Insights On Gating Functions Of Cytosolic Domains Of Connexin26 Hemichannels Revealed By A Human Pathogenic Mutation (N14k)

Connexin 26 (Cx26) is a hexameric, transmembrane protein. At the cell membrane, it can be found forming hemichannels or gap junction channels (GJCs) with opposing cells. The N-terminal domain (NT) of these channels is folded into the pore, playing an important role in permeability and gating. A group of mutations within the NT that produce aberrant hemichannels with increased basal activity is responsible for Keratitis-Ichthyosis-Deafness (KID) syndrome. In this study we focus on N14K, an NT KID mutant. Structural data reveals an interaction between the NT and cytoplasmic loop (CT) of the channel, where position 14 is located. Here, we explored how the N14K mutant affects the interactions between these two regions and consequently, promotes gain in function. Assessing macroscopic and single-channel recordings, we observed that the N14K mutant shows an increase in the energy barrier for the transition between open and closed states, shifting calcium sensitivity, voltage sensitivity, and deactivation time constants. Correlation analysis of Cx26 WT molecular dynamics (MD) simulations identified several sites of NT-CL interaction: These include interactions between N14 and residues H100 and Y97, and between K15 and E101. Interestingly, the same analysis performed on the Cx26 N14K MD simulations showed that the insertion of a Lys at position 14 completely disrupted the NT-CL interactions. To test this, we used double mutant cycle analysis, which showed that the NT-CL interaction does occur in the WT channel and is disrupted in the N14K mutant. Our data suggest that disruption of NT-CL interaction facilitates hemichannel opening and stabilizes the open state. In addition, it provides a mechanistic understanding of how mutations at position 14 cause human disease.