Gephyrin regulates the cell surface dynamics of synaptic GABAA receptors.
The efficacy of fast synaptic inhibition is critically dependent on the accumulation of GABAA receptors at inhibitory synapses, a process that remains poorly understood. Here, we examined the dynamics of cell surface GABAA receptors using receptor subunits modified with N-terminal extracellular ecliptic pHluorin reporters. In hippocampal neurons, GABAA receptors incorporating pHluorin-tagged subunits were found to be clustered at synaptic sites and also expressed as diffuse extrasynaptic staining. By combining FRAP (fluorescence recovery after photobleaching) measurements with live imaging of FM4-64-labeled active presynaptic terminals, it was evident that clustered synaptic receptors exhibit significantly lower rates of mobility at the cell surface compared with their extrasynaptic counterparts. To examine the basis of this confinement, we used RNAi to inhibit the expression of gephyrin, a protein shown to regulate the accumulation of GABAA receptors at synaptic sites. However, whether gephyrin acts to control the actual formation of receptor clusters, their stability, or is simply a global regulator of receptor cell surface number remains unknown. Inhibiting gephyrin expression did not modify the total number of GABAA receptors expressed on the neuronal cell surface but significantly decreased the number of receptor clusters. Live imaging revealed that clusters that formed in the absence of gephyrin were significantly more mobile compared with those in control neurons. Together, our results demonstrate that synaptic GABAA receptors have lower levels of lateral mobility compared with their extrasynaptic counterparts, and suggest a specific role for gephyrin in reducing the diffusion of GABAA receptors, facilitating their accumulation at inhibitory synapses.
Jacob
Tija C
TC
Department of Pharmacology, University College London, London WC1E 6BT, United Kingdom.
Bogdanov
Yury D
YD
Magnus
Christopher
C
Saliba
Richard S
RS
Kittler
Josef T
JT
Haydon
Philip G
PG
Moss
Stephen J
SJ
D000818:Animals; D019921:Biotinylation / Q000379:methods; D015153:Blotting, Western / Q000379:methods; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D003001:Cloning, Molecular / Q000379:methods; D004305:Dose-Response Relationship, Drug; D004558:Electric Stimulation / Q000379:methods; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D009433:Neural Inhibition / Q000502:physiology*; D009474:Neurons / Q000378:metabolism; D017711:Nonlinear Dynamics; D018408:Patch-Clamp Techniques / Q000379:methods; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D038761:Photobleaching; D017729:Presynaptic Terminals / Q000378:metabolism; D021122:Protein Subunits / Q000378:metabolism; D011726:Pyridinium Compounds / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D034622:RNA Interference / Q000502:physiology; D051381:Rats; D018091:Receptors, AMPA / Q000378:metabolism; D011963:Receptors, GABA-A / Q000235:genetics / Q000378:metabolism*; D013569:Synapses / Q000502:physiology*; D058570:TOR Serine-Threonine Kinases; D014162:Transfection / Q000379:methods; D050495:Vesicular Inhibitory Amino Acid Transport Proteins / Q000378:metabolism
D002352:Carrier Proteins; D049452:Green Fluorescent Proteins; D008565:Membrane Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D021122:Protein Subunits; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D018091:Receptors, AMPA; D011963:Receptors, GABA-A; D058570:TOR Serine-Threonine Kinases; D050495:Vesicular Inhibitory Amino Acid Transport Proteins; C092350:FM 4-64; C073441:gephyrin; C546842:MTOR protein, human