LacSwitch II regulation of connexin43 cDNA expression enables gap-junction single-channel analysis.
Metabolic and electrical coupling through gap junction channels is implicated in cell differentiation, tissue homeostasis, and electrotonic propagation of signals in excitable tissues. The characterization of gating properties of these channels requires electrophysiological recordings at both single- and multiple-channel levels. Hence, a system that is able to control connexin expression by external means would provide a useful tool. To regulate the expression of connexins in cells, plasmids encoding a transactivator and/or a lac-operon IPTG response-dependent Cx43 target gene were transfected into communication-deficient N2a neuroblastoma cells. Immunoblotting, dye coupling, and electrophysiological methods revealed that expression of Cx43 in selected clones could be tightly regulated. After 15-20 h of acute induction with IPTG, cell-to-cell communication reached its peak with junctional conductances of 15-30 nS. Chronic induction at specific doses of IPTG produced constant, controlled levels of Cx43 expression, which were reflected by predictable junctional coupling levels. These conditions allowed prolonged recordings from either lowly or highly coupled cells, making lac operon an ideal regulatory system for channel gating studies at a single-channel level.
Zhong
G
G
Indiana University School of Medicine, Indianapolis, IN, USA.
Mantel
P L
PL
Jiang
X
X
Jarry-Guichard
T
T
Gros
D
D
Labarrere
C
C
Moreno
A P
AP
D000818:Animals; D045744:Cell Line, Tumor; D003001:Cloning, Molecular / Q000379:methods; D018031:Connexin 43 / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D004553:Electric Conductivity; D017629:Gap Junctions / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics*; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology*; D007763:Lac Operon / Q000235:genetics*; D009447:Neuroblastoma / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D051381:Rats
D018031:Connexin 43; D007544:Isopropyl Thiogalactoside