Intracellular Domains of Mouse Connexin26 and -30 Affect Diffusional and Electrical Properties of Gap Junction Channels |
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Authors: | D Manthey K Banach T Desplantez CG Lee CA Kozak O Traub R Weingart K Willecke |
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Institution: | Institut für Genetik, Abt. Molekulargenetik, Universit?t Bonn, R?merstr. 164, 53117 Bonn, Germany, DE Dept. of Physiology, University of Bern, Bühlplatz 5, 3012 Bern, Switzerland, CH National Inst. of Allergy and Infectious Diseases, 9000 Rockville Pike, BLDG. 4, Room 329, Bethesda, MD 20892, USA, US
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Abstract: | To evaluate the influence of intracellular domains of connexin (Cx) on channel transfer properties, we analyzed mouse connexin
(Cx) Cx26 and Cx30, which show the most similar amino acid sequence identities within the family of gap junction proteins.
These connexin genes are tightly linked on mouse chromosome 14. Functional studies were performed on transfected HeLa cells
stably expressing both mouse connexins. When we examined homotypic intercellular transfer of microinjected neurobiotin and
Lucifer yellow, we found that gap junctions in Cx30-transfected cells, in contrast to Cx26 cells, were impermeable to Lucifer
yellow. Furthermore, we observed heterotypic transfer of neurobiotin between Cx30-transfectants and HeLa cells expressing
mouse Cx30.3, Cx40, Cx43 or Cx45, but not between Cx26 transfectants and HeLa cells of the latter group. The main differences
in amino acid sequence between Cx26 and Cx30 are located in the presumptive cytoplasmic loop and C-terminal region of these
integral membrane proteins. By exchanging one or both of these domains, using PCR-based mutagenesis, we constructed Cx26/30
chimeric cDNAs, which were also expressed in HeLa cells after transfection. Homotypic intercellular transfer of injected Lucifer
yellow was observed exclusively with those chimeric constructs that coded for both cytoplasmic domains of Cx26 in the Cx30
backbone polypeptide chain. In contrast, cells transfected with a construct that coded for the Cx26 backbone with the Cx30
cytoplasmic loop and C-terminal region did not show transfer of Lucifer yellow. Thus, Lucifer yellow transfer can be conferred
onto chimeric Cx30 channels by exchanging the cytoplasmic loop and the C-terminal region of these connexins. In turn, the
cytoplasmic loop and C-terminal domain of Cx30 prevent Lucifer yellow transfer when swapped with the corresponding domains
of Cx26. In chimeric Cx30/Cx26 channels where the cytoplasmic loop and C-terminal domains had been exchanged, the unitary
channel conductance was intermediate between those of the parental channels. Moreover, the voltage sensitivity was slightly
reduced. This suggests that these cytoplasmic domains interfere directly or indirectly with the diffusivity, the conductance
and voltage gating of the channels.
Received: 26 July 2000/Revised: 15 February 2001 |
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Keywords: | : Lucifer yellow — Neurobiotin — Electrophysiology — Chimeric connexins — Structure-function relationship |
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