The connexin26 human mutation N14K disrupts cytosolic intersubunit interactions and promotes channel opening

Abstract

A group of human mutations within the N-terminal (NT) domain of connexin 26 (Cx26) hemichannels produce aberrant channel activity, which gives rise to deafness and skin disorders, including keratitis-ichthyosis-deafness (KID) syndrome. Structural and functional studies indicate that the NT of connexin hemichannels is folded into the pore, where it plays important roles in permeability and gating. In this study, we explore the molecular basis by which N14K, an NT KID mutant, promotes gain of function. In macroscopic and single-channel recordings, we find that the N14K mutant favors the open conformation of hemichannels, shifts calcium and voltage sensitivity, and slows deactivation kinetics. Multiple copies of MD simulations of WT and N14K hemichannels, followed by the Kolmogorov–Smirnov significance test (KS test) of the distributions of interaction energies, reveal that the N14K mutation significantly disrupts pairwise interactions that occur in WT hemichannels between residue K15 of one subunit and residue E101 of the adjacent subunit (E101 being located at the transition between transmembrane segment 2 [TM2] and the cytoplasmic loop [CL]). Double mutant cycle analysis supports coupling between the NT and the TM2/CL transition in WT hemichannels, which is disrupted in N14K mutant hemichannels. KS tests of the α carbon correlation coefficients calculated over MD trajectories suggest that the effects of the N14K mutation are not confined to the K15–E101 pairs but extend to essentially all pairwise residue correlations between the NT and TM2/CL interface. Together, our data indicate that the N14K mutation increases hemichannel open probability by disrupting interactions between the NT and the TM2/CL region of the adjacent connexin subunit. This suggests that NT–TM2/CL interactions facilitate Cx26 hemichannel closure.

Opposing modulation of Cx26 gap junctions and hemichannels by CO2

“…KIDS mutations are thought to cause the syndrome through gain-of-function. Several lines of evidence suggest that KIDS mutated Cx26 hemichannels are leaky: either they are less sensitive to Ca 2+ blockade (Sanchez et al 2010;Mese et al 2011;Lopez et al 2013;Zhang & Hao, 2013;Sanchez et al 2014) or have an altered voltage sensitivity such that they spend more time in the open state at transmembrane potentials closer to the resting potential (Sanchez et al 2016;Valdez Capuccino et al 2019). Our finding that three KIDS mutations studied here prevent gap junctions from closing to CO 2 is effectively a further gain of function caused by these mutations: they will remain open under conditions of elevated CO 2 .…”

Opposing modulation of Cx26 gap junctions and hemichannels by CO2

“…KIDS mutations are thought to cause the syndrome through gain-of-function. Several lines of evidence suggest that KIDS mutated Cx26 hemichannels are leaky: either they are less sensitive to Ca 2+ blockade (Sanchez et al, 2010;Mese et al, 2011;Lopez et al, 2013;Zhang & Hao, 2013;Sanchez et al, 2014) or have an altered voltage sensitivity such that they spend more time in the open state at transmembrane potentials closer to the resting potential (Sanchez et al, 2016;Valdez Capuccino et al, 2019). Our finding that three KIDS mutations studied here prevent gap junctions from closing to CO2 is effectively a further gain of function caused by these mutations: they will remain open under conditions of elevated CO2.…”

A heterozygous mutation in GJB2 (Cx26F142L) associated with deafness and recurrent skin rashes results in connexin assembly deficiencies

“…[1,[26][27][28][29] "Leaky" hemichannels, with altered gating characteristics, are proposed to be a causative event in the progression of KID syndrome. [8,9,16,[30][31][32] Each KID mutation studied to date has unique characteristics rendering the channels more sensitive to changes in calcium, zinc and other ions and are influenced by pro-inflammatory mediators such as peptidoglycan a key component of gram-positive bacterial cell wall, [8,9,16,31,32] with several mutations resulting in lethal phenotypes. [10,[33][34][35] Overexpression of CX26 (for example in psoriasis) or these leaky KID hemichannels release ATP from the cells feeding into purinergic signalling pathways and associated pro-inflammatory signalling cascades leading to hyperproliferation loss of epidermal integrity and inflammation.…”