REVERSIBLE INTERCELLULAR COUPLING BY REGULATED EXPRESSION OF A GAP JUNCTION CHANNEL GENE

Direct intercellular coupling through gap junction channels has been i mplicated in diverse processes including cellular differentiation, gro wth control, metabolic cooperativity and electronic coupling and natur al and induced mutations in connexin genes have been described in huma n 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-controlle d transactivator and a tetracycline-responsive connexin32 target gene were introduced in the communication-deficient SKHep1 cell line. Quant itative immunoblotting and confocal immunofluorescence microscopy with connexin32-specific antibodies demonstrated that expression of connex in32 in stable transfectants was tightly regulated by tetracycline tre atment. Moreover, transfectants exhibited a highly coupled phenotype w hich was rapidly and reversibly converted to the communication deficie nt parental state after tetracycline treatment. Time constants for dec ay of the messenger RNA, protein and functional coupling were similar (similar to 4 hrs), implying that transcription was rate-limiting and that separate long-lived pools of connexin32 protein were absent. In c ontrast to other approaches in which the extent of coupling is pharmac ologically regulated by altering channel gating characteristics or by generalized blockade of transcription or translation, in this system i ntercellular communication is regulated by directly controlling connex in gene expression.