title: Specific amino-acid residues in the N-terminus and TM3 implicated in channel function and oligomerization compatibility of connexin43.
abstract: To identify signals that convey connexin oligomerization compatibility, we have aligned amino-acid sequences of alpha and beta group connexins (Cx) and compared the physico-chemical properties of each homologous amino-acid residue. Four positions were identified that consistently differed between alpha and beta-type connexins; two are located in the N-terminal domain (P1 and P2, corresponding to residues 12 and 13 of the Cx43 sequence), and two in the third trans-membrane-spanning domain TM3 (P3 and P4, corresponding to residues 152 and 153 of the Cx43 sequence). Replacement of each of these residues in Cx43 (an alpha-type connexin) with the corresponding residues of Cx32 (a beta-type connexin) resulted in the assembly of all variants into gap junctions; however, only the P4 variant was functional, as indicated by lucifer yellow dye transfer assays. The other three variants exerted a moderate to severe dose-dependent, dominant-negative effect on co-expressed wild-type (wt) Cx43 channel activity. Moreover, a significant dose-dependent, trans-dominant inhibition of channel activity was observed when either one of the N-terminal variants was co-expressed with wt Cx32. Assembly analyses indicated that dominant and trans-dominant inhibitory effects appeared to be based on the oligomerization of wt and variant connexins into mixed connexons. Interestingly, the identified N-terminal amino acids coincide with the position of naturally occurring, disease-causing missense mutations of several beta-connexin genes (Cx26, Cx30, Cx31, Cx32). Our results demonstrate that three of the identified discriminative amino-acid residues (positions 12, 13 and 152) are crucial for Cx43 channel function and suggest that the N-terminal amino-acid residues at position 12/13 are involved in the oligomerization compatibility of alpha and beta connexins.
authors.affiliation: Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Valerie
authors.initials: V
authors.lastname: Lagree
authors.forename: Karin
authors.initials: K
authors.lastname: Brunschwig
authors.forename: Patricia
authors.initials: P
authors.lastname: Lopez
authors.forename: Norton B
authors.initials: NB
authors.lastname: Gilula
authors.forename: Gabriele
authors.initials: G
authors.lastname: Richard
authors.forename: Matthias M
authors.initials: MM
authors.lastname: Falk
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002450:Cell Communication; D002478:Cells, Cultured; D018031:Connexin 43 / Q000378:metabolism*; D017630:Connexins / Q000378:metabolism; D006224:Cricetinae; D005455:Fluorescent Antibody Technique; D017629:Gap Junctions / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D020033:Protein Isoforms / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D016415:Sequence Alignment
chemical_list: D018031:Connexin 43; D017630:Connexins; D020033:Protein Isoforms