Correlating Structural and Energetic Changes in Glycine Receptor Activation*

Background: Interactions between Gln−26′ (in M1 domain), Arg19′ (in M2 domain), and Lys24′ (in M2-M3 linker) may reveal molecular mechanisms of glycine receptor activation. Results: α1Q−26′E-containing receptors have longer active periods and lower conductances. Conclusion: The energy for activation is distributed broadly at the transduction zone. Significance: These energetic interactions are likely present in multiple pentameric ligand-gated ion channels. Pentameric ligand-gated ion channels (pLGICs) mediate fast chemoelectrical transduction in the nervous system. The mechanism by which the energy of ligand binding leads to current-conducting receptors is poorly understood and may vary among family members. We addressed these questions by correlating the structural and energetic mechanisms by which a naturally occurring M1 domain mutation (α1Q−26′E) enhances receptor activation in homo- and heteromeric glycine receptors. We systematically altered the charge of spatially clustered residues at positions 19′ and 24′, in the M2 and M2-M3 linker domains, respectively, which are known to be critical to efficient receptor activation, on a background of α1Q−26′E. Changes in the durations of single receptor activations (clusters) and conductance were used to determine interaction coupling energies, which we correlated with conformational displacements as measured in pLGIC crystal structures. Presence of the α1Q−26′E enhanced cluster durations and reduced channel conductance in homo- and heteromeric receptors. Strong coupling between α1−26′ and α119′ across the subunit interface suggests an important role in receptor activation. A lack of coupling between α1−26′ and α124′ implies that 24′ mutations disrupt activation via other interactions. A similar lack of energetic coupling between α1−26′ and reciprocal mutations in the β subunit suggests that this subunit remains relatively static during receptor activation. However, the channel effects of α1Q−26′E on α1β receptors suggests at least one α1-α1 interface per pentamer. The coupling-energy change between α1−26′ and α119′ correlates with a local structural rearrangement essential for pLGIC activation, implying it comprises a key energetic pathway in activating glycine receptors and other pLGICs.

The glycine receptor channel (GlyR) 2 is an anion-selective member of the pentameric ligand-gated ion-channel (pLGIC) family. pLGICs are comprised of modular domains (Fig. 1A). The ligand binding pockets are found in the extracellular domain, at subunit interfaces, whereas the permeation gate is located at the transmembrane domain (TD). Between the ligand binding pockets and the gate lies a transduction zone (TZ), made of interacting loops that originate from the extracellular domain, such as loops 2 and 7, and the TD, such as the M2-M3 linker (Fig. 1A, inset). Channel activation proceeds as a wave-like progression of structural rearrangements, initiated by the ligand binding reaction (1,2) and conveyed to the gate via interactions across the TZ (3)(4)(5).
Structure-function studies of the GlyR, and indeed other pLGICs, have benefitted greatly from the study of naturally occurring mutations that perturb channel activation. These perturbations give rise to impaired neurotransmission at glycinergic synapses, resulting in movement disorders such as hyperekplexia (6). Mutations in the ␣1 subunit have been most informative in this respect (7). Although this subunit readily forms homomeric receptors, synaptic GlyRs mainly comprise heteromers of ␣1 and ␤ subunits (8).
Three hyperekplexia-causing point mutations in the ␣1 subunit TZ are vital for efficient signal transduction. Two of these produce impaired receptor activation; ␣1 R271Q (␣1 R19ЈQ ), which is situated at the extracellular end of M2, and ␣1 K276E (␣1 K24ЈE ) in the M2-M3 linker (Fig. 1A). The ␣1 R19ЈQ mutation gives rise to a reduced single channel conductance and a marked decrease in glycine sensitivity when expressed as homomeric receptors (9,10). Similarly, the ␣1 K24ЈE mutation reduces glycine sensitivity in both homo-and heteromeric GlyRs, although no change in single channel conductance was reported (5,11,12). A recently identified third mutation, ␣1 Q226E (␣1 QϪ26ЈE ), located near the top of M1, gives rise to spontaneous channel opening, a reduction in single channel conductance, but no significant change in whole cell agonist sensitivity (13).
