Ρ2 Subunits Research Articles

Endocytosis of GABA(C) receptors depends on subunit composition and is regulated by protein kinase C-ζ and protein phosphatase 1.

Neuronal excitability depends on the surface concentration of neurotransmitter receptors. Type C gamma-aminobutyric acid receptors (GABA(C)R) are composed of ρ subunits that are highly expressed in the retina. Molecular mechanisms that guide the surface concentration of this receptor type are largely unknown. Previously, we reported physical interactions of GABA(C)R ρ subunits with protein kinase C-ζ (PKCζ) via adapter proteins of the ZIP protein family, as well as of protein phosphatase 1 (PP1) via PNUTS. Here, we demonstrate that co-expressing ρ1 with ZIP3 and PKCζ enhanced basal internalization of GABA(C)R, while receptor internalization was reduced in the presence of PNUTS and PP1. Co-expression of ρ1 with individual binding partners showed no alterations, except for PP1. Heterooligomeric GABA(C)R composed of ρ1 and ρ2 subunits had a significant higher endocytosis rate than ρ1 containing homooligomeric receptors. Mutant constructs lacking binding sites for protein interactions ensured the specificity of our data. Finally, substitution of serine and threonine residues with alanines indicated that GABA(C)R internalization depends on serine/threonine kinases and phosphatases, but not on tyrosine phosphorylation. We conclude that GABA(C)R internalization is reciprocally regulated by PKCζ and PP1 that are anchored to the receptor via ZIP3 or PNUTS respectively.

Role of Neurotransmitter Receptors in Mediating Light-Evoked Responses in Retinal Interplexiform Cells

Interplexiform (IP) cells are a long-neglected group of retinal neurons the function of which is yet to be determined. Anatomical study indicates that IP cells are located in the inner nuclear layer, juxtaposed with the third-order neurons. However, the synaptic transmission of IP cells in the inner retina is poorly understood. Using whole cell patch-clamp and pharmacological techniques, we extensively studied synaptic receptors in IP cells. The IP cells in amphibian retinal slices were identified by electrical and morphological properties with voltage-clamp recording and Lucifer yellow dialysis. We find that light-evoked excitatory postsynaptic currents (L-EPSCs) are mediated by AMPA and N-methyl-d-aspartate receptors in IP cells. Although both receptors contributed to the amplitude and kinetics of L-EPSCs, AMPA receptor desensitization substantially shaped L-EPSCs in the neurons, similar to those found in the third-order neurons. The light-evoked inhibitory postsynaptic currents (L-IPSCs) in IP cells were primarily mediated by strychnine-sensitive glycine receptors with a small component of GABA(C) receptors. GABA(C) receptor rho2 subunits were detected in IP cells with single-cell RT-PCR assays. Expression of GABA(C) receptors is one of the special features for IP cells, distinct from most of the third-order neurons that depend on GABA(A) and glycine receptors to relay the inhibitory signals. However, GABA(A) receptors in IP cells acted like nonsynaptic receptors that were activated by exogenous GABA application. Furthermore, L-IPSCs in IP cells were inhibited by the serial inhibitions between amacrine cells in the inner retina. In addition, application of neurotransmitters on the axon terminals of IP cells had no significant current generated in the cells, indicating that the synaptic inputs of IP cells are mainly from the inner retina. This study demonstrates the important role that light signals are encoded by both experiment of inhibitory receptors in IP cells.

Focus on Molecules: The GABAC Receptor

γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the retina and CNS, exerts its effects by activating three pharmacologically distinct types of GABA receptor (GABAR), each of which is expressed on retinal neurons. The GABACR is a member of the Cys-loop superfamily of ligand-gated ion channels that include the nicotinic acetylcholine, glycine and 5-HT3 receptors. The pentomeric structure of native GABAC receptors is composed of GABA ρ subunits, and three of which (ρ1 – ρ3) have been cloned from mammalian retinal cDNA libraries (NCBI accession number for human GABA ρ1, ρ2, and ρ3 subunit: NM_002042, NM_002043, and NM_001105580, respectively). In humans, the ρ1 and ρ2 subunits are located on chromosome 6q15; the ρ3 subunit is on chromosome 3q11. Each of the subunits is capable of forming a GABA-sensitive homooligomeric receptor when expressed in Xenopus oocytes, and co-assembly with the γ subunit of the GABAAR results in the formation of a receptor with functional properties that closely mimic the behavior of GABACR on bipolar cells (Qian and Ripps, 1999).

