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doi: 10.1242/10.1242/jcs.00604

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Specific amino-acid residues in the N-terminus and TM3 implicated in channel function and oligomerization compatibility of connexin43

Valérie Lagrée^1,*, Karin Brunschwig^1,, Patricia Lopez¹, Norton B. Gilula^1,, Gabriele Richard² and Matthias M. Falk^1,¶

¹ Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
² Department of Dermatology and Cutaneous Biology, and Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, PA, USA

^¶ Author for correspondence (e-mail: mfalk{at}scripps.edu)

Accepted 9 April 2003

To identify signals that convey connexin oligomerization compatibility, we have aligned amino-acid sequences of and ß group connexins (Cx) and compared the physico-chemical properties of each homologous amino-acid residue. Four positions were identified that consistently differed between and ß-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 -type connexin) with the corresponding residues of Cx32 (a ß-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 ß-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 and ß connexins.

Key words: Gap junction diseases, Gap junctions, Green fluorescent protein, Membrane channels, Oligomeric proteins, Connexin subunit assembly

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