We noticed that the spontaneous openings of the D R

We noticed that the spontaneous openings of the D97R α1 GlyR appeared to be quite similar to those elicited by a maximally-effective glycine concentration on wildtype GlyR; both generated similar open and shut dwell-time histograms, both exhibited openings grouped into clusters with Popen values greater than 0.9 and in both, clusters of channel-opening events appeared to conclude with either desensitization or deactivation (Todorovic et al., 2010). We reasoned that since the breakage of the D97-R119 electrostatic bond would be consistent with clusters of spontaneous activity with a very high Popen, this breakage could represent an important initial step in determining an agonist’s efficacy. D97 mutant GlyR can be found in three major classes of states: those that are spontaneously open, those that are found in a shut but activatable state and those in a desensitized state, all of which are illustrated in the tracing shown in Fig. 6. Spontaneous channel opening results in whole-cell holding currents typically between 300 and 500nA in magnitude, increasing to 3000–5000nA in the presence of saturating concentrations of glycine or taurine. Since spontaneously-opening individual D97 mutant netilmicin mg exhibit intra-burst Popen values of ∼0.95 (Figs. 2A & 7A) it is inconceivable that the currents observed after applying agonists are due to an increase in the channel Popen of these already activated channels and must instead be due to the activation of channels that were previously in the shut state, as exemplified by the large vertical deflection in current seen in Fig. 6 when glycine is applied. This suggests that the breakage of the putative D97-R119 electrostatic bond would not be the sole determinant of channel opening, but the loss of this intersubunit interaction could decrease the energy barrier that must be surmounted in order for channels to open. Individual D97 mutant GlyR may thus open spontaneously or remain in a shut state until either glycine or taurine bind. Interestingly, even the conservative mutation to E97, differing from D97 by only a methyl group, led to channels which exhibited some spontaneous activity as well as an increased relative efficacy of taurine. This suggests the possibility that charge is not the sole factor determining the nature of an interaction between the 97 and 119 residues, and that molecular volumes or shapes of the two amino acids may also play a role. The binding regions for glycine and taurine overlap in the α1 homomeric receptor (Schmieden et al., 1992) and, in particular, the I111 residue is involved in taurine activation. For example, mutations of amino acids (104, 108, and 112) near I111 enhanced the efficacy of β-amino acids (Schmieden et al., 1999). Taurine and β-ABA displayed significantly increased efficacies in these three mutants while efficacy for β-AIBA was enhanced to a lesser degree (Fig. 5). Even the conservative D97E mutation leads to greatly enhanced taurine efficacy (Fig. 4). This supports the idea that ligand efficacy is exquisitely sensitive to an electrostatic interaction involving D97. One possible explanation is that ligands could be acting as partial agonists on wildtype GlyR because they are not as effective as glycine in breaking the D97-R119 bond and this idea is supported by the crystal structure data showing that D97 and R119 are located further apart when glycine is bound than when strychnine is bound (Du et al., 2015). In mutants, this bond would be expected to be weaker or absent, thereby enhancing likelihoods of channel activation and increasing the relative efficacies of partial agonists. It should be noted that mutations at D97 also decrease agonist potency (Table 1), which is not surprising given that the D97 residue is located close to other residues previously implicated in glycine and taurine binding. In these mutants, an increase in agonist efficacy coupled with an increase in EC50 implies that agonist affinity is decreased.