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Picrotoxin is a pore blocker that can differentiate ligand-gated inhibitory chloride channels. Even within one receptor type, such as the glycine receptor, picrotoxin block differs between subunits. The effect of subunit gating properties on block of the inhibitory glycine receptor (GlyR) was explored using heteromeric α subunit expression in voltage-clamped HEK293 cells. The α2 GlyR is more sensitive to picrotin block than the α1 GlyR, and this difference was used to explore whether mutations that interfered with gating of the α2 subunit would also interfere with picrotin block. Two mutations were used: one that decreased the glycine sensitivity of α2 by almost two log units and the other that was unresponsive to glycine. In both cases, the sensitivity to picrotin was essentially unaltered. The results indicated that α2 subunits can determine the picrotin sensitivity of α1α2-heteromeric receptors and that direct gating of the α2 subunit is not required for this picrotin inhibition.

Glycine is the lone fast neurotransmitter for which a metabotropic pathway has not been identified. In retina, we found a strychnine-insensitive glycine response in bipolar and ganglion cells. This glycine response reduced high voltage-activated calcium current. It was G-protein mediated and protein kinase A dependent. The EC(50) of the metabotropic glycine response is 3 mum, an order of magnitude lower than the ionotropic glycine receptor in the same retina. The bipolar cell glutamatergic input to ganglion cells was suppressed by metabotropic glycine action. The synaptic output of about two-thirds of bipolar cells and calcium current in two-thirds of ganglion cells are sensitive to the action of glycine at metabotropic receptors, suggesting this signal regulates specific synaptic pathways in proximal retina. This study resolves the curious absence of a metabotropic glycine pathway in the nervous system and reveals that the major fast inhibitory neurotransmitters, GABA and glycine, both activate G-protein-coupled pathways as well.

Phasic and tonic light responses provide a fundamental division of visual information that is thought to originate in the inner retina. However, evidence presented here indicates that this duality originates in the outer retina. In response to a steady light stimulus, the temporal responses of On-bipolar cells fell into two groups. In one group, the light response peaked and then rapidly declined (tau approximately 400 msec) close to the resting membrane potential. At light offset, these cells exhibited a transient afterhyperpolarization. In the second group of On-bipolar cells, the light response declined 10-fold more slowly and reached a steady depolarization that was approximately 40% of the peak response. These neurons had a slowly decaying afterhyperpolarization at light offset. A metabotropic glutamate antagonist, (RS)-alpha-cyclopropyl-4-phosphonophenylyglycine (CPPG), blocked light responses in both types of On-bipolar cell. CPPG only slightly depolarized transient On-bipolar cells, whereas sustained On-bipolar cells were significantly depolarized. Inorganic calcium channel blockers disclosed that these distinct On-bipolar responses were inherent to the bipolar cell and not attributable to synaptic feedback. CPPG had distinct effects on sustained and transient ganglion cells, similar to its action on bipolar cells. The antagonist depolarized and blocked the light responses of sustained ganglion cells. In transient ganglion cells, CPPG suppressed the On light response but did not depolarize the cell or block the Off light response. These results suggest that transient and sustained light responses in ganglion cells result from selective bipolar cell input and that these two fundamental visual channels originate at the dendritic terminals of bipolar cells.

1. Protein kinase modulation of glycine-activated currents was examined in acutely dissociated ganglion cells from tiger salamander retina using whole-cell voltage-clamp techniques. 2. Glycine (100 microM) induced an outward chloride current in cells clamped at 0 mV. Co-application of 50 microM forskolin made the glycine-induced current more transient. The combination of forskolin and glycine reduced the later portion of current response without changing the initial peak amplitude. 3. 3-Isobutyl-1-methylxanthine (IBMX) or 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) produced effects similar to those of forskolin. H-89, a protein kinase A (PKA) inhibitor, blocked the effect of forskolin. 4. A protein kinase C (PKC) activator, OAG (1-oleoyl-2-acetyl-sn-glycerol), also made the glycine response more transient. Unlike PKA analogues, OAG enhanced the glycine peak response without changing the glycine late response. OAG effects were blocked by 1 microM GF-109203X, a PKC inhibitor. 5. Nanomolar concentrations of strychnine selectively blocked the fast phase of the glycine current and reversed the effect of OAG, but not that of forskolin. Conversely, forskolin occluded the effect of 5,7-dichlorokynurenic acid, which selectively suppresses the late phase of the glycine current. The action of OAG was not blocked by 5,7-dichlorokynurenic acid. 6. Thus, through a differential modulation, both protein kinase A and C shorten the decay time of the glycine current. PKA suppresses the slow component, while PKC potentiates the fast component.