Expression of GABAρ subunits during rat cerebellum development

In the present study, we provide evidence for the expression of all three GABA C receptor ρ subunits through development of the rat cerebellum. Injection of cerebellum mRNA into frog oocytes gave rise to the expression of both GABA A and GABA C receptors. qRT-PCR of RNA isolated from postnatal developing cerebella showed that the expression of each ρ subunit is relatively low, with a relative comparative expression of ρ3 > ρ1 > ρ2. In situ hybridization and immunohistochemistry revealed a limited distribution of GABA C receptors in the Purkinje and Golgi neurons whereas electron microscopy detected the ρ1 and ρ2 subunits in the soma and dendritic tree of the Purkinje cells. The expression of GABA C receptors in the cerebellum adds a new dimension to the regulation of GABAergic neurotransmission and suggests further experiments to determine their functional consequences.

A single amino acid in the second transmembrane domain of GABA ρ receptors regulates channel conductance

GABA C receptors, expressed predominately in vertebrate retina, are thought to be formed mainly by GABA ρ subunits, each of which exhibits distinct physiological and pharmacological properties. In this study, the receptors formed by perch GABA ρ subunits were expressed in HEK cells, and their single channel conductances were determined using noise analysis techniques. The receptors formed by the perch ρ1A subunit gate a channel with a conductance of 0.2 pS, whereas the receptors formed by GABA ρ2 subunits exhibit much higher channel conductances, i.e., 3.2 and 3.5 pS for perch ρ2A and ρ2B receptors, respectively. A comparison of the amino acid sequences of the channel-forming TMII regions of the various subunits suggested that a single amino acid at position 2′ was a potential site for the large differential in conductance. We found that switching the serine residue at that site in the GABA ρ2 subunit to the proline residue present in the ρ1 subunit reduced the channel conductance to a level similar to that of the wild type ρ1 receptor. Conversely, mutating proline to serine in the amino acid sequence of the ρ1 receptor significantly increased its unitary conductance. These results indicate that a single amino acid in the TMII region plays an important role in determining the single channel conductance of the GABA C receptors.

Cloning and functional expression of the bovine GABA C ρ2 subunit : Molecular evidence of a widespread distribution in the CNS

GABA C receptors were first described as a non-desensitizing, bicuculline- and baclofen-insensitive component in Xenopus oocytes expressing bovine retina mRNA. However, the expression, tissue distribution and functional properties of GABA C receptors from other areas of the CNS still remain controversial. In previous experiments, the injection of rat cerebellum mRNA into Xenopus oocytes induced the expression of receptors that generated currents with both GABA A and GABA C characteristics; the latter component apparently being given by the ρ2 subunit, suggesting the expression of GABA C receptors in the CNS and the formation of homooligomeric receptors. In this study, using RT-PCR, we found that the ρ1 and ρ2 subunits are widely expressed in the CNS including areas where they have not been previously described such as the bulb, pons and the caudate nucleus. To determine if the GABA C component of the GABA-currents elicited by oocytes expressing cerebellum mRNA was caused by activation of homomeric GABA ρ2 receptors, we cloned the corresponding cDNA and expressed it in Xenopus oocytes. It was found that oocytes injected with ρ2 cDNA, efficiently formed GABA-gated homooligomeric receptors. The GABA-dose–current response gave an EC50 = 1.19 μM and the currents were resistant to bicuculline and reversibly antagonized by the specific GABA C receptor antagonist TPMPA. Altogether, our results indicate a widespread distribution of both ρ1 and ρ2 subunits in the bovine CNS and show further that the ρ2 subunit cDNA isolated from cerebellum, forms fully functional receptors when expressed in Xenopus oocytes.