Recently published crystal structures of prokaryotic and eukaryotic pLGIC homologues (14 -21) reveal close proximity * This work was supported by the National Health and Medical Research of residues at 19Ј, 24Ј, and Ϫ26Ј. A high degree of sequence conservation is also evident at these positions, especially among anion-selective pLGICs, suggesting a common functional relevance. In particular, the Gln Ϫ26Ј side group from one subunit lies in close apposition to Arg 19Ј and Lys 24Ј in the adjacent subunit (Fig. 1A, inset). Given this proximity, and the observation that all three hyperekplexia-causing mutations involve a potential alteration in charge, it is reasonable to postulate that this discrete TZ subregion constitutes a functional unit with a key role in receptor activation. The recent demonstration, based on whole-cell measurements of ␣1 QϪ26ЈE homomeric GlyRs, that a glutamic acid occupying position Ϫ26Ј interacts with the arginine at 19Ј during channel activation (22), supports this idea.
With the exception of ␣1 K24ЈE -containing receptors, investigations into the effects of mutations at the 19Ј, 24Ј, and Ϫ26Ј positions have mainly been confined to homomeric receptors examined via whole cell patch clamp analysis. This affords only limited mechanistic insights into the channel activation mechanism and, of course, avoids the physiologically relevant ␣1␤ GlyR isoform.
To address these deficiencies we conducted a single channel study that examines the effects of charge-altering mutations to 19Ј, 24Ј in the ␣1 and ␤ subunits, co-expressed on a background of the newly described ␣1 QϪ26ЈE mutation. We employed two key functional parameters of single channel currents to infer the presence of charge and residue interactions. The first of these was the main unitary conductance state of the channel, which was taken as an index of net charge strength near the central conduction pathway (23,24). The second parameter was the mean duration that a receptor remains active for (cluster duration) in the presence of agonist. These parameters were used alone or in combination to quantitate the strength of interactions between the 19Ј, 24Ј, and Ϫ26Ј positions in both homomers and heteromers. Our data suggest that receptor activation involves multiple energetic pathways at the TZ.

EXPERIMENTAL PROCEDURES
Cell Culture-HEK AD293 cells were seeded onto poly-Dlysine-coated glass coverslips and transfected with cDNAs encoding human ␣1 (pCIS) and ␤ (pcDNA 3.1ϩ) GlyR subunits using a calcium phosphate-DNA co-precipitate method. The cDNA encoding the CD4 surface antigen was also added to the transfection mixture and acted as a marker of transfected cells. For some transfections, empty (non-coding) pCIS cDNA plasmid was included to reduce expression levels and facilitate the resolution of single-channel activations (25). To minimize the formation of homomeric receptors, heteromeric GlyRs were expressed by cotransfecting the ␣1 and ␤ cDNAs at an ␣1:␤ ratio of 1:100. We will provisionally assume a stoichiometry and subunit arrangement of ␤-␣1-␤-␣1-␤ (26, 27) but will consider other possibilities, see "Discussion." Cells were used in experiments 2-3 days after transfection. Point mutations were incorporated into the subunits using the QuikChange site-directed mutagenesis method. The homomeric ␣1 K24ЈC cross-linking experiments were done on an ␣1 C41A background. Successful incorporation of mutations was confirmed by sequencing the mutated DNA.
Electrophysiology-All experiments were carried out at room temperature (21-24°C). Single-channel and macropatch currents were recorded from outside-out excised patches at a clamped potential of Ϫ70 mV, unless indicated otherwise. The cells were continuously perfused via a gravity-fed plastic tube with an extracellular bath solution containing (in mM), 140 NaCl, 5 KCl, 1 MgCl 2 , 2 CaCl 2 , 10 HEPES, and 10 D-glucose and titrated to pH 7.4. Glass electrodes were pulled from borosilicate glass (G150F-3; Warner Instruments), coated with a silicone elastomer (Sylgard-184; Dow Corning), and heat-polished to a final tip resistance of 4 -15 M⍀ when filled with an intracellular solution containing (in mM) 145 CsCl, 2 MgCl 2 , 2 CaCl 2 , 10 HEPES, and 5 EGTA, pH 7.4. Excised patches were directly perfused with extracellular solution by placing them in front of one barrel of a double-barreled glass tube. Single channel currents were elicited by exposing the patch continuously to glycine containing solution, flowing through the adjacent barrel, by lateral switching of the tube. Experiments were recorded using an Axopatch 200B amplifier (Molecular Devices), filtered at 5 kHz (2 kHz for the ␣1 QϪ26ЈE/R19ЈA double mutant) and digitized at 20 (ensemble currents) or 50 kHz (single-channel currents) using Clampex (pClamp 10 suite, Molecular Devices) via a Digidata 1440A digitizer.
