Studies of glycine-activated ionotropic receptors in retina

Ionotropic glycine receptors (GlyRs) serve important inhibitory roles throughout the central nervous system, including the retina. They are ligand-gated chloride ion channels that belong to the same cysteine-loop ligand-gated ion channel superfamily as ionotropic GABA receptors. There are four types of ionotropic glycine receptors based on the alpha subunit composition. Unlike its close relative GABAA/C receptors, there are no selective pharmacological agents for different subtypes of glycine receptors. In the first half of my study, I focused on the search of possible antagonists that show selectivity for glycine receptor subtypes.;I found that caffeine is a structural analog of strychnine and a competitive antagonist at ionotropic glycine receptors. Docking simulations indicate that caffeine and strychnine may bind to similar sites at the GlyR. The R131A GlyR mutation, which reduces strychnine antagonism without suppressing activation by glycine, also reduces caffeine antagonism. GlyR subtypes show different caffeine sensitivity. Tested against the EC50 of each GlyR subtype, the order of caffeine potency (IC50) is: alpha2beta (248 +/- 32 muM) ≈ alpha3beta (255 +/- 16muM) > alpha4beta (517 +/- 50 muM) > alpha1beta (837 +/- 132 muM). However, considering that the alpha3beta GlyR is the least sensitive to glycine than the other GlyR subtypes, this receptor is most effectively blocked by caffeine. The glycine dose-response curves and the effects of caffeine in retina suggest that amphibian retinal ganglion cells are dominated by the alpha1beta and possibly alpha4beta GlyRs, but are unlikely to utilize the alpha3beta or alpha4beta GlyRs. Comparing the effects of caffeine on glycinergic spontaneous and light-evoked post-synaptic currents indicates that evoked release may be a result of either elevation of glycine concentration at the synapse or recruitment of more synapses. The caffeine IC50 values for bath applied 100 muM glycine and for the synaptic glycinergic-currents are both 1.7 mM, thus the synaptic glycine concentration may be estimated to be around 100 muM in salamander retinal ganglion cells. As high millimolar concentrations of caffeine can completely inhibit glycinergic synaptic transmission, the use of caffeine to stimulate ryanodine receptors should be under discretion.;NMDA receptors (NMDAR), along with AMPA/Kainate receptors, are excitatory ionotropic glutamate receptors possessing integral cation channels. NMDARs are abundantly expressed in the second synaptic layer of the retina; however, previous experiments suggest that the excitatory light responses of retinal ganglion cells are driven almost exclusively by AMPA/Kainate receptors. NMDA receptor activation in retina seems to rely on glutamate spillover to perisynaptic sites where NMDARs are located. In the second part of my study, I found evidence for synaptic NMDA receptor activation in retinal ganglion cells, by changing some of the commonly employed experimental protocols. If the retina was exposed to an adapting light, then there is a progressive enhancement of the NMDA synaptic current. If glycine inhibition was blocked then a large NMDA receptor current can contribute to the ganglion cell light response. Similarly, when magnesium was removed from the extracellular solution then the light response became dominated by the NMDAR receptor current. These experiments indicate the synaptic existence of NMDAR in retina, and that the activation of NMDA receptors is state-dependent.