Structure of native lens connexin 46/50 intercellular channels by cryo-EM

Abstract

Gap junctions establish direct pathways for cell-to-cell communication, through the assembly of twelve subunits (connexins) that form intercellular channels connecting neighboring cells. Co-assembly of different connexin isoforms produces channels with unique properties, and enables communication across cell-types. To gain access into the structural underpinnings of connexin co-assembly, we used single particle CryoEM to determine the structure of native lens gap junction channels, composed of connexin-46 and connexin-50 (Cx46/50). We provide the first comparative analysis to connexin-26 (Cx26), which together with computational studies elucidates key energetic features governing gap junction perm-selectivity. Cx46/50 adopts an open-state conformation that is unique from the Cx26 crystal structure, yet appears to be stabilized by a conserved set of hydrophobic anchoring residues. ‘Hot spots’ of genetic mutations linked to hereditary cataract formation map to the core structural-functional elements identified in Cx46/50, rationalizing many of the disease-causing effects.

Connexin-46/50 in a dynamic lipid environment resolved by CryoEM at 1.9 Å

“…Each monomer consists of four transmembrane helices (TM1-4), two extracellular loops (EC1-2) that form the sites of docking interaction and an amphipathic n-terminal helix (NTH), implicated in channel selectivity/gating, is well resolved in the stabilized open-state, as previously described 16 (Fig. 1c,d and Supplemental Movie 3).…”

“…Native (heteromeric/heterotypic) connexin-46/50 intercellular channels were purified from mammalian lens tissue (obtained from sheep), as previously described 16 . Freshly purified channels were reconstituted into self-assembling lipid nanodiscs containing pure dimyristoyl phosphatidylcholine (DMPC) at room temperature (∼25° C), supported by the membrane scaffold protein MSP1E1 17 (see Methods).…”

“…Cx46/50 is captured in the stabilized open-state, as previously described 16 (backbone Cα r.m.s.d. = 0.49–0.56 Å and Extended Data Fig.…”

“…EC1 and the TM1 parahelix contribute to the selectivity, conductance and slow (loop) voltage-gating mechanisms of Cx46/50 16,21-23 and other connexins 24-29 , and are implicated in Ca 2+ -regulation in Cx26 by X-ray crystallography 19 , MD studies 27,30 , and by functional mutation studies of Cx46 30 . As such, these pore-lining waters may functionally contribute to these mechanisms, for example, by orienting or extending the hydrogen-bonding potential of amino-acid sidechains involved in the coordination of substrates (or regulatory ions), buffering the electrostatic properties of the channel pore, or integrating the electrostatic network that is proposed to couple EC1/TM1 to the fast (NTH) voltage-gating domain 31,32 .…”

A novel voltage clamp/dye uptake assay reveals saturable transport of molecules through CALHM1 and connexin channels

“…They are ubiquitously expressed in almost all human tissues. Based on structural data and permeability profiles, it is thought that the pore diameter of hemichannels is ~12-15 Å (Harris, 2007;Maeda et al, 2009;Sáez and Leybaert, 2014;Myers et al, 2018). Thus, connexin hemichannels have been described as conduits not only for atomic ions, but also for small biological molecules such as ATP, IP3, cAMP, glutamate, prostaglandins, and NAD + (Harris, 2007).…”