The N terminus of connexin37 contains an alpha-helix that is required for channel function.
The cytoplasmic N-terminal domain of connexins has been implicated in multiple aspects of gap junction function, including connexin trafficking/assembly and channel gating. A synthetic peptide corresponding to the first 23 amino acids of human connexin37 was prepared, and circular dichroism and nuclear magnetic resonance studies showed that this N-terminal peptide was predominantly alpha-helical between glycine 5 and glutamate 16. The importance of this structure for localization of the protein at appositional membranes and channel function was tested by expression of site-directed mutants of connexin37 in which amino acids leucine 10 and glutamine 15 were replaced with prolines or alanines. Wild type connexin37 and both substitution mutants localized to appositional membranes between transfected HeLa cells. The proline mutant did not allow intercellular transfer of microinjected neurobiotin; the alanine mutant allowed transfer, but less extensively than wild type connexin37. When expressed alone in Xenopus oocytes, wild type connexin37 produced hemichannel currents, but neither of the double substitution mutants produced detectable currents. The proline mutant (but not the alanine mutant) inhibited co-expressed wild type connexin37. Taken together, our data suggest that the alpha-helical structure of the connexin37 N terminus may be dispensable for protein localization, but it is required for channel and hemichannel function.
Kyle
John W
JW
Department of Medicine (Section of Cardiology), University of Chicago, Chicago, Illinois 60637-1470, USA.
Berthoud
Viviana M
VM
Kurutz
Josh
J
Minogue
Peter J
PJ
Greenspan
Michael
M
Hanck
Dorothy A
DA
Beyer
Eric C
EC
D000595:Amino Acid Sequence; D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives / Q000378:metabolism; D002942:Circular Dichroism; D017630:Connexins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017629:Gap Junctions / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D017433:Protein Structure, Secondary; D014981:Xenopus; D000097006:Gap Junction alpha-4 Protein
D001710:Biotin; D017630:Connexins; D010455:Peptides; D000097006:Gap Junction alpha-4 Protein; C070614:neurobiotin