Abstract
A procedure for rapidly determining the functionality of gap junctions constructed of recombinant connexins in communication-deficient HeLa cells is described. Nuclear microinjection of cDNA encoding wild-type connexins (Cx) 26, 32, 43, and a range of connexin-aequorin (Cx-Aeq) chimerase resulted in generation of gap junction intercellular communication channels. Expression of recombinant protein was detected in > 95% of cells 18-72 h following nuclear microinjection, and the functionality of the channels generated was determined according to their ability to transfer the fluorescent dye tracers Lucifer yellow and propidium iodide. The dye transfer results obtained correlated closely with other published studies using stably transfected cells and yet are obtained as rapidly as 18 h following microinjection of cDNA. Expression of a truncated form of Cx43 (CX43(Δ244)) by this new method indicated diminished intercellular transfer of both dyes and supports a channel gating mechanism that postulates interaction between the carboxyl tail and the intracellular loop.
Original language | English |
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Pages (from-to) | 785-789 |
Number of pages | 5 |
Journal | Biochemical and Biophysical Research Communications |
Volume | 247 |
Issue number | 3 |
DOIs | |
Publication status | Published - 29 Jun 1998 |
Externally published | Yes |
Keywords
- Connexin-aequorin
- Dye coupling
- Gap junctions
- HeLa cells
- Microinjection
ASJC Scopus subject areas
- Biophysics
- Biochemistry
- Molecular Biology
- Cell Biology