TY - JOUR

T1 - Length of C-terminus of rCx46 influences oligomerization and hemichannel properties

AU - Zeilinger, Carsten

AU - Steffens, Melanie

AU - Kolb, Hans-Albert

N1 - Funding information: The authors are grateful to Dr. D.L. Paul for the supply of wtrCx46 DNA and I. Forster for improvements. Dr. C. Zeilinger was supported by the Helmholtz-Gemeinschaft, Institut für Biologische Strukturforschung, VIBS (VH-VI-013) and M. Steffens by the EU project “REFLEX”.

PY - 2005/12/30

Y1 - 2005/12/30

N2 - 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.

AB - 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.

KW - Amino Acid Sequence

KW - Animals

KW - Connexin 26

KW - Connexins/chemistry

KW - Electric Conductivity

KW - Escherichia coli/metabolism

KW - Gene Expression

KW - Humans

KW - Ion Channels/physiology

KW - Lipid Bilayers/chemistry

KW - Molecular Sequence Data

KW - Peptide Fragments/chemistry

KW - Protein Structure, Quaternary

KW - Rats

KW - Recombinant Proteins/chemistry

KW - Sequence Alignment

KW - Xenopus

U2 - 10.1016/j.bbamem.2005.10.015

DO - 10.1016/j.bbamem.2005.10.015

M3 - Article

C2 - 16388779

VL - 1720

SP - 35

EP - 43

JO - Biochimica et biophysica acta

JF - Biochimica et biophysica acta

SN - 0006-3002

IS - 1-2

ER -