Gabab receptor mechanisms

Depending on cell type and the location of the receptor on neurons, GABAB receptors act via G-proteins to affect the activity of either Ca2+ channels, K+ channels or adenylate cyclase (Bowery and Enna 2000). For example, in dorsal root ganglion (DRG) neurons baclofen was found to inhibit the Ca2+-dependent phase of the DRG action potential, an effect attributed to block of voltage-activated Ca2+ currents. A similar action on presynaptic Ca2+ channels was presumed to underlie the block of neurotransmitter release by baclofen. This has now been demonstrated by recording Ca2+ currents from presynaptic terminals directly (Takahashi, Kajikawa and Tsujimoto 1998). In the CNS such presynaptic GABAB receptors are found on terminals releasing a variety of transmitters, as well as on GABAergic terminals themselves, where they may act as autoreceptors.

In several neuronal types baclofen was also shown to cause a postsynaptic hyperpolarisation due to the opening of K+ channels (EK is more negative than Vm). These postsynaptic GABAB receptors can be activated by synaptically released GABA and give rise to a delayed and long-lasting (hundreds of milliseconds) hyperpolarisation which, following as it does the GABAA receptor-mediated fast IPSP, is often referred to as the 'late IPSP' (Dutar and Nicoll 1988). In general, both types of GABAB response can be blocked by the same antagonists or by treatment with Pertussis toxin (which blocks the activation of G-proteins of the Gi/o class). However, at some sites presynaptic effects of baclofen are not blocked by all agents, indicating that multiple types of GABAB receptor may exist (see below).

STRUCTURE OF GABAB RECEPTORS

GABAB receptors long resisted attempts at expression cloning of the type used to identify GABAA receptor subunits, partly because the requirement for G-protein coupling to ion channels made functional assays in cell lines or oocytes difficult to devise. Recently, however, a GABAB receptor (GABABR1) was successfully isolated from a rat cDNA library by screening transfected cells with a high-affinity radiolabelled GABAB receptor antagonists, binding of which does not require the presence of G-proteins (Kaupmann et al. 1997). Two isoforms of the receptor were identified which differed only in the length of their amino-terminals — these were termed GABABR1a (960 amino acids) and GABABR1b (844 amino acids). Additional isoforms, both termed GABABRIc, have also been identified in rat (Pfaff et al. 1999) and human (Ng et al. 2001). Each protein has a predicted structure consisting of a large N-terminal and seven transmembrane domains, similar to metabotropic glutamate receptors.

The cloned receptors, when expressed in cell lines and studied by radioligand binding assays, showed some of the expected pharmacology of GABAB receptors. However, the affinity of agonists was much lower than seen with native receptors and not all expected coupling to effector systems could be demonstrated (possibly because of inappropriate or inefficient linkage to G-proteins). Subsequently, a second receptor protein, GABABR2, was identified (Jones et al. 1998; Kaupmann et al. 1998; White et al. 1998), and shown to interact with GABABR1, through the coiled regions of their intracellular C-termini, to form fully functional heterodimers (Fig. 11.8). GABABR1

GABAbR1 GABAbR2

GABAbR1 GABAbR2

Figure 11.8 Pharmacology and structure of GABAb receptors, (a) Various GABAb agonists and antagonists described in the text, (b) A GABAb receptor shown as a dimer containing one copy of GABAbR1 and one copy of GABAbR2, joined by their coiled intracellular carboxy-terminals. The large extracellular amino-terminals are the proposed sites of GABA binding. A G-protein is shown linked to each of the GABAbR proteins. The activated fS/y subunits trigger the opening of a K+ channel while the a subunit is shown inhibiting the activity of adenylate cyclase (AC). At presynaptc sites the ply subunits would inhibit a Ca2+ channel (after Bowery and Enna 2000) w

Figure 11.8 Pharmacology and structure of GABAb receptors, (a) Various GABAb agonists and antagonists described in the text, (b) A GABAb receptor shown as a dimer containing one copy of GABAbR1 and one copy of GABAbR2, joined by their coiled intracellular carboxy-terminals. The large extracellular amino-terminals are the proposed sites of GABA binding. A G-protein is shown linked to each of the GABAbR proteins. The activated fS/y subunits trigger the opening of a K+ channel while the a subunit is shown inhibiting the activity of adenylate cyclase (AC). At presynaptc sites the ply subunits would inhibit a Ca2+ channel (after Bowery and Enna 2000) w and GABABR2 are found in areas of the brain known to contain GABAB receptors, including hippocampus, cerebellum and cortex, but some differences in their distribution suggest that in certain cases homomeric GABABRs may be functional or that dimerisation may occur with other unidentified GABABRs (Bowery and Enna 2000). The existence of structurally and pharmacologically distinct pre- and postsynaptic GABAB receptors is supported by the recent demonstration that gabapentin, an anticonvulsant GABA analog, is a selective agonist for postsynaptic GABABR1a/R2 heterodimers coupled to K+ channels (Ng et al. 2001).

