Clathrin-independent endocytosis of GABA(A) receptors in HEK 293 cells

The endocytosis of GABAA receptors was investigated in HEK 293 cells expres sing receptor alpha1 beta2- and alpha1 beta2 gamma2-subunit combinations. F or assessment of internalized receptors by radioimmunoassay or immunofluore scence, a triple c-myc epitope was introduced into the amino terminus of th e beta2 subunit. An assay based on biotin inaccessibility was used for alph a1 subunits. GABA(A) alpha1 beta2- and alpha1 beta2 gamma2-subunit receptor s were internalized with a t(1/2) of 5.5 min at 37 degreesC. With both subu nit combinations, phorbol 12-myristate 3-acetate enhanced internalization b y nearly 100%. Treatment of the cells with hypertonic sucrose prevented bot h the basal and phorbol ester-induced endocytosis of GABAA receptors. GF 10 9203X, an inhibitor of protein kinase C, blocked the stimulation by phorbol ester but had no detectable effect on basal receptor endocytosis. Coexpres sion with a dominant-negative mutant of dynamin (K44A) led to a 100% enhanc ement of GABAA receptor internalization, while the endocytosis of beta (2)- adrenergic receptors was completely prevented. The results indicate that th e endocytosis of GABA(A) alpha1 beta2-subunit receptors in HEK cells is con stitutive, positively modulated by activation of protein kinase C, and occu rs by a mechanism that requires neither the participation of a GABA(A) rece ptor gamma2 subunit nor a clathrin-mediated pathway.