Nontransformed cells can normalize gap junctional communication with transformed cells
Section snippets
Materials and methods
Cell culture. Nontransformed and v-Src transformed cells from homozygous null connexin 43 knockout mice (KoA and KoASrc) were used as described [2]. For all analyses, cells were maintained in DMEM supplemented with 10% FBS at 37 °C in 100% humidity. Src transformed cells were identified in coculture by staining with DiI or DiD as described, while nontransformed cells were stained with cell tracker green (5-chloromethyl-fluorescein diacetate; Molecular Probes) as described [2].
Evaluation of gap
Results
We have previously shown that nontransformed and Src transformed cells from Cx43 knockout mice express low levels of Cx45 [2]. We have verified this by RT-PCR with primers specific for Cx26, Cx30, Cx32, Cx36, Cx37, Cx40, Cx43, and Cx45. As shown in Fig. 1, both cell types expressed mRNA encoding Cx36, Cx37, and Cx45. Cx26 was expressed in nontransformed cells, but was not detected in Src transformed cells. Cx30, Cx32, Cx40, and Cx43 were not detected in either cell type.
As presented in Table 1,
Discussion
The cells used in this study express Cx45 but not Cx43, Cx40, or Cx30 [2]. In addition to 28 pS unitary events arising from the activity of Cx45 in these cells, another unitary conductance of 38 pS was also observed. However, the frequency of occurrence (0.18 versus 0.70) for this channel type was significantly increased by transformation with the Src tyrosine kinase.
Our results indicate that the Src tyrosine kinase affects the activity of gap junction channels by either increasing the gating
Acknowledgments
This work was supported by grants from the United States National Institutes of Health CA88805 and EY014479 to G.S.G. and GM555263 to P.R.B., the Canadian Institutes of Health Research MOP-14358 to C.C.N., and the American Heart Association AHA0335236N to V.V.
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