| Abstract: | Direct intercellular coupling through gap junction channels has been implicated in diverse processes including cellular differentiation, growth control, metabolic cooperativity and electronic coupling and natural and induced mutations in connexin genes have been described in human and experimental disease states. Genetic systems in which the extent of coupling could be reversibly regulated would provide an important approach for examining these potential functional roles, both in vitro and in vivo. Here we describe the generation and characterization of cell lines in which the extent of coupling is reversibly controlled at the transcriptional level. Plasmids encoding a tetracycline-controlled trans-activator and a tetracycline-responsive connexin32 target gene were introduced in the communication-deficient SKHepl cell line. Quantitative immunoblotting and confocal immunofluorescence microscopy with connexin32-specific antibodies demonstrated that expression of connexin32 in stable transfectants was tightly regulated by tetracycline treatment. Moreover, transfectants exhibited a highly coupled phenotype which was rapidly and reversibly converted to the communication deficient parental state after tetracycline treatment. Time constants for decay of the messenger RNA, protein and functional coupling were similar (4 hrs), implying that transcription was rate-limiting and that separate long-lived pools of connexin32 protein were absent. In contrast to other approaches in which the extent of coupling is pharmacologically regulated by altering channel gating characteristics or by generalized blockade of transcription or translation, in this system intercellular communication is regulated by directly controlling connexin gene expression. |
