Biomedical subjects

David R Serwanski

Publications and source records attributed to David R Serwanski.

5 recordsLinked to original sources

PubMed2006

Synaptic and nonsynaptic localization of GABAA receptors containing the alpha5 subunit in the rat brain.

The alpha5 subunit of the GABA(A) receptors (GABA(A)Rs) has a restricted expression in the brain. Maximum expression of this subunit occurs in the hippocampus, cerebral cortex, and olfactory bulb. Hippocampal pyramidal cells show high expression of alpha5 subunit-containing GABA(A)Rs (alpha5-GABA(A)Rs) both in culture and in the intact brain. A large pool of alpha5-GABA(A)Rs is extrasynaptic and it has been proposed to be involved in the tonic GABAergic inhibition of the hippocampus. Nevertheless, there are no studies on the localization of the alpha5-GABA(A)Rs at the electron microscope (EM) level. By using both immunofluorescence of cultured hippocampal pyramidal cells and EM postembedding immunogold of the intact hippocampus we show that, in addition to the extrasynaptic pool, there is a pool of alpha5-GABA(A)Rs that concentrates at the GABAergic synapses in dendrites of hippocampal pyramidal cells. The results suggest that the synaptic alpha5-GABA(A)Rs might play a role in the phasic GABAergic inhibition of pyramidal neurons in hippocampus and cerebral cortex.

Animals↗

PubMed2006

Identification and characterization of two novel splice forms of GRIP1 in the rat brain.

We cloned two novel alternatively-spliced mRNA isoforms of glutamate receptor interacting protein 1 (GRIP1) which we named GRIP1d and GRIP1e 4-7. GRIP1d is a 135 kDa, 7-PDZ-domain variant of GRIP1, containing the 12 amino acid C-terminus originally described for the 4-PDZ-domain GRIP1c 4-7. GRIP1e 4-7 is a 75 kDa 4-PDZ-domain variant of GRIP1, containing the 12 amino acid C-terminus originally described for the 7-PDZ-domain GRIP1a/b. Northern blots indicated that GRIP1d mRNA is 5.1 kb long and abundant in brain. An antibody to the C-terminus of the 75 kDa GRIP1c 4-7 also recognized an abundant 135 kDa protein, consistent with the predicted size of GRIP1d. Similarly, an antibody to the C-terminus of the 135 kDa GRIP1a/b also recognized a low abundance 75 kDa protein, consistent with the predicted size of GRIP1e 4-7. Immunocytochemistry of hippocampal cultures and intact brain using these antibodies showed that (i) these isoforms are present in both GABAergic and glutamatergic synapses, and (ii) the isoforms co-localize in individual synapses. While GRIP1a/b isoforms are abundant in interneurons and highly concentrated in GABAergic presynaptic terminals, the isoforms recognized by the antibody to the C-terminus common to GRIP1c 4-7 and GRIP1d are much less abundant in interneurons and preferentially concentrate at the postsynaptic complex.

Alternative Splicing↗

PubMed2005

GRIP1 in GABAergic synapses.

The glutamate receptor-interacting protein GRIP1 is present in glutamatergic synapses and interacts with the GluR2/3/4c subunits of the AMPA receptors. This interaction plays important roles in trafficking, synaptic targeting, and recycling of AMPA receptors as well as in the plasticity of glutamatergic synapses. Although GRIP1 has been shown to be present at GABAergic synapses in cultured neurons, the use of EM (electron microscopy) immunocytochemistry in the intact brain has failed to convincingly reveal the presence of GRIP1 in GABAergic synapses. Therefore, most studies on GRIP1 have focused on glutamatergic synapses. By using mild tissue fixation and embedding in EM, we show that in the intact brain the 7-PDZ domain GRIP1a/b is present not only in glutamatergic synapses but also in GABAergic synapses. In GABAergic synapses GRIP1a/b localizes both at the presynaptic terminals and postsynaptically, being frequently localized on the synaptic membranes or the synaptic junctional complex. Considerably higher density of GRIP1a/b is found in the presynaptic GABAergic terminals than in the glutamatergic terminals, while the density of GRIP1a/b in the postsynaptic complex is similar in both types of synapses. The results also show that the 7-PDZ and the shorter 4-PDZ domain splice forms of GRIP1 (GRIP1c 4-7) frequently colocalize with each other in individual GABAergic and glutamatergic synapses. The results suggest that GRIP1 splice forms might play important roles in brain GABAergic synapses.

