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

Hyuk-Joon Lee,Hyeongseop Jeong,Jaekyung Hyun,Bumhan Ryu,Jae-Sung Woo,Kunwoong Park,Jejoong Yoo,Hyun-Ho Lim

doi:10.1126/sciadv.aba4996

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.

Highlights

Gap junction facilitates direct communication between neighboring cells and plays key roles in numerous cellular processes such as cardiac contraction, ionic transmission in electrical synapse, development, and differentiation
The hemichannel structure showed that the entrance-covering N-terminal helix (NTH) conformation forms a small pore slightly bigger than hydrated K+ or Cl− ions, suggesting that the transport of metabolites much bigger than ions would require a conformational change of NTH
We propose that the entrance- covering NTH conformation represents a partially closed state of Cx31.3 hemichannel, which would prevent the movement of metabolites through and selectively allows small anions to pass through the hemichannel

Summary

INTRODUCTION

Gap junction facilitates direct communication between neighboring cells and plays key roles in numerous cellular processes such as cardiac contraction, ionic transmission in electrical synapse, development, and differentiation. In a more recent electrophysiological study on Cx26 hemichannel, D50 and E47 were found to be major Ca2+-binding sites [13], suggesting that the Ca2+-dependent closing mechanism of hemichannel is different from that of GJICh. Clear explanation of the causal relationship between the Ca2+ binding to these residues and Cx26 channel closing is precluded because of the lack of high-resolution structure. We used single-particle cryo-EM to determine three high-resolution structures of Cx31.3 hemichannels in the presence and absence of Ca2+ ions and the R15G-mutant hemichannel, respectively These structures show the NTH conformation distinct from the previous GJICh structures, putative lipid-binding sites in the inner surface of the pore, and Ca2+-binding tunnels in the interprotomer interface and between transmembrane and extracellular regions. Together with molecular dynamics (MD) simulations and electrophysiological experiments, reveals the selective permeability mechanism of Cx31.3 hemichannel

RESULT

DISCUSSION

MATERIALS AND METHODS