Constitutive GABAA receptor endocytosis is dynamin-mediated and dependent on a dileucine AP2 adaptin-binding motif within the beta 2 subunit of the receptor.
Receptor endocytosis is an important mechanism for regulating the synaptic efficacy of neurotransmitters. There is strong evidence that GABA(A) receptor endocytosis is clathrin-dependent; however, this process is not well understood. Here we demonstrate that in HEK 293 cells, endocytosis of GABA(A) receptors composed of either alpha1beta2gamma2Lor alpha1beta2 subunits is blocked by the dominant negative dynamin construct K44A. Furthermore, we identify a dileucine AP2 adaptin-binding motif within the receptor beta2 subunit that is critical for endocytosis. Internalization of GABAA receptors lacking this motif is dramatically inhibited, and the receptors appear to accumulate on the cell surface. Patch clamp analysis of receptors lacking the dileucine motif show that there is an increase in the peak amplitude of GABA-gated chloride currents compared with wild-type receptors. Additionally, GABA-gated chloride currents in HEK 293 cells expressing wild-type receptors are increased by introduction of a peptide corresponding to the dileucine motif region of the receptor beta2 subunit but not by a control peptide containing alanine substitutions for the dileucine motif. In mouse brain cerebral cortical neurons, the dileucine motif peptide increases GABA-gated chloride currents of native GABA(A) receptors. This is the first report to our knowledge that an AP2 adaptin dileucine recognition motif is critical for the endocytosis of ligand-gated ion channels belonging to this superfamily.
Herring
Dina
D
Department of Pharmacology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130, USA.
Huang
RenQi
R
Singh
Meharvan
M
Robinson
Lucy C
LC
Dillon
Glenn H
GH
Leidenheimer
Nancy J
NJ
D033962:Adaptor Protein Complex 2; D020816:Amino Acid Motifs; D000818:Animals; D001923:Brain Chemistry; D002460:Cell Line; D034281:Dynamins / Q000502:physiology*; D004705:Endocytosis; D005260:Female; D006801:Humans; D007930:Leucine; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D009474:Neurons / Q000166:cytology* / Q000502:physiology; D018408:Patch-Clamp Techniques; D011247:Pregnancy; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011963:Receptors, GABA-A / Q000737:chemistry* / Q000502:physiology*
D033962:Adaptor Protein Complex 2; D021122:Protein Subunits; D011963:Receptors, GABA-A; D034281:Dynamins; D007930:Leucine