Structural insights into the gating mechanism of human Cx43/GJA1 gap junction channel

Abstract

Connexin family proteins assemble into hexameric hemichannels in a cell membrane, which dock together between two adjacent membranes to form gap junction intercellular channels (GJIChs). The most ubiquitously expressed connexin Cx43 plays important roles in numerous biological processes. Here we report cryo-EM structures of Cx43 GJIChs at 3.1–3.6 Å resolutions, which show dynamic conformational changes of N-terminal helices (NTHs) caused by pH change or C-terminal truncation. Cx43 GJIChs in a channel-closing condition contain 12 protomers in gate-covering NTH (GCN) conformation, while those in opening conditions have varying compositions of GCNs and pore-lining NTHs (PLNs) resulting in various pore dimensions and electrostatic surface potentials. GCN-to-PLN transition accompanies π-helix formation in the first transmembrane helix (TM1), movement of TM2-4 that creates a side opening to the membrane, and structural stabilization of the cytoplasmic loop. Our study provides structural insights into the intercellular ion/metabolite transfer and the lateral lipid transport through Cx43 GJICh.

Light-activated assembly of connexon nanopores in synthetic cells

“…With a functional Cx43 synthetic cell system, we turned our attention to suppressing Cx43 nanopore activity. Previous work in cells showed that Cx43 function is sensitive to cytoplasmic domain modification, 61 and recent Cryo-EM structures of connexon pores, 44,45,48 including Cx43’s, 46,47 present an opportunity to use structure-guided design to engineer inhibition of Cx43. For Cx43, two solved structures show that the N-terminus provides key contacts at the bottom of the pore (cytoplasmic side) and faces inward toward the vacant channel (Fig.…”

“…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

“…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.…”

Light-Activated Assembly of Connexon Nanopores in Synthetic Cells

“…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) , 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.…”