Structure of the connexin-43 gap junction channel in a putative closed state

Abstract

Gap junction channels (GJCs) mediate intercellular communication by connecting two neighboring cells and enabling direct exchange of ions and small molecules. Cell coupling via connexin-43 (Cx43) GJCs is important in a wide range of cellular processes in health and disease, yet the structural basis of Cx43 function and regulation has not been determined until now. Here we describe the structure of a human Cx43 GJC solved by cryo-EM and single particle analysis at 2.26 Å resolution. The pore region of Cx43 GJC features several lipid-like densities per Cx43 monomer, located close to a putative lateral access site at the monomer boundary. We found a previously undescribed closed sieve-like gate on the cytosolic side of the pore, formed by the N-terminal domain and the transmembrane helix 2 of Cx43 and stabilized by a small molecule. Structures of the Cx43 GJC and hemichannels in nanodiscs confirm the sieve-like gate arrangement. The features of the Cx43 GJC and hemichannel cryo-EM maps and the channel properties revealed by molecular dynamics simulations suggest that the captured states of Cx43 are closed.

Insulin docking within the open hemichannel of connexin 43 may reduce risk of amyotrophic lateral sclerosis

“…Because astrocytes may be responsible for the progression of ALS, drugs that block Cx43 hemichannels might be therapeutic [5]. One analysis of the Cx43 structure revealed a closed sieve-like molecular gate [16]. The Cx43 N-terminal domain is involved in channel gating and oligomerization and may control the switch between the channel’s open and closed states.…”

Light-activated assembly of connexon nanopores in synthetic cells

“…Fortunately, several pore structures of connexins have recently been solved by Cryo-EM, opening the door for structureguided design of an activatable pore. [44][45][46][47][48] Here we report the light-activated assembly of a connexon nanopore in synthetic cells and the subsequent release of internal contents. We re-engineer connexin-43 (Cx43) 46,47 with a bulky protease recognition domain, which allows membrane-protein association but prevents nanopore function in the membrane.…”

“…Here we report the light-activated assembly of a connexon nanopore in synthetic cells and the subsequent release of internal contents. We re-engineer connexin-43 (Cx43) 46,47 with a bulky protease recognition domain, which allows membrane-protein association but prevents nanopore function in the membrane. After enzymatic digestion, Cx43 rapidly assembles into a functional nanopore, releasing contents into the extracellular space.…”

Connexins and Pannexins: Important Players in Neurodevelopment, Neurological Diseases, and Potential Therapeutics

“…For instance, comparative analysis between Cx26 [ 33 ] and Cx46/50 gap junctions [ 34 ], as well as the structure of Cx31.3 hemichannels [ 35 ] showed that the N-terminal domain appears to be involved in the gating mechanisms of gap junctions and hemichannels, adopting several conformations in different Cxs [ 36 ]. Disease-related mutations seem to be located in the extracellular loops, the pore region and the gating region.…”

“…At a pH of 6.9, the open state is favored: the transition between the closed and open state involves structural changes (in the secondary structure) in the first transmembrane domain, movement of the second to the fourth transmembrane domain and structural stabilization of the cytoplasmic loop [ 36 , 38 ]. The pharmacological inhibition of hemichannels formed by Cx43 have been tested in cerebral ischemia and in myocardial ischemia models, in which a large number of hemichannels are usually opened.…”