Clathrin-independent endocytosis of GABAA receptors in HEK 293 cells

The endocytosis of GABAA receptors was investigated in HEK 293 cells expressing receptor alpha1 beta2- and alpha1 beta2 gamma2-subunit combinations. For assessment of internalized receptors by radioimmunoassay or immunofluorescence, a triple c-myc epitope was introduced into the amino terminus of the beta2 subunit. An assay based on biotin inaccessibility was used for alpha1 subunits. GABA(A) alpha1 beta2- and alpha1 beta2 gamma2-subunit receptors were internalized with a t(1/2) of 5.5 min at 37 degreesC. With both subunit combinations, phorbol 12-myristate 3-acetate enhanced internalization by nearly 100%. Treatment of the cells with hypertonic sucrose prevented both the basal and phorbol ester-induced endocytosis of GABAA receptors. GF 109203X, an inhibitor of protein kinase C, blocked the stimulation by phorbol ester but had no detectable effect on basal receptor endocytosis. Coexpression with a dominant-negative mutant of dynamin (K44A) led to a 100% enhancement of GABAA receptor internalization, while the endocytosis of beta (2)-adrenergic receptors was completely prevented. The results indicate that the endocytosis of GABA(A) alpha1 beta2-subunit receptors in HEK cells is constitutive, positively modulated by activation of protein kinase C, and occurs by a mechanism that requires neither the participation of a GABA(A) receptor gamma2 subunit nor a clathrin-mediated pathway.