Preferential accumulation of GABA_A receptor γ2L, not γ2S, cytoplasmic loops at rat spinal cord inhibitory synapses

Abstract

Alternative splicing generates two variants of the GABA_AR γ2-subunit, γ2S and γ2L, which differ by insertion of the amino acid sequence LLRMFSFK into the large cytoplasmic loop between transmembrane domains 3 and 4. This additional sequence within the GABA_AR γ2L-subunit contains the potential protein kinase C (PKC) phosphorylation site serine 343 (Ser343). In the present study we intended to determine the capacity of these two splice variants to accumulate at inhibitory synaptic terminals and to colocalize with gephyrin, and to find out whether phosphorylation of Ser343 has any effect on GABA_AR distribution. Green fluorescent protein (GFP)-tagged large cytoplasmic loops of GABA_AR γ2S and γ2L (GFP::γ2S/L) were used as surrogates for full-length receptors to study the function of the individual γ2S and γ2L peptides in transfected spinal cord neurones (SCNs) and COS-7 cells. It was found that GFP::γ2L displayed a significantly higher capacity to accumulate at inhibitory synapses than GFP::γ2S. GABA_AR GFP::γ2S accumulation at inhibitory postsynaptic sites was suppressed to the extent that GFP::γ2S assumed a diffuse cytosolic distribution. PKC activation facilitated the postsynaptic clustering of GFP::γ2L but not of GFP::γ2S. This required the Ser343 residue, since substituting Ala343 for Ser343 produced a diffuse cytosolic localization pattern, like that of GFP::γ2S. Furthermore, upon PKC activation Discosoma Red2-tagged GABA_AR γ2L (DsRed 2::γ2L) colocalized with gephyrin in transfected COS-7 cells. These results support the idea that alternative splicing regulates the access of GABA_ARs to inhibitory postsynaptic sites in a Ser343 phosphorylation-regulated way.