Data Analysis-Single-channel current amplitudes were measured in Clampfit. The current amplitude of most receptors was estimated using amplitude histograms. However, for receptors that produced activations containing many unresolved openings (see Fig. 3A) we opted for direct cursor measurement of expanded sections of record that showed fully resolved levels. Multiple measurements of openings of the largest amplitude were made from at least three patches for each channel type held at Ϫ70 mV. In current-voltage (i-V) experiments, the amplitude was measured at voltages of Ϯ70, Ϯ35, Ϯ15, and 0 mV. The data were fit to a polynomial function in SigmaPlot (Systat Software) and the reversal potential was read directly off the plots. Single-channel conductance (␥) was calculated from the single-channel amplitude (i) using Ohm's law, where V hold is the holding potential (Ϫ70 mV), V ljp is the liquid junction potential, and V rev is the reversal potential. V ljp was calculated to be 4.7 mV for the solutions used (28). We confined our analysis to the largest, main conductance level.
The QuB suite was used to analyze the kinetic properties of GlyR activations. Segments of single-channel activity separated by long periods of baseline were selected by eye and idealized into noise-free open and shut events using a temporal resolution of 70 s (29). Idealized data were fit with a simple model in which open and shut states were connected to a central shut state. Rate constants for transitions in the model were optimized using Maximum Likelihood Fitting (30), and states were added, to the central shut state, and re-fit until the log likelihood improved by less than 10 units. This fit was used to determine the critical time (t crit ), which was taken for the shut durations and used to divide the idealized segments into clusters (or bursts at 2 M glycine). t crit varied between 9 and 51 ms and generally preserved three shut components when the divided (chopped) data were re-fit in the manner described above. This analysis yielded mean cluster durations, intra-cluster open probabilities (P o ), and number of events per cluster. In addition, we monitored the effects of the mutations on the time constant of the main open exponential component (MOC) in the open dwell histograms of the re-fitted data. The MOC accounted for 51-96% of the current.
We used cluster duration and conductance to assay channel function. Cluster duration is a function of the net affect of all the equilibrium constants in the underlying activation mechanism and, as such, is an integrated index of the entire activation process. A point mutation will affect multiple state transitions along the activation coordinate, and it is experimentally intractable to determine which state-to-state transitions mutations will be affected. Because it is not possible to know, a priori, what the physical correlate of a particular equilibrium constant is, we avoided using any particular equilibrium constant as an index of receptor function, including in the pairwise interaction energy calculations.
Any mutation-induced change in conductance was taken to indicate that a given residue is charged. The magnitude of the change in conductance reflects the strength of the charge. Because we are only interested in factors affecting the activation process, we made no inferences regarding the determinants of permeation to avoid conflating the processes of activation and ion permeation. We used cluster duration and conductance as measures of the electrostatic topography of the gating landscape, exploiting the fact that the region of interest happens to be near the permeation pathway, thus giving us a measurable way of ascertaining the effective charge of relevant residues. As a measure of open state stability we monitored the shutting frequency within active periods. This proved to be a more sensitive stand-alone parameter than more conventional ones, such as intracluster P o . P o was only used in conjunction with cluster duration as an alternative method of calculating coupling energies.
Parameter measurements were plotted in Prism 6 (Graph-Pad). Data were analyzed by ordinary one-way analysis of variance in Prism, and Sidak's multiple comparisons test was used in the case of significance. Because cluster and burst durations, MOCs, and shutting frequencies are exponentially distributed, we analyzed these data on a patch-to-patch basis. Mean values for cluster duration, MOC, and shutting frequency were determined for each patch for a given receptor (these are normally distributed). Analysis of variance tests were applied to these means. The overall mean was then determined for plotting and data interpretation. Statistical significance was set at p Ͻ 0.01, and all data are presented as mean Ϯ S.E.