Expression and dendritic mRNA localization of GABAC receptor rho1 and rho2 subunits in developing rat brain and spinal cord.

The cellular distribution of GABAC receptor rho1 and rho2 subunits in the rat central nervous system remains controversial. We investigated how these subunits were distributed in cerebellum, hippocampus and spinal cord at postnatal day 1, 7 or in adult life. We found that in the adult cerebellum rho1 and rho2 mRNAs were expressed in Purkinje cells and basket-like cells only. In the hippocampus both subunits were expressed throughout the CA1 pyramidal layer, dentate gyrus and scattered interneurons with maximum staining intensity at P7. In the adult hippocampus in situ staining was predominantly found on interneurons. GABAC antibody labelling in P7 and adult hippocampus was largely overlapping with the in situ staining. Western blot analysis showed GABAC receptor in retina, ovary and testis. In the spinal cord the rho2 signal was consistently stronger than rho1 with overlapping expression patterns. At P1, the most intensely labelled cells were the motoneurons while on P7 and adult sections, interneurons and motoneurons were likewise labelled. On spinal neurons both rho1 and rho2 mRNAs showed somatodendritic localization, extending out for >100 microm with punctate appearance especially in adult cells. A similar spinal distribution pattern was provided with polyclonal antibody labelling, suggesting close correspondence between mRNA and protein compartmentalization. Electrophysiological experiments indicated that P1 spinal motoneurons did possess functional GABAC receptors even though GABAC receptors played little role in evoked synaptic transmission. Our results suggest a pattern of rho1 and rho2 subunit distribution more widespread than hitherto suspected with strong developmental regulation of subunit occurrence.

Elimination of the ρ1 Subunit Abolishes GABACReceptor Expression and Alters Visual Processing in the Mouse Retina

Inhibition is crucial for normal function in the nervous system. In the CNS, inhibition is mediated primarily by the amino acid GABA via activation of two ionotropic GABA receptors, GABAAand GABAC. GABAAreceptor composition and function have been well characterized, whereas much less is known about native GABACreceptors. Differences in molecular composition, anatomical distributions, and physiological properties strongly suggest that GABAAreceptors and GABACreceptors have distinct functional roles in the CNS. To determine the functional role of GABACreceptors, we eliminated their expression in mice using a knock-out strategy. Although native rodent GABACreceptors are composed of ρ1 and ρ2 subunits, we show that after ρ1 subunit expression was selectively eliminated there was no GABACreceptor expression. We assessed GABACreceptor function in the retina because GABACreceptors are highly expressed on the axon terminals of rod bipolar cells and because this site modulates the visual signal to amacrine and ganglion cells. In GABACρ1 null mice, GABA-evoked responses, normally mediated by GABACreceptors, were eliminated, and signaling from rod bipolar cells to third order cells was altered. These data demonstrate that elimination of the GABACρ1 subunit, via gene targeting, results in the absence of GABACreceptors in the retina and selective alterations in normal visual processing.

Developmentally regulated expression of GABA receptor ρ1 and ρ2 subunits, L7 and cone–rod homeobox (CRX) genes in mouse retina

In this study, we compared the temporal expression pattern of four retinal genes; ρ1 and ρ2 that encode subunits of GABA c receptors, L7 that encodes Purkinje cell protein and CRX that encodes the cone–rod homeobox transcription factor. A reverse-transcription-polymerase chain reaction (RT-PCR) strategy that generated a linear correlation between the amount of retinal RNA and the amount of amplified product was used to quantify transcripts from each gene. Results with this method showed that the ρ1 and L7 have similar developmental patterns. Both exhibit basal level expression before P7. From P7 to P20, the RNA levels for both genes were increased about 12-fold. After P20, the RNA levels remained unchanged. Compared to ρ1 and L7, expression of ρ2 began later, since the ρ2 RNA could not be detected until P10. At P10, the ρ2 RNA level was about 10% of its level at P35. Expression of ρ2 reached its peak at a later developmental stage compared to that of ρ1 and L7. The different temporal patterns were confirmed by co-amplification of ρ1, ρ2, and L7 in a single PCR tube. CRX RNA was detected at embryonic day 15 (E15) and increased progressively, in agreement with a prior study using in situ hybridization. These data, combined with evidence that the tissue distribution of ρ1 and L7 RNA in the CNS are similar, indicates that ρ1 and L7 may share common transcriptional regulatory elements. Furthermore, the difference in the timing of ρ subunit expression suggests that the subunit composition of GABA c receptors vary during retinal development.

