Cryo-EM structure of human Cx31.3/GJC3 connexin hemichannel

Abstract

Connexin family proteins assemble into hexameric channels called hemichannels/connexons, which function as transmembrane channels or dock together to form gap junction intercellular channels (GJIChs). We determined the cryo–electron microscopy structures of human connexin 31.3 (Cx31.3)/GJC3 hemichannels in the presence and absence of calcium ions and with a hearing-loss mutation R15G at 2.3-, 2.5-, and 2.6-Å resolutions, respectively. Compared with available structures of GJICh in open conformation, Cx31.3 hemichannel shows substantial structural changes of highly conserved regions in the connexin family, including opening of calcium ion–binding tunnels, reorganization of salt-bridge networks, exposure of lipid-binding sites, and collocation of amino-terminal helices at the cytoplasmic entrance. We also found that the hemichannel has a pore with a diameter of ~8 Å and selectively transports chloride ions. Our study provides structural insights into the permeant selectivity of Cx31.3 hemichannel.

Cryo-EM structure of an open conformation of a gap junction hemichannel in lipid bilayer nanodiscs

“…In the Ca 2+ -bound Cx26 GJC structure (PDB: 5ERA), Ca 2+ binding occurs near the extracellular end of the M1 helix, with coordination mediated between adjacent protomers. This binding site overlaps with the tunnel of Ca 2+ ions identified in the Cx31.3/ Cx3 hemichannel structure (Lee et al, 2020). This finding suggests that Ca 2+ -mediated regulation of hemichannels and GJCs may achieve the same ends by similar means.…”

“…Nearly all moderate to high-resolution structures of Cx channels have been limited to the dodecameric GJC (Bennett et al, 2016;Fleishman et al, 2004;Flores et al, 2020;Khan et al, 2020;Maeda et al, 2009;Myers et al, 2018;Unger et al, 1997Unger et al, , 1999. One exception is the recently published cryo-EM structure of Cx31.3/GJC3, an obligate hemichannel expressed in oligodendrocytes in the CNS, and it will be discussed below (Lee et al, 2020).…”

“…During preparation of our revised manuscript, a high-resolution cryo-EM structure of the Cx31.3/GJC3 hemichannel solubilized in the detergent LMNG was published (Lee et al, 2020). A unique feature of Cx31.3/GJC3 hemichannels is that it is an obligate hemichannel and does not form GJCs (Flores et al, 2020;Liang et al, 2011).…”

“…This diameter is substantially smaller compared with our crystal structures of the Cx26 GJC, with a pore diameter of $20 Å (Bennett et al, 2016), and to the cryo-EM structure of the Cx26 hemichannel presented here. Thus, the Cx31.3 hemichannel structure was deemed to be ''partially closed,'' as molecular dynamics (MD) simulations and electrophysiological recordings revealed that small anions (such as Cl À ) could still permeate the pore (Lee et al, 2020).…”

“…Based on electrophysiology of Cx26 hemichannels, Ca 2+ may destabilize the open state and trigger pore attenuation, and this has been implicated to occur by a steric closure mechanism (Lopez et al, 2016). The cryo-EM structure of Cx31.3/Cx3 in the presence of Ca 2+ revealed a ''tunnel'' of Ca 2+ ions weakly bound at an acidic region of residues across protomers at the transmembrane/extracellular loop interface (Lee et al, 2020). Many of the residues participating in this Ca 2+ coordination network are highly conserved across the Cx family.…”