Declusterization of GABAA Receptors Affects the Kinetic Properties of GABAergic Currents in Cultured Hippocampal Neurons

Enrica Maria Petrini,Paola Zacchi,Andrea Barberis,Jerzy W Mozrzymas,Enrico Cherubini

doi:10.1074/jbc.m213081200

Abstract

Speed and reliability of synaptic transmission are essential for information coding in neuronal networks and require the presence of clustered neurotransmitter receptors at the plasma membrane in precise apposition to presynaptic terminals. Receptor clusterization is the result of highly regulated processes involving functional and structural proteins. Among the structural elements, microtubules are known to play a crucial role in anchoring of gamma-aminobutyric acid, type A (GABA(A)) receptors. Here we show that microtubule depolymerization with nocodazole induces the declusterization of GABA(A) receptors and modifies the kinetic properties of GABAergic currents in cultured hippocampal neurons. In particular, this drug, applied either in the bath or via the patch pipette, induced the acceleration of the onset kinetics of miniature inhibitory postsynaptic currents (mIPSCs) without significantly affecting their frequency, thus suggesting a main postsynaptic site of action. After nocodazole treatment, current responses to ultrafast applications of GABA exhibited a faster rise time and an accelerated onset of desensitization. A quantitative analysis of GABA-evoked currents and model simulations suggest that declusterization affects the gating properties of GABA(A) receptors. In particular, a faster entry into the desensitized state of declustered GABA(A) receptors may account for the changes in the kinetic properties of mIPSCs after nocodazole treatment. Hence it appears that the clustered condition of GABA(A) receptors contributes in shaping GABAergic currents.

Highlights

Efficient GABAergic1 synaptic transmission requires the presence of clustered postsynaptic GABAA receptors localized in precise apposition to presynaptic releasing sites
Nocodazole Treatment Induces the Depolymerization of Microtubules and the Declusterization of GABAA Receptors— GABAA receptor clusterization is known to be dependent on the presence of the intact cytoskeleton
Model Simulations—The present findings demonstrate that declusterization of GABAA receptors following microtubule disruption produces similar effects on both miniature inhibitory postsynaptic currents (mIPSCs) and GABAevoked currents, namely it accelerates the current onset without significantly affecting the current decay or the peak amplitude

Summary

Introduction

Efficient GABAergic synaptic transmission requires the presence of clustered postsynaptic GABAA receptors localized in precise apposition to presynaptic releasing sites. To form and maintain postsynaptic clusters, neurons must possess the ability to appropriately sort, target, cluster, recycle, and degrade GABAA receptors (1). Does it show tubulinbinding activity (5) and interaction with the ␥2 subunit of GABAA receptors (4), but it binds N-ethylmalemide sensitive factor, a protein that plays an essential role in intracellular membrane trafficking (6). One of the major candidate molecules for synaptic GABAA receptor clustering is gephyrin (11–14), a tubulin-binding protein that has been shown to co-localize with GABAA receptors at postsynaptic sites (15). Additional proteins must be involved in the clustering process (3), because biochemical approaches failed to show a direct interaction between gephyrin and GABAA receptors (16). Despite the efforts to clarify the cellular and molecular mechanisms regulating cytoskeleton-receptor interactions, this topic is still a matter of debate

Results

Discussion

Conclusion