Expression of GABA(C) receptor rho1 and rho2 subunits during development of the mouse retina.

Retinal gamma-aminobutyric acid type C (GABA(C)) receptors consist of rho subunits. Here we report our results from a competitive PCR and patch-clamp electrophysiology study quantifying rho subunit message and characterizing GABA(C) receptor-mediated currents at different stages of mouse retinal development. Mouse rho1 message is first detected at postnatal day 6 (P6), increases significantly until P9 and remains at this level through adulthood, whereas mouse rho2 message does not appear until P9, peaks at P15 and remains at this level through adulthood. There is an approximate twofold excess of rho1 compared to rho2 message at most stages of development, which persists in adulthood. Functional GABA(C) receptors are detected in acutely dissociated bipolar cells of P9 or older mouse retina. Early in development (P9-10), GABA(C) receptors are composed solely of rho1 subunits, but subsequently contain rho1 and/or rho2 subunits (by P11 and later). These findings are intriguing because the onset and rapid increase in rho subunit transcription and functional expression match the initiation and active period of bipolar cell differentiation in retinal development as well as the stage of eye opening and initial visual experience in the rodent. The investigation of mouse rho subunits here forms a basis for future studies on the role of GABA(C) receptors in retinal development.

Expression of GABA Receptor ρ1 and ρ2 Subunits in the Retina and Brain of the Rat

We have investigated the distribution of GABA receptor rho 1 and rho 2 subunits in the rat central nervous system. Cloning of rat rho 1 and rho 2 cDNA fragments revealed similarities to the corresponding human sequences of 99% (rho 1) and 88% (rho 2) at the protein level. Whereas the human rho 2 subunit has no consensus sequence for phosphorylation by protein kinase C, the cytoplasmic loop of the rat sequence contains two such sites. Use of the polymerase chain reaction with reverse-transcribed total RNA (RT-PCR) from different brain tissues revealed that transcript for the rho 1 subunit was present in the retina only. The rho 2 mRNA was detected in all brain regions, with the highest level of expression in the retina. In situ hybridization of retinal sections revealed that rho 1 and rho 2 transcripts are present in the inner nuclear layer. RT-PCR and in situ hybridization of isolated retinal cells showed that both rho subunits are present in rod bipolar cells. Since these cells express bicuculline-insensitive GABA receptors, our results further support the idea that rho subunits are part of the GABAc receptor.

Localization of the ρ1- and ρ2-subunit messenger RNAs in chick retina by in situ hybridization predicts the existence of γ-aminobutyric acid type C receptor subtypes

We have amplified partial complementary DNAs for the chicken γ-aminobutyric acid type C (GABAC) receptor ρ1 and ρ2 subunits using the polymerase chain reaction. These nucleotide sequences have been utilized to design specific oligonucleotide probes for the in situ hybridization localization of the corresponding messenger RNAs (mRNAs) in the 1-day-old chick retina. Although both transcripts are found almost exclusively in the inner nuclear layer, their distributions differ markedly. From the locations of the hybridization signals, we deduce that the ρ1-subunit mRNA is present mainly in bipolar cells and that the ρ2-subunit mRNA is present in both amacrine and horizontal cells. These results suggest that the ρ1 and ρ2 subunits frequently occur in different receptor complexes and, therefore, that subtypes of the GABAC receptor exist.