# Lipid dependence of connexin-32 gap junction channel conformations

## TL;DR

The study reveals how specific lipids and sterols control the opening and closing of Cx32 gap junction channels, which are linked to a genetic disease.

## Contribution

The novel finding is that phospholipid and sterol binding directly regulates Cx32 channel conformation through the N-terminal gating helix.

## Key findings

- Phospholipids block the Cx32 gap junction channel pore by binding to a site formed by N-terminal helices.
- Sterol molecules are required for phospholipid binding, as their absence disrupts the phospholipid binding site.
- The CMT1X-linked W3S mutation prevents phospholipid binding due to an incompatible N-terminal conformation.

## Abstract

Connexin-32 (Cx32) gap junction channels (GJCs) mediate intercellular coupling in various tissues, including myelinating Schwann cells. Mutations in Cx32, such as W3S, are associated with X-linked Charcot-Marie-Tooth (CMT1X) disease. Lipids regulate Cx32 GJC permeation, although the regulatory mechanism is unclear. Here, we determine the cryo-EM structures of Cx32 GJCs reconstituted in nanodiscs, revealing that phospholipids block the Cx32 GJC pore by binding to the site formed by N-terminal gating helices. The phospholipid-bound state is contingent on the presence of a sterol molecule in a hydrophobic pocket formed by the N-terminus: the N-terminal helix of Cx32 fails to sustain a phospholipid binding site in the absence of cholesterol hemisuccinate. The CMT1X-linked W3S mutant which has an impaired sterol binding site adopts a conformation of the N-terminus incompatible with phospholipid binding. Our results indicate that different lipid species control connexin channel gating directly by influencing the conformation of the N-terminal gating helix.

Gap junction channels formed by Cx32 enable intercellular communication, and their disruption causes CMT1X disease. Here, authors use cryo-EM and molecular dynamics simulations to show how specific lipids and sterols may directly control Cx32 channel gating