Cluster (or burst) durations were determined for the largest conductance level for each receptor. The energy of interaction between two residue positions incorporating mutations in the receptors was determined using, where ⌬⌬G int is the first-order coupling free energy (in kJ mol Ϫ1 ) in a mutant cycle, R is the gas constant, T is absolute temperature, and ⍜ is the product of the channel conductance and cluster duration at saturating concentrations of glycine for wild-type (wt), double mutant (dm), single mutant 1 (sm1), and single mutant 2 (sm2). Alternative parameters for ⍜ are shown in Table 2. Ensemble currents were recorded in response to brief (Ͻ1 ms) exposure to a saturating concentration of glycine by rapidly moving the double-barreled tube by means of a piezoelectric translator (Siskyou MXPZT-300), controlled by a voltage-step protocol in Clampex. The exchange time (rise and decay ϳ200 s) was verified for each recording session by monitoring changes in the liquid junction potential using an open-tip electrode. Ten to fifteen responses from each patch were peaknormalized and averaged. The rise and decay times constants were measured by fitting 10 -100% of the rising phase of the current to, and the 20 -80% of the decay phase of the current using, where A is the amplitude, t is time, is the time constant, and C is a constant. The number of components (j) ranged from one to three to give the best fit as determined by eye. To facilitate comparison between the decay time constants, the weighted average time constant ( w ) was calculated using the following equation.

RESULTS
Current-Voltage (i-V) Plots and Conductance-Our first experiments were aimed at accurately calculating the conductance of each receptor at Ϫ70 mV. Single channel current amplitude was measured over a voltage range of Ϯ70 mV for three GlyRs to establish if the reversal potential varied between receptors incorporating a change of charge near the extracellular pore entrance. We tested the homomeric ␣1 wild-type, ␣1 QϪ26ЈE , and ␣1 R19ЈQ receptors, the latter two involving a potential alteration in charge near the pore lumen. The largest, predominant current level was considered for ␣1 wild-type and ␣1 QϪ26ЈE receptors, elicited by glycine concentrations of 20 M (␣1 wild-type) and 1 M (␣1 QϪ26ЈE ) (Fig. 1B, Table 1). Previous reports suggest that the homomeric ␣1 R19ЈQ GlyR exhibits either a single conductance of 18 pS (10) or two conductances, with one being predominant (15 pS, Ͼ99%) and the other being rare (37 pS, Ͻ1%) (9). We found that ␣1 R19ЈQ receptors exhibited multiple levels when exposed to glycine concentrations from 2 to 300 mM. We analyzed the smallest and largest of these levels (Fig. 1C). The i-V for all three channels intersected the voltage axis at 4.0 mV (Fig. 1D), allowing us to correct for the driving force according to Equation 1. The corresponding conductance calculations yielded values of 92.5 Ϯ 0.3 pS for ␣1 wild-type and 60.6 Ϯ 0.4 pS for ␣1 QϪ26ЈE receptors. For the ␣1 R19ЈQ receptors the conductance values were 14.9 Ϯ 0.5 and 52.7 Ϯ 0.8 pS for the small and large amplitude currents, respectively. As all four plots reversed current at 4.0 mV, irrespective of the receptor type or conductance level measured, we infer that mutations at the subregion under investigation do not affect the reversal potential. For calculations of conductance for other receptors we will assume that they too reverse current at 4.0 mV.