Physiological and pharmacological properties of possible subtypes of the native glycine receptor were investigated in retinal neurons using whole-cell voltage-clamp techniques. Two discrete inhibitory glycine responses were identified in ganglion cells. The responses could be distinguished pharmacologically: one was sensitive to strychnine and the other to 5,7-dichlorokynurenic acid. The two responses had different kinetics: the former had a fast onset and fast desensitization, whereas the latter had a slower onset and was much more sustained. The physiological and pharmacological distinctions suggest that the responses are mediated by different receptors. These receptors transduce glycinergic synaptic signals to ganglion cells, where they serve as low- and high-pass filters, respectively, of EPSPs.

Whole cell voltage and current clamp recordings were obtained from third order neurons isolated from the salamander retina. Using cross desensitization, the structure-function relationship of short chain amino acids on the glycine receptor were examined. L-Serine, L-alanine, beta-alanine and taurine all cross desensitized with glycine, but did not show significant cross desensitization with GABA. This indicates that these amino acids act at the glycine receptor. The order of potency was glycine >> beta-alanine > taurine >> L-alanine > L-serine. TAG, a reputed selective taurine antagonist, was equally effective in blocking taurine and glycine currents. There is no evidence for distinct receptors for taurine. Amino acids with larger moieties at the alpha carbon, such as threonine and valine, produced inactive ligands. Placing a methyl group on the amine of glycine or esterification of the carboxyl group also greatly reduced activity. Based on these modifications of the glycine molecule, it appears that selectivity at the glycine receptor results in part from steric restrictions at all three sites in the glycine chain. The steric interference is most critical at the carboxyl and amino ends, and less limiting at the alpha carbon. Doses of L-serine that had only slight effects in voltage clamp experiments, nevertheless produced large effects in current clamp experiments. This indicates that several endogenous amino acids can have significant effects on membrane voltage, even when their shunting activity may be small. High concentrations of agonists produced desensitization in the voltage clamp records, but there was little evidence of desensitization in the current clamp experiments. These results indicate that several endogenous amino acids can activate the glycine receptor, but there is no evidence for a discrete receptor for taurine, beta-alanine, L-alanine or L-serine. Since all these endogenous amino acids have similar amino and acid terminals, reduction in potency results from steric interference around the alpha carbon. This graded potency may have functional significance in mediating inhibition.

1. The effects of inhibitory amino acid transmitters on horizontal cells in the superfused amphibian retina were studied by the use of conventional intracellular recording techniques. 2. Gamma-aminobutyric acid (GABA) caused a calcium-independent depolarization of horizontal cells in mud puppy and tiger salamander. This action was mimicked by muscimol but not baclofen (BAC) and blocked by bicuculline and picrotoxin (PTX), matching the GABAa receptor profile. 3. The purported GABA uptake inhibitors nipecotate (NPA) and guvacine (GUV) acted as GABAa agonists, having pharmacological properties very similar to GABA itself. These agents also activated receptors of amacrine and ganglion cells, causing membrane polarizations similar to GABA. Concentrations of these analogues that did not activate the GABAa receptor (submillimolar) did not lower the effective dose of GABA, even after prolonged application. 4. Glycine (GLY) also depolarized horizontal cells, but only in approximately 25% of the horizontal cells was the amplitude of the depolarization as great as GABA. The glycine response was blocked by both strychnine (STR, 10 microM) and PTX (100 microM). In contrast, the action of GABA was unaffected by STR. 5. Ion substitution and channel-blocking agents indicated that the effects of applied GABA and GLY were independent of both external sodium and calcium. 6. The results suggest that GABA receptors on horizontal cells may act 1) as a positive feedback system to modulate the light response and 2) as a mechanism for chemical coupling between horizontal cells.