Abstract
Understanding the mechanisms that control synaptic efficacy through the availability of neurotransmitter receptors depends on uncovering their specific intracellular trafficking routes. gamma-Aminobutyric acid type B (GABA(B)) receptors (GABA(B)Rs) are obligatory heteromers present at dendritic excitatory and inhibitory postsynaptic sites. It is unknown whether synthesis and assembly of GABA(B)Rs occur in the somatic endoplasmic reticulum (ER) followed by vesicular transport to dendrites or whether somatic synthesis is followed by independent transport of the subunits for assembly and ER export throughout the somatodendritic compartment. To discriminate between these possibilities we studied the association of GABA(B)R subunits in dendrites of hippocampal neurons combining live fluorescence microscopy, biochemistry, quantitative colocalization, and bimolecular fluorescent complementation. We demonstrate that GABA(B)R subunits are segregated and differentially mobile in dendritic intracellular compartments and that a high proportion of non-associated intracellular subunits exist in the brain. Assembled heteromers are preferentially located at the plasma membrane, but blockade of ER exit results in their intracellular accumulation in the cell body and dendrites. We propose that GABA(B)R subunits assemble in the ER and are exported from the ER throughout the neuron prior to insertion at the plasma membrane. Our results are consistent with a bulk flow of segregated subunits through the ER and rule out a post-Golgi vesicular transport of preassembled GABA(B)Rs.
Highlights
We visualized the mobility of the subunits in live hippocampal neurons by fluorescence recovery after photobleaching (FRAP) using GABABR1 and GABABR2 fused to EGFP
Over a period of 30 min the majority of GABABR1-mRFP- and GABABR2EGFP-containing structures moved independently and in bidirectional fashion. These results indicate that GABABR1 and GABABR2 are transported in dendrites and suggest that they reside in segregated compartments, which move with different kinetics
The endoplasmic reticulum (ER) retention of GABABR1 and the necessary assembly with GABABR2 have been shown in a variety of cellular contexts [9]
Summary
Animals—Adult pregnant female Sprague-Dawley rats were purchased from the Central Animal Facility at Universidad Catolica de Chile and killed by asphyxia in a CO2 chamber according to the Guide for Care and Use of Laboratory Animals (National Academy of Sciences, 1996). GABABR1a-enhanced green fluorescent protein (EGFP) was generated by subcloning the SpeI/PstI fragment of rat GABABR1a into pEGFP-N1 (Clontech, Mountain View, CA) and adding the extreme C-terminal sequence amplified by PCR to provide an in-frame fusion at the C terminus GABABR1a-monomeric red fluorescent protein (mRFP) was generated by subcloning the SpeI/BamHI fragment of rat GABABR1a into a modified version of pEGFP-N1 containing mRFP and subsequently adding the BamHI fragment from GABABR1a-EGFP containing the extreme C-terminal sequence of GABABR1a to provide an in-frame fusion at the C terminus. GABABR2-EGFP was generated by PCR amplification of rat GABABR2 (primers: forward, 5Ј-GCGAATTCATGGCTTCCCCGCCGAGCTC-3Ј; reverse, 5Ј-GCGGTACCAGGCCCGAGACCATGACTC-3Ј) and subcloned into pEGFP-N1 (Clontech). Guinea pig GABABR1 (which recognize GABABR1a and GABABR1b), GABABR2, and microtubuleassociated protein 2 antibodies were purchased from Chemicon (Temecula, CA). We calibrated the x-y shift with a grid and corrected all images before the calculation of colocalization coefficients
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