DAPI hydrochloride br Discussion The GlyR is responsible for

Discussion The GlyR is responsible for mediating much of the neuronal inhibition in the brainstem and spinal cord, although these receptors are also found in a number of higher brain regions (Baer et al., 2009, Jonsson et al., 2012, Jonsson et al., 2009, Lynch, 2004). A variety of structurally-diverse allosteric modulators are known to affect GlyR function including divalent cations, alcohols, anesthetics and numerous drugs of abuse (Beckstead et al., 2000, Harvey et al., 1999, Kirson et al., 2013, Kirson et al., 2012, Mihic et al., 1997). Although glycine has long been thought to be the endogenous agonist acting at these receptors, taurine, the second most abundant amino DAPI hydrochloride in the brain, may also play a role in many brain regions (Albrecht and Schousboe, 2005, Mori et al., 2002). Allosteric modulation of glycine-activated receptors has been quite extensively studied, but not as much is known about modulation of GlyR activated by taurine. Ethanol, inhalants and zinc leftshift or rightshift glycine concentration–response curves, depending on whether they act as positive or negative allosteric modulators of the GlyR (Beckstead et al., 2000, Miller et al., 2005, Welsh et al., 2010). The greatest percent enhancing effects of these agents are thus seen when low concentrations of agonists are tested, and this is likely due to an enhancement of glycine affinity for its receptors. However, these allosteric modulators have minimal effects when applied with saturating concentrations of glycine (Fig. 4B). In contrast, Kirson et al. (2012) showed that ethanol, volatile anesthetics and inhaled drugs of abuse produce marked enhancement of currents elicited by maximally-effective concentrations of taurine. The same phenomenon was later also shown using 100nM zinc (Kirson et al., 2013). The effects of these modulators at saturating concentrations of taurine cannot be due to their enhancement of taurine affinity but must instead be due to their increasing probability of channel opening (Po) subsequent to binding. In contrast, since a saturating concentration of glycine produces a Po of approximately 0.95 (Lape et al., 2008), there is little opportunity for enhancement of Po by modulators at glycine-activated receptors. Even when very low concentrations of glycine (3μM) are used, the intra-burst Po is still about 0.7 (Welsh et al., 2009). We first compared how 100nM zinc affected GlyR responses to low concentrations of glycine vs. taurine by determining the concentrations of glycine and taurine that produced the same currents. These concentrations corresponded to markedly different agonist EC values on their respective concentration–response curves (Fig. 1). We expected that the degree of zinc potentiation would be lower for taurine-activated GlyR than receptors activated by glycine as the concentration of taurine used fell much higher on its concentration–response curve than glycine did on its. Surprisingly, our data showed the same degree of enhancement for both agonists (Fig. 2B). This also suggested that, even at lower effective concentrations of taurine, allosteric modulators might have greater effects on taurine-activated than on glycine-activated GlyR. A direct comparison of zinc enhancement of taurine- and glycine-activated receptors showed that considerably greater enhancement was seen in the former (Fig. 4). The most parsimonious explanation is that allosteric modulators have minimal effects on intra-burst Po at all concentrations of glycine, since polygenic inheritance are already high, instead acting primarily to decrease the rate of glycine unbinding and thus prolonging burst durations (Laube et al., 2000; Welsh et al., 2009). This is no longer a factor at a saturating glycine concentration, since a glycine molecule would bind almost immediately upon the unbinding of another, explaining the lack of ethanol and zinc effects at maximally-effective glycine concentrations. When a saturating concentration of taurine is applied the same reasoning holds true: modulators are still not able to exert any effects via enhancing taurine affinity. However, by affecting intra-burst properties, they are able to affect Po, which they clearly do as shown in Fig. 4A and C. At lower taurine concentrations the modulators may affect both taurine unbinding to enhance burst durations and also enhance intra-burst Po. This could explain why zinc produces greater enhancement of taurine-activated GlyR than glycine-activated GlyR at low agonist concentrations (Fig. 4C) and also why similar degrees of ethanol enhancement are seen with EC8 glycine and EC34 taurine (Fig. 5).