Wild-type and ␣1 QϪ26ЈE Homo-and Heteromeric GlyRs-Having established an accurate method for calculating channel conductance, we compared single channel currents recorded from homomeric ␣1 wild-type and ␣1 QϪ26ЈE receptors, with those of the same receptors incorporating the ␤ subunit ( Table  1). As previously noted, wild-type homo-and heteromeric receptors exhibit negligible spontaneous (agonist-free) activations (12, 31)( Fig. 2A, above). At a saturating (1 mM) glycine concentration, active periods occurred as clusters of openings flanked by quiet periods that correspond to receptor desensitization ( Fig. 2A, below). We also observed spontaneous channel activity in ␣1 QϪ26ЈE homomeric receptors ( Fig. 2B above), as previously reported (13). As evidence was adduced for an interaction between the introduced glutamic acid at Ϫ26Ј in one subunit and the Arg 19 Ј position in the adjacent subunit (22) we investigated the effect of introducing the wild-type ␤ subunit, which has an alanine at 19Ј. To our surprise, ␣1 QϪ26ЈE -␤ heteromers were also constitutively active (Fig. 2B, above). This suggests that there might be additional residues that can interact with ␣1 QϪ26ЈE in ␣1 and ␤ subunits that give rise to spontaneous openings, or that at least one ␣1-␣1 interface exists in ␣1␤ heteromeric receptors.
In homomeric ␣1 QϪ26ЈE receptors, the introduction of a glutamic acid decreased the channel conductance to 60.6 Ϯ 0.4 pS, dramatically increased the mean cluster length to 3161 Ϯ 262 ms, and decreased the intracluster shutting frequency to 15 Ϯ 3 Hz. A comparable pattern of changes was observed in ␣1 QϪ26ЈE -␤ heteromers. There was a significant decrease in conductance to 30.8 Ϯ 1.5 pS (Fig. 2C), which is proportional to the decrease measured in the homomeric receptors. The mean cluster duration for ␣1 QϪ26ЈE ␤ heteromers was 893 Ϯ 283 ms, representing a proportional increase relative to the homomeric mutant receptor. The mean frequency of shutting within clusters mediated by ␣1 QϪ26ЈE -␤ heteromers was 11 Ϯ 1 Hz.
We infer that the ␣1 QϪ26ЈE mutation confers four effects on GlyRs. First, receptors bearing this mutation are constitutively active. Second, the decrease in conductance demonstrates that the introduced glutamic acid side group carries a negative charge at pH 7.4, and is close enough to the permeation pathway to influence conductance. Third, the negative charge induces longer duration active periods (and greater MOC time constants) within which the conducting states are more stable. Finally, the ␤ subunit dilutes the effect of the ␣1 QϪ26ЈE mutation on cluster properties, but not on conductance. In saturating glycine, ␣1 QϪ26ЈE -containing receptors activate in clusters of longer duration, lower conductance, and a reduced intra-cluster shutting frequency compared with their wild-type counterparts. C, group data summarizing the effects on channel conductance, cluster duration, and intra-cluster shutting frequency for wild-type and ␣1 QϪ26ЈE -containing receptors. The number of clusters used in the analysis was 175 (␣1 wild-type, 10 patches), 69 (␣1␤ wild-type, 8 patches), 124 (␣1 QϪ26ЈE , 9 patches), and 72 (␣1 QϪ26ЈE -␤, 9 patches). ***, p Ͻ 0.0001; **, p Ͻ 0.001; *, p Ͻ 0.01. FEBRUARY 27, 2015 • VOLUME 290 • NUMBER 9

JOURNAL OF BIOLOGICAL CHEMISTRY 5625
19Ј Mutations on an ␣1 QϪ26ЈE Background-The above results indicate that ␣1 QϪ26ЈE induces spontaneous openings, but that its effects on ligand-induced cluster duration are reduced when the ␤ subunit is present. This suggests that ␤ subunit residues proximal to ␣1 QϪ26ЈE may account for the attenuation. To investigate this further, we introduced mutations at 19Ј in the ␣1 and ␤ subunits ( Table 1). As with ␣1 R19ЈQ receptors, the homomeric ␣1 R19ЈA receptors, in the presence of a saturating glycine concentration (50 mM) also showed a significantly reduced conductance (75.0 Ϯ 0.8 pS), a marked decrease in cluster duration to 257 Ϯ 54 ms, a marked leftward shift in the MOC (0.7 Ϯ 0.2 ms, 77 Ϯ 9%), and a substantial increase in shutting frequency (395 Ϯ 18 Hz) compared to the homomeric wild-type (Fig. 3, A and C). Expressing the ␣1 R19ЈA on an ␣1 QϪ26ЈE background produced the ␣1 QϪ26ЈE/R19ЈA homomeric receptor. Functional impairment was most severe in this double mutant receptor (Fig. 3A). Notably, all spontaneous activity was ablated. Indeed, little activity was observed for these receptors at glycine concentrations below 100 mM. We used 300 mM glycine to induce enough channel activity for analysis. These receptors opened to a conductance of 8.6 Ϯ 0.7 pS, with a mean cluster length of 89 Ϯ 25 ms, an MOC of 1.2 Ϯ 0.2 ms (fraction 83 Ϯ 5%), and a shutting frequency of 292 Ϯ 41 Hz (Fig. 3C).