GABAC RECEPTORS

Early studies of the action of GABA and its analogues on spinal neurons revealed that the depressant action of one of these, ci's-4-aminocrotonic acid (CACA), was not blocked by bicuculline. Several analogues of GABA shared the same properties and did not interact with the then newly described GABAB receptors. In 1984, the term GABAC was introduced to distinguish this third class of GABA receptor (Johnston 1996). Like GABAA receptors, GABAC receptors activate anion channels permeable to Cl" (and HCO3) and the responses are similarly governed by the distribution of Cl" across the neuronal membrane.

GABAC RECEPTOR PHARMACOLOGY

GABAC receptors are defined by their insensitivity to bicuculline and their activation by conformationally restricted analogues of GABA such as CACA and (+)-CAMP (1S,2^-2-(aminomethyl)cyclopropanecarboxylic acid). They are blocked by picrotoxin but can be selectively antagonised by TPMPA (1,2,5,6-tetrahydropyridin4-ylphosphinic acid). Unlike GABAA receptors, they are not affected by benzodiazepines, barbiturates or anaesthetics (Barnard et al. 1998; Bormann 2000; Chebib and Johnston 2000).

STRUCTURE OF GABAC RECEPTORS

The best evidence for the existence of functional GABAC receptors and the clearest indication as to their molecular identity comes from work on the retina. Expression of retinal mRNA in Xenopus oocytes produces GABA-gated chloride channels with conventional GABAA receptor pharmacology as well as channels with characteristics of GABAC receptors (i.e. blocked by picrotoxin but not bicuculline). The basis of this distinction was made clear with the cloning from a retinal cDNA library of a new GABA receptor subunit termed p (Cutting et al. 1991). To date, three p subunits have been identified in mammals (p1-p3). Originally classed as GABAA subunits, with which they have ~ 35% sequence identity, they are now accepted as a distinct group of subunits, forming the basis of the relatively simple, and evolutionarily older, GABAC receptors. Unlike subunits of the GABAA receptor, p subunits form fully functional homomeric receptors and do not co-assemble with a or ¿6 subunits. These homomeric receptors are similar to native GABAC receptors, in that they are activated by GABA and CACA, blocked by picrotoxin and TPMPA but not bicuculline, and unaffected by barbiturates, benzodiazepines or anaesthetics (Fig. 11.9). Receptors formed from p subunits have a higher affinity for GABA than many GABAA receptors formed from afty combinations, have a lower single-channel conductance and produce currents that decay more slowly after removal of GABA.

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Figure 11.9 GABAC receptor pharmacology and structure, (a) Various GABAC agonists and antagonists described in the text, Picrotoxinin is the active component of picrotoxin and also acts at GABAa receptors. (b) Presumed subunit structures of GABAC receptors. The receptors can form as homomeric assemblies of p subunits but native receptors may be heteromeric assemblies of p subunits (e.g. pi and p2) or may contain both p and y subunits

Figure 11.9 GABAC receptor pharmacology and structure, (a) Various GABAC agonists and antagonists described in the text, Picrotoxinin is the active component of picrotoxin and also acts at GABAa receptors. (b) Presumed subunit structures of GABAC receptors. The receptors can form as homomeric assemblies of p subunits but native receptors may be heteromeric assemblies of p subunits (e.g. pi and p2) or may contain both p and y subunits

In the retina, electrophysiological data indicate that GABAC receptors are present on horizontal and bipolar cells, and p subunits have been localised to subsets of synapses formed by amacrine cells onto the axon terminals of rod bipolar cells. Activation of these presynaptic receptors inhibits glutamate release from the bipolar cells. However, the true molecular composition of native GABAC receptors is still under investigation. While homomeric receptors formed from p subunits share many features of retinal GABAC receptors, a number of discrepancies have been noted in the details of ion permeability, single-channel conductance and channel open time (Wotring, Chang and Weiss 1999). Thus, it has been suggested that native GABAC receptors may be composed of heteromeric assemblies of p subunits or, in certain cases, that such assemblies may also contain a y2 subunit (Qian and Ripps 1999). All three p subunits have been identified in brain, but their precise location and the functional significance of this expression is unclear. In particular, the basis of GABAC receptor-like responses seen, for example, in the spinal cord, cerebellum, optic tectum and hippocampus is yet to be determined.

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