Length of C-terminus of rCx46 influences oligomerization and hemichannel properties.
Wild type connexin 46 of rat (wtrCx46), and human connexin 26 (wthCx26) and derivates from rCx46 elongated at the C-terminus by 25 amino acids (rCx46Ct) as well as C-terminal truncated constructs (rCx28.1, rCx45.3) were expressed in frog oocytes of Xenopus laevis. Single oocyte voltage-clamp analysis revealed that connexons or hemichannels of rCx46Ct exhibit similar conducting properties as those of wtrCx46. Insertion of a stop codon at C-terminal domains at position 243 and 409 resulted in a significant reduction in the corresponding hemichannel conductance. This result was also found for wthCx26, the shortest human connexin. Tagged connexin constructs rCx46Ct and hCx26Ct could be expressed in E. coli as monomers. The monomers of rCx46Ct and hCx26Ct were purified and electro-eluted from corresponding SDS gels. Studies of in vitro oligomerization showed that hexamers of these connexins were formed in presence of kinase and specific lipids. Purified rCx46Ct formed some oligomers in vitro if a lipid mixture of POPE/POPG and casein kinase I (CKI) was added, but in the presence of POPC, phosphorylated rCx46Ct monomers preferentially formed hexamers. Purified hCx26Ct formed hexamers in the presence of POPE/POPG. In addition, N-terminal truncated rCx46 (Cx35) oligomerized after phosphorylation. Reconstitution of purified recombinant connexin rCx46Ct in planar lipid bilayers mediated Ca(2+)-sensitive single channel activity. It is discussed whether the specific C-terminal end of the expressed connexins are responsible for hexamer formation as well as channel opening.
Zeilinger
Carsten
C
Universität Hannover, Institut für Biophysik, Herrenhäuserstr. 2, D-30419 Hannover, Germany. carsten@biop.uni-hannover.de
Steffens
Melanie
M
Kolb
Hans-Albert
HA
D000595:Amino Acid Sequence; D000818:Animals; D000072259:Connexin 26; D017630:Connexins / Q000737:chemistry* / Q000235:genetics; D004553:Electric Conductivity; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007473:Ion Channels / Q000502:physiology; D008051:Lipid Bilayers / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D020836:Protein Structure, Quaternary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D014981:Xenopus
D017630:Connexins; C000607045:GJB2 protein, Xenopus; C487370:Gja3 protein, rat; C000607044:Gjb2 protein, rat; D007473:Ion Channels; D008051:Lipid Bilayers; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000072259:Connexin 26