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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).
GABA ρ subunits have four transmembrane domains (Fig. 1): a short extracellular loop connects TM II and III, whereas the long intracellular loop that joins TM III and IV is thought to mediate receptor localization, anchoring, and clustering at the neuronal membrane through its interaction with the cytoskeletal protein MAP1b (Hanley et al., 1999). In addition, each ρ subunit contains a very short extracellular C-terminus and an extremely long extracellular N-terminal domain that contains two cystine residues thought to form a disulfide bond. The extracellular domain constitutes the GABA binding site (marked in purple in Fig. 1), formed at the junction between two neighboring subunits. The TM II is located at the innermost side of the receptor, and a cluster of these domains, contributed by five subunits of the pentomeric structure, forms the Cl− permeable channel in the center of the receptor (Fig. 1, inset). Although this region exhibits a high degree of homology among all GABA ρ subunits, there is a strikingly consistent difference at the 2′ position, i.e., the presence of a proline residue in ρ1 receptors, and a serine residue at the same location in ρ2 receptors. Our earlier studies demonstrated that the amino acid at position 2′ of GABA ρ subunits influences response kinetics, receptor pharmacology, and ion selectivity and conductance (Qian et al., 1999).
The GABACR is expressed predominantly in the retina, although its distribution is also detected in other parts of the CNS. In retina, the GABACR is expressed mainly on bipolar cells of every subtype, with extensive distribution in the axon terminal regions and with minor expression in the dendritic region of the cell. Consequently, GABACR play an important role in controlling visual signaling from retinal bipolar cells, which link photoreceptors to the amacrine and ganglion cells in the retina.
The inhibitory action of the GABACR is mediated by the gated chloride channel. In retinal bipolar cells, there is a high concentration gradient for chloride ions, and opening of chloride channels will clamp the membrane potential of the cell close to the resting potential, increase the membrane conductance, and produce shunting inhibition. The unique physiological properties of the GABACR (high GABA sensitivity and slow kinetics of activation and deactivation) indicate that the inhibitory action mediated by this receptor will have a very specific function in the retina. Indeed, the slow response kinetics of GABACR-mediated inhibition allows the early phase of the signal to pass the bipolar cell terminal relatively unattenuated, but diminishes the late phase of the response. In other words, inhibition mediated by the GABACR at the bipolar cell terminal acts as a high pass filter to make the electrical signal more transient.
In addition to the inhibitory effect mediated by the gated chloride channel, the ρ subunits of the GABACR also exhibit metabotropic activity through the interaction of the large intracellular loop with cellular retinoic acid binding protein (CRABP). Since CRABP is a carrier protein that plays important roles in modulating retinoic acid - sensitive gene expression, interaction between the GABACR and CRABP provides the link that connects neuronal activity and gene expression in the retina.
Thus far no disease entities have been directly associated with mutations in the GABACR, and GABACR knock-out mice do not exhibit observable defects in visual function. Nevertheless, the receptor is clearly a potential target for various ocular disorders because of its key location in the visual pathway. For example, GABACR antagonists have been implicated in the prevention of form-deprivation-induced myopia (Stone et al., 2003). Although the molecular machinery is unclear, it is possible that genetic mechanisms involved in the development of myopia are governed in part by the metabotropic activity of the GABACR.
The inhibitory action mediated by the gated chloride channel of GABACR could also serve as a target to control neurotransmitter (glutamate) release from retinal bipolar cells, and lessen the activity of inner retinal neurons. Reducing the discharge of glutamate and the level of neuronal excitability may serve as a form of therapy for a number of eye diseases. For example, glaucoma, whose clinical hallmark is the loss of retinal ganglion cells, is thought to be caused in large part by glutamate-induced excitotoxicity. In addition, reducing neuronal excitation can diminish metabolic demand in the retina and relieve tissue hypoxia, one of the leading pathogenic factors in several common forms of retinopathy, e.g., diabetic retinopathy, retinopathy of prematurity (ROP), and retinal vasculature occlusions. Thus, reducing neural activity by activating GABACR in the retina may prove beneficial for preserving visual function under these pathological conditions.
In summary, a number of unique features that characterize GABACR make this type of receptor a particularly suitable target for regulating retinal glutamate level and neuronal activities; (i) The GABACR is expressed mainly on the axon terminals of bipolar cells in the mammalian retina, and the GABACR -mediated current constitutes a majority of the inhibitory response of these cells. Activation of the GABACR will efficiently reduce the excitatory output of the bipolar cells, i.e., glutamate release to the third order neurons in the retina. (ii) GABACR mediate sustained responses with little sign of desensitization. Therefore, activation by externally applied agonists will be able to provide constant inhibition to bipolar cell activity, which will, in turn, reduce excitability for third order neurons in the retina. (iii) The GABACR exhibits significantly higher agonist sensitivity, and consequently relatively low drug doses will be required to achieve the desired inhibition of inner retinal neurons. (iv) The restricted distribution of GABACR in the CNS minimizes the chances of side effects from local application of GABACR agonists.
The unique properties of the GABACR suggest that it plays a special role in retinal function. Although it has been demonstrated that GABACR affect synaptic input from amacrine cells to the bipolar cell terminals in the retina, delineating the exact neuronal circuitry will be an important step in revealing GABACR function in visual signal processing. In addition, the study of the metabotropic function of the GABACR in retinal neuron is still in its infancy, but could afford a better understanding of the long-term effects of any aberrations in this receptor. The investigation of GABACR function will also greatly benefit from the development of GABACR-specific agents, which may provide novel therapeutic approaches in treating various eye disorders.
We thank Dr. L. Adamian for providing a structural model of the N-terminal region of the GABA ρ subunit, and Ms. Lisa Birmingham for technical help in composing the figure. This work was supported by an NIH research grant EY12028 (HQ), core grant EY01792, an Alcon Research Institute Award (HR), an RPB Senior Scientific Investigator Award (HR) and an unrestricted departmental award from Research to Prevent Blindness, Inc.