Isoelectric points and post-translational modifications of connexin26 and connexin32.
The isoelectric points of the gap junction proteins connexin26 (Cx26) and connexin32 (Cx32) were determined by isoelectric focusing in free fluids. The isoelectric points were significantly more acidic than predicted from amino acid sequences and different from each other, allowing homomeric channels to be resolved separately. The isoelectric points of the homomeric channels bracketed the isoelectric points of heteromeric Cx26/Cx32 channels. For heteromeric channels, Cx26 and Cx32 were found in overlapping, pH-focused fractions, indicating quaternary structure was retained. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to identify post-translational modifications of Cx26 and Cx32 cytoplasmic domains, including the first reported post-translational modifications of Cx26. Suspected modifications were hydroxylation and/or phosphorylation near the amino terminus of both connexins, gamma-carboxyglutamate residues in the cytoplasmic loop of both connexins, phosphorylation in the carboxyl-terminal domain of Cx32, and palmitoylation at the carboxyl-terminus of Cx32. These modifications contribute to the measured acidic isoelectric points of Cx26 and Cx32, whereas their low molecular masses would not appreciably change connexin SDS-PAGE mobility. Most of these modifications have not previously been identified for connexins and may be instrumental in guiding and understanding novel aspects of channel trafficking and molecular mechanisms of channel regulation.
Locke
Darren
D
Department of Pharmacology and Physiology, New Jersey Medical School, 185 South Orange Ave., University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103, USA. lockeda@umdnj.edu
Koreen
Irina V
IV
Harris
Andrew L
AL
D000818:Animals; D000072259:Connexin 26; D017630:Connexins / Q000737:chemistry* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D051379:Mice; D011499:Protein Processing, Post-Translational; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000097002:Gap Junction beta-1 Protein
D000072259:Connexin 26; D017630:Connexins; D000097002:Gap Junction beta-1 Protein; C000607042:GJB2 protein, human; C000607044:Gjb2 protein, rat