We then investigated the effect of the reciprocal ␤ subunit mutation in ␣1␤ A19ЈR GlyRs. When activated by a saturating (1 mM) glycine concentration, this receptor exhibited a small (ϳ7-8 pS) reduction in conductance (53.7 Ϯ 1.5 pS) and no change to cluster duration (511 Ϯ 179 ms), MOC (30 Ϯ 6 ms, 78 Ϯ 14%), or the frequency of shut events (37 Ϯ 4 Hz) relative to wild-type ␣1␤ receptors (Fig. 3, B and D). This demonstrates a functional asymmetry between ␣1 and ␤ subunits, in accord with a previous report based on whole cell experiments (34). That is, when an arginine residue occupies all five 19Ј positions in the mutant heteromeric receptors, the functional parameters of conductance, cluster duration, and number of shut events within clusters are nearly indistinguishable from wild-type heteromers. This is in stark contrast to the effects of removing the arginine in the ␣1 subunit.
The heteromeric ␣1 QϪ26ЈE -␤ A19ЈR GlyR also exhibited minimal changes relative to ␣1 QϪ26ЈE -␤ receptors. These receptors were constitutively active (Fig. 3B) with a conductance of 29.0 Ϯ 1.5 pS. Glycine (1 mM) elicited clusters with a mean duration of 639 Ϯ 106 ms, an MOC of 69 Ϯ 5 ms (fraction 91 Ϯ 6%), and a shutting frequency of 22 Ϯ 2 Hz. The parameters of conductance, cluster duration, and MOC were not significantly different from the ␣1 Q-26ЈE -␤ receptors (Fig. 3D), whereas there was a small but significant difference in shutting frequency. Together, these data demonstrate the significance of the arginine residue at 19Ј in the ␣1 subunit on receptor activation, in contrast to the ␤ subunit, where the functional influence of this residue is minimal. The existence of spontaneous activity in ␣1 QϪ26ЈE -␤ and ␣1 QϪ26ЈE -␤ A19ЈR receptors implies the presence of at least one ␣1-␣1 interface. Alternatively, ␣1 QϪ26ЈE may interact with different residues in ␣1 versus ␤ subunits, giving rise to the observed effects.
24Ј Mutations on an ␣1 QϪ26ЈE Background-A possible candidate residue that might interact with the ␣1 QϪ26ЈE residue is Lys 24 Ј. This residue is highly conserved, like Arg 19 Ј, has a basic side group, and is critical to efficient channel activation in the ␣1 subunit (5,35), especially when mutated to a hyperekplexiacausing glutamic acid (11,12). Moreover, Lys 24 Ј sits on a segment (the M2-M3 linker) that is mobile enough to permit K24ЈC cross-linking between ␣1 subunits in functional GlyRs. This mobility was tested on ␣1 K24ЈC homomeric channels (Fig.  4A, Table 1). In the presence of glycine alone, the activations were sporadic and short-lived, as illustrated by the single ϳ2 ms open dwell-time component (Fig. 4B, left). Following application of the reducing agent, DTT (4 mM), for a duration of 3-6 min in three patches, the mean burst duration increased dramatically to ϳ90 ms and the open dwell-time distributions showed multiple components (Fig. 4B, right). This observation, and the demonstration of H 2 O 2 -induced reversibility (22) suggests that DTT reduces pre-existing disulfide bonds formed between ␣1 K24ЈC residues. No significant difference was detected in conductance for this mutant (90.0 Ϯ 0.5 pS) compared with wild-type, either before or after DTT application. We reasoned that the high amplitude movement of the M2-M3 linker during the activation process could bring Lys 24 Ј close enough to ␣1 QϪ26ЈE to facilitate interaction between the two. This is also consistent with the pLGIC crystal structures that show proximity between the Lys 24 Ј and Ϫ26Ј positions (Fig.  1A).
