Molecular Dynamics Simulations of the Cx26 Hemichannel: Insights into Voltage-Dependent Loop-Gating

Abstract

Loop-gating is one of two voltage-dependent mechanisms that regulate the open probability of connexin channels. The loop-gate permeability barrier is formed by a segment of the first extracellular loop (E1) (the parahelix) and appears to be accompanied by straightening of the bend angle between E1 and the first transmembrane domain (TM1). Here, all-atom molecular dynamics simulations are used to identify and characterize interacting van der Waals and electrostatic networks that stabilize the parahelices and TM1/E1 bend angles of the open Cx26 hemichannel. Dynamic fluctuations in an electrostatic network in each subunit are directly linked to the stability of parahelix structure and TM1/E1 bend angle in adjacent subunits. The electrostatic network includes charged residues that are pore-lining and thus positioned to be voltage sensors. We propose that the transition to the closed state is initiated by voltage-driven disruption of the networks that stabilize the open-state parahelix configuration, allowing the parahelix to protrude into the channel pore to form the loop-gate barrier. Straightening of the TM1/E1 bend appears to be a consequence of the reorganization of the interacting networks that accompany the conformational change of the parahelix. The electrostatic network extends across subunit boundaries, suggesting a concerted gating mechanism.

Mechanism of gating by calcium in connexin hemichannels

“…Nevertheless, the initial events seen in the present simulations indicate that the subsequent rearrangements of the network of electrostatic interactions below D50-K61 are among the steps that enhance the probability of transition to a Ca 2+ -induced closed state. The previously equilibrated Cx26 hemichannel and other connexin structural models also associate these residues with electrostatic interactions in the open conformation (21,22).…”

“…Several charged residues in this region (D46, E47, D50, K61, R75, R184) participate in electrostatic networks (21). Those that are negatively charged are pore-lining, which may favor accumulation of permeant cations in this region.…”

“…S2). It has been proposed that reorganization of this network plays a key role in gating (21). The molecular dynamics simulations show that the electrostatic network involving these residues is rearranged in A C D several subunits in response to Ca 2+ coordination by D50 in two subunits.…”

A gap junction docking mechanism revealed by functional rescue of a human disease-linked connexin mutant

“…When sufficient E2-E2 hydrogen bonds ($4 per pair or $24 per gap junction channel) had been formed, the two hemichannels would be docked properly and ready to 'open' as a functional gap junction channel. The open transition probably requires further conformational change at the E1 domains, part of which could be the previously described 'loop-gating' process (Kwon et al, 2012). Our earlier structural studies Nakagawa et al, 2011) indicated that the inter E1-E1 hydrogen bonds in Cx26 and/or Cx32 channels are also formed and might help to stabilize the gap junction channel, but they are unlikely to serve for docking recognition because the hydrogen-bond-forming residues are almost identical for both compatible and non-compatible groups of Cxs.…”

Functional Requirement for a Highly Conserved Charged Residue at Position 75 in the Gap Junction Protein Connexin 32

“…In addition, mutations in this residue (or its homologous residue at position 76) have been associated with at least four different human disorders. Furthermore, the crystal structure of Cx26, a connexin closely related to Cx32, shows that the Arg 75 residue to be a key component of an electrostatic cluster having a central role in interprotomeric interactions of the Cx26 hemichannel (23,31). These facts suggest that the arginine at position 75 is critical for the normal functioning of Cx32.…”