lipids play an important role in the locking mechanism. To determine whether their structure is ion-open or closed, they performed molecular dynamics studies and showed that ions do not pass through the channel under molecular dynamics conditions when lipids are present.

   powers
   The high-resolution cryo-EM density maps clearly show the protein structure with N-termini in lateral position and the density of gap-blocking lipids between adjacent helices. The conformation they observe when they resolve the protein's structure in detergent is also revealed when they convert the protein into nanodiscs, which present a more membrane-like environment. So you seem to have trapped the protein in a stable conformational state.
   Molecular dynamics simulations are consistent with channel closure in the presence of lipids, suggesting the possibility that lipids are involved in regulation.
   Comparison of this structure with other structures of connexins and hemicanals provides another illustration of how the N-terminal helix of connexins may be differentially involved in regulating channel opening.

   weaknesses
   Although the authors captured the relatively stable state of the protein and showed that ions do not pass through the protein under molecular dynamics simulation conditions, it is more difficult to understand whether this is physiologically relevant. Determining this would go beyond the scope of this article. As far as I know, there is no direct evidence that lipids are involved in regulating connexins in this way, but it is also an interesting area for future research. It is also possible that the lipids became trapped in the pores during the solubilization process, rendering them non-physiological. The authors recognize this and describe the structure as an "implicitly" closed state.
   The mutation positions in the disease shown in Figure 4 are interesting. However, the authors do not discuss/speculate how these mutations might affect lipid binding or the conformational state of the protein.

   It should also be noted that the structure of the same protein has recently been published. This shows a very similar N-terminal conformation with lipids bound in the same way, although dissolved in a different detergent.

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3. 

   eLife

   22. May 2023 r

   Reviewer #2 (public review):

   Manuscript by Qi et. Aluminum. provide new structures for connexin 43 (Cx43) gap junction channels (GJCs) and hemichannels that are thought to correspond to the closed conformations of these channels. This leads the authors to propose a gating mechanism involving the presence of lipids in the pore that can stabilize the N-terminal domain as the gate region in the pore. The authors performed a lipid analysis of their structures and identified dehydroepiandrosterone (DHEA), a sterol compound that is particularly enriched in their purified Cx43 samples. However, with the current structure resolution, they cannot say whether DHEA is a low-density lipid-like substance found in the pores of closed channels. Further research, including functional studies, is needed to determine if DHEA is a gating...

   Reviewer #2 (public review):

   Manuscript by Qi et. Aluminum. provide new structures for connexin 43 (Cx43) gap junction channels (GJCs) and hemichannels that are thought to correspond to the closed conformations of these channels. This leads the authors to propose a gating mechanism involving the presence of lipids in the pore that can stabilize the N-terminal domain as the gate region in the pore. The authors performed a lipid analysis of their structures and identified dehydroepiandrosterone (DHEA), a sterol compound that is particularly enriched in their purified Cx43 samples. However, with the current structure resolution, they cannot say whether DHEA is a low-density lipid-like substance found in the pores of closed channels. Further research, including functional studies, is needed to determine if DHEA is a gating mediator. Interestingly, other recently published structures of large-pore channels support the assumption that the pores contain lipids. However, this evidence is only supported by cryo-EM structures and is a topic that generates considerable controversy in the field, especially when these molecules are involved in gating mechanisms. The discovery of putative lipid-pore interactions is a very exciting observation, but should be interpreted with caution. The main problem is that channel reconstitution occurs with an excess of lipids and detergents, which can lead to artifacts. Therefore, these lipid-like densities observed in Cx43 (and other structures) after single-particle analysis could not represent native lipid-protein interactions. Then any conclusions about the role of lipids in gating can be based on a possible artifact caused by protein purification. In addition, it is difficult to visualize how lipids can move in and out of the pores during gating, particularly from this putative lipid pore conformation to an open conformation.

   Another important aspect of this work is the provision of structures for both GJC Cx43 and half channels. As expected, there are differences in the rearrangement of the extracellular loops between the two structures. A problem, however, is that the resolution of the Cx43 hemichannels is still low (3.98 Å), so interpretations should be treated with caution. In addition, intracellular domains important for the control and regulation of Cx43, including the intracellular loop and the carboxy-terminal domain, were not resolved in these structures. Nevertheless, this is a common problem with other cryo-EM connexin structures reported in the literature.

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4. 

   Version 3 published on bioRxiv

   22. May 2023 r

5. 

   Version 2 published on bioRxiv

   March 9, 2023 r

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   Version 1 published on bioRxiv

   March 27, 2022 r