TY - JOUR
T1 - pH regulation of connexin43
T2 - Molecular analysis of the gating particle
AU - Ek-Vitorín, José F.
AU - Calero, Guillermo
AU - Morley, Gregory E.
AU - Coombs, Wanda
AU - Taffet, Steven M.
AU - Delmar, Mario
N1 - Funding Information:
The authors wish to thank Dr. Jose Jalife for his advice and support throughout the project. We also thank Ms. Kathleen Stergiopoulos for her critical reading of the manuscript. The technical assistance of Christine McElwain, Christine Kapuscinski, and Anne Caron, and the secretarial skills of Mrs. LaVeme Gilbert are greatly appreciated. This work was supported by grants POI-HL39707 and RO1-HL52812 from the National Institutes of Health, Heart, Lung and Blood Institute, and by a NASPE Fellowship to J.F.E-V. Mario Delmar is an Established Investigator of the American Heart Association.
PY - 1996/9
Y1 - 1996/9
N2 - Gap junction channels allow for the passage of ions and small molecules between neighboring cells. These channels are formed by multimers of an integral membrane protein named connexin. In the heart and other tissues, the most abundant connexin is a 43-kDa, 382-amino acid protein termed connexin43 (Cx43). A characteristic property of connexin channels is that they close upon acidification of the intracellular space. Previous studies have shown that truncation of the carboxyl terminal of Cx43 impairs pH sensitivity. In the present study, we have used a combination of optical, electrophysiological, and molecular biological techniques and the oocyte expression system to further localize the regions of the carboxyl terminal that are involved in pH regulation of Cx43 channels. Our results show that regions 261-300 and 374-382 are essential components of a pH-dependent 'gating particle,' which is responsible for acidification-induced uncoupling of Cx43-expressing cells. Regions 261-300 and 374-382 seem to be interdependent. The function of region 261-300 may be related to the presence of a poly-proline repeat between amine acids 274 and 285. Furthermore, site- directed mutagenesis studies show that the function of region 374-382 is not directly related to its net balance of charges, although mutation of only one amine acid (aspartate 379) for asparagine impairs pH sensitivity to the same extent as truncation of the carboxyl terminal domain (from amine acid 257). The mutation in which serine 364 is substituted for proline, which has been associated with some cases of cardiac congenital malformations in humans, also disrupts the pH gating of Cx43, although deletion of amine acids 364- 373 has no effect on acidification-induced uncoupling. These results provide new insight into the molecular mechanisms responsible for acidification- induced uncoupling of gap junction channels in the heart and in other Cx43- expressing structures.
AB - Gap junction channels allow for the passage of ions and small molecules between neighboring cells. These channels are formed by multimers of an integral membrane protein named connexin. In the heart and other tissues, the most abundant connexin is a 43-kDa, 382-amino acid protein termed connexin43 (Cx43). A characteristic property of connexin channels is that they close upon acidification of the intracellular space. Previous studies have shown that truncation of the carboxyl terminal of Cx43 impairs pH sensitivity. In the present study, we have used a combination of optical, electrophysiological, and molecular biological techniques and the oocyte expression system to further localize the regions of the carboxyl terminal that are involved in pH regulation of Cx43 channels. Our results show that regions 261-300 and 374-382 are essential components of a pH-dependent 'gating particle,' which is responsible for acidification-induced uncoupling of Cx43-expressing cells. Regions 261-300 and 374-382 seem to be interdependent. The function of region 261-300 may be related to the presence of a poly-proline repeat between amine acids 274 and 285. Furthermore, site- directed mutagenesis studies show that the function of region 374-382 is not directly related to its net balance of charges, although mutation of only one amine acid (aspartate 379) for asparagine impairs pH sensitivity to the same extent as truncation of the carboxyl terminal domain (from amine acid 257). The mutation in which serine 364 is substituted for proline, which has been associated with some cases of cardiac congenital malformations in humans, also disrupts the pH gating of Cx43, although deletion of amine acids 364- 373 has no effect on acidification-induced uncoupling. These results provide new insight into the molecular mechanisms responsible for acidification- induced uncoupling of gap junction channels in the heart and in other Cx43- expressing structures.
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U2 - 10.1016/S0006-3495(96)79328-1
DO - 10.1016/S0006-3495(96)79328-1
M3 - Article
C2 - 8874002
AN - SCOPUS:0029781699
SN - 0006-3495
VL - 71
SP - 1273
EP - 1284
JO - Biophysical Journal
JF - Biophysical Journal
IS - 3
ER -