Animals↗

PubMed2004

A four PDZ domain-containing splice variant form of GRIP1 is localized in GABAergic and glutamatergic synapses in the brain.

We have isolated, from a rat brain cDNA library, a clone corresponding to a 2779-bp cDNA encoding a novel splice form of the glutamate receptor interacting protein-1 (GRIP1). We call this 696-amino acid splice form GRIP1c 4-7 to differentiate it from longer splice forms of GRIP1a/b containing seven PDZ domains. The four PDZ domains of GRIP1c 4-7 are identical to PDZ domains 4-7 of GRIP1a/b. GRIP1c 4-7 also contains 35 amino acids at the N terminus and 12 amino acids at the C terminus that are different from GRIP1a/b. In transfected HEK293 cells, a majority of GRIP1c 4-7 was associated with the plasma membrane. GRIP1c 4-7 interacted with GluR2/3 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor. In low density hippocampal cultures, GRIP1c 4-7 clusters colocalized with GABAergic (where GABA is gamma-aminobutyric acid) and glutamatergic synapses, although a higher percentage of GRIP1c 4-7 clusters colocalized with gamma-aminobutyric acid, type A, receptor (GABA(A)R) clusters than with alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor clusters. Transfection of hippocampal neurons with hemagglutinin-tagged GRIP1c 4-7 showed that it could target to the postsynaptic complex of GABAergic synapses colocalizing with GABA(A)R clusters. GRIP1c 4-7-specific antibodies, which did not recognize previously described splice forms of GRIP1, recognized a 75-kDa protein that is enriched in a postsynaptic density fraction isolated from rat brain. EM immunocytochemistry experiments showed that in intact brain GRIP1c 4-7 concentrates at postsynaptic complexes of both type I glutamatergic and type II GABAergic synapses although it is also presynaptically localized. These results indicate that GRIP1c 4-7 plays a role not only in glutamatergic synapses but also in GABAergic synapses.

5' Untranslated Regions↗

PubMed2004

The brefeldin A-inhibited GDP/GTP exchange factor 2, a protein involved in vesicular trafficking, interacts with the beta subunits of the GABA receptors.

We have found that the brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2) interacts with the beta subunits of the gamma-aminobutyric acid type-A receptor (GABA(A)R). BIG2 is a Sec7 domain-containing guanine nucleotide exchange factor known to be involved in vesicular and protein trafficking. The interaction between the 110 amino acid C-terminal fragment of BIG2 and the large intracellular loop of the GABA(A)R beta subunits was revealed with a yeast two-hybrid assay. The native BIG2 and GABA(A)Rs interact in the brain since both coprecipitated from detergent extracts with either anti-GABA(A)R or anti-BIG2 antibodies. In transfected human embryonic kidney cell line 293 cells, BIG2 promotes the exit of GABA(A)Rs from endoplasmic reticulum. Double label immunofluorescence of cultured hippocampal neurons and electron microscopy immunocytochemistry of rat brain tissue show that BIG2 concentrates in the trans-Golgi network. BIG2 is also present in vesicle-like structures in the dendritic cytoplasm, sometimes colocalizing with GABA(A)Rs. BIG2 is present in both inhibitory GABAergic synapses that contain GABA(A)Rs and in asymmetric excitatory synapses. The results are consistent with the hypotheses that the interaction of BIG2 with the GABA(A)R beta subunits plays a role in the exocytosis and trafficking of assembled GABA(A)R to the cell surface.

Amino Acid Sequence↗