Details
| Original language | English |
|---|---|
| Pages (from-to) | 35-43 |
| Number of pages | 9 |
| Journal | Biochimica et biophysica acta |
| Volume | 1720 |
| Issue number | 1-2 |
| Publication status | Published - 30 Dec 2005 |
Abstract
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.
Keywords
- Amino Acid Sequence, Animals, Connexin 26, Connexins/chemistry, Electric Conductivity, Escherichia coli/metabolism, Gene Expression, Humans, Ion Channels/physiology, Lipid Bilayers/chemistry, Molecular Sequence Data, Peptide Fragments/chemistry, Protein Structure, Quaternary, Rats, Recombinant Proteins/chemistry, Sequence Alignment, Xenopus
Cite this
- Standard
- Harvard
- Apa
- Vancouver
- BibTeX
- RIS
In: Biochimica et biophysica acta, Vol. 1720, No. 1-2, 30.12.2005, p. 35-43.
Research output: Contribution to journal › Article › Research › peer review
}
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 -