Rapid determination of gap junction formation using HeLa cells microinjected with cDNAs encoding wild-type and chimeric connexins

C. H. George*, P. E.M. Martin, W. H. Evans

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

36 Citations (Scopus)

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 languageEnglish
Pages (from-to)785-789
Number of pages5
JournalBiochemical and Biophysical Research Communications
Volume247
Issue number3
DOIs
Publication statusPublished - 29 Jun 1998
Externally publishedYes

Keywords

  • Connexin-aequorin
  • Dye coupling
  • Gap junctions
  • HeLa cells
  • Microinjection

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint

Dive into the research topics of 'Rapid determination of gap junction formation using HeLa cells microinjected with cDNAs encoding wild-type and chimeric connexins'. Together they form a unique fingerprint.

Cite this