Identification of a pore lining segment in gap junction hemichannels

Area of Science:

• Molecular biology
• Cellular biophysics
• Structural biology

Background:

• Connexins form gap junction channels and hemichannels, crucial for intercellular communication.
• Understanding connexin hemichannel pore structure is vital for studying channel function and regulation.
• Previous studies have utilized cysteine scanning mutagenesis to map protein structures.

Purpose of the Study:

• To investigate the pore structure of cx46 and cx32E(1)43 hemichannels.
• To identify amino acid residues accessible from the extracellular space within the hemichannel pore.
• To elucidate the contribution of transmembrane segments to the hemichannel pore lining.

Main Methods:

• Cysteine scanning mutagenesis was performed on cx46 and a chimeric connexin (cx32E(1)43).
• Mutated connexins were expressed in Xenopus oocytes to form functional hemichannels.
• The thiol-reactive reagent maleimido-butyryl-biocytin was used to probe cysteine accessibility.

Main Results:

• Maleimido-butyryl-biocytin inhibited hemichannel activity when specific cysteines were introduced at positions I33C/M34C (cx32E(1)43) and I34C/L35C (cx46).
• These identified positions in the first transmembrane segment are accessible from the extracellular environment.
• The findings suggest these residues contribute to the lining of the hemichannel pore.

Conclusions:

• The first transmembrane segment of cx46 and cx32E(1)43 hemichannels contains residues lining the pore and accessible from the extracellular space.
• The hemichannel pore structure is complex and likely involves contributions from multiple transmembrane segments.
• This study provides insights into the structural organization of connexin hemichannels.