We tested mutant channels that incorporated the K24ЈA substitution in ␣1 and ␤ subunits. Representative current traces are shown in Fig. 4, C and D. The first of these was the ␣1 K24ЈA homomeric GlyR (Fig. 4C). A saturating (50 mM) glycine concentration induced clusters with a conductance of 89.1 Ϯ 1.6 pS, representing a nonsignificant change compared with ␣1 wild-type homomers. Greater differences were observed in the kinetic properties of the receptors. Cluster duration and the MOC were an order of magnitude briefer at 214 Ϯ 23 and 3.0 Ϯ 1 ms (fraction 51 Ϯ 1%), whereas the intracluster shutting frequency was an order of magnitude higher at 324 Ϯ 88 Hz (Fig.  4E). The ␣1 K24ЈA mutation was then combined with the ␣1 QϪ26ЈE to produce the double mutant ␣1 QϪ26ЈE/K24ЈA homomeric receptor. These were active in the absence of glycine (Fig. 4C) and had a similar channel conductance (56.4 Ϯ 1.6 pS) to the ␣1 QϪ26ЈE homomers. Clusters of activity induced by 50 mM glycine had a mean duration of 882 Ϯ 158 ms and the receptor shut at a frequency of 23 Ϯ 3 Hz while active (Fig. 4F). The MOC for this receptor was 124 Ϯ 26 ms (fraction 96 Ϯ 3%).

Structure and Energy in Glycine Receptors
minor effects on channel conductance suggest that whereas the channel is conducting current the Lys 24 Ј position is either too far from the permeation pathway to affect conductance or is otherwise neutralized. The Energetics of ␣1 R19Ј , ␣1 K24Ј , and ␣1 QϪ26ЈE Interactions in Homo-and Heteromeric GlyRs-Our data reveal that ␣1 QϪ26ЈE enhances channel function by conferring spontaneous activity, increasing the time constant of the main open dwell component, lengthening the duration of glycine-induced clusters, and decreasing the transition frequency to shut states while the channel is active. We have assessed the contributions of local electrostatic interactions with 19Ј and 24Ј residues in both the ␣1 and ␤ subunits in mediating these effects. We next employed channel conductance and cluster duration in an attempt to define the relative strengths of these interactions in the channel activation process. These parameters were used in mutant cycle calculations for four potential interaction partner combinations involving the ␣1 QϪ26ЈE residue (Fig. 5). The first of these was the ␣1 QϪ26Ј and ␣1 R19Ј cycle. This revealed an interaction energy of 9.43 kJ mol Ϫ1 (2.3 kcal mol Ϫ1 ) (Fig. 5A). The energy of interaction between ␣1 QϪ26Ј and ␣1 K24Ј was Ϫ0.97 kJ mol Ϫ1 (Ϫ0.23 kcal mol Ϫ1 ) (Fig. 5B). The coupling energies involving heteromers were lower than for homomeric receptors. The ␣1 QϪ26Ј -␤ A19Ј cycle yielded an interaction energy of 1.60 kJ mol Ϫ1 (0.38 kcal mol Ϫ1 ) (Fig. 5C), whereas the interaction energy for the ␣1 QϪ26Ј and ␤ K24Ј was the lowest, being Ϫ0.32 kJ mol Ϫ1 (Ϫ0.08 kcal mol Ϫ1 ) (Fig. 5D). Table 2 shows ⌬⌬G int calculations using other kinetic parameters, in addition to the product of cluster duration and conductance. These include the use of cluster duration and conductance, separately, the product of the cluster duration and open state probability (P o ), and the MOC time constant for each receptor. Overall, these data show that interactions between Ϫ26Ј and the 19Ј and 24Ј positions are strongest in ␣1 homomeric receptors, especially the interaction between ␣1 QϪ26Ј and ␣1 R19Ј . Introducing the ␤ subunit, even one bearing an arginine at 19Ј did not increase the strength of interactions with ␣1 QϪ26ЈE , even though we predict a majority of ␣1-␤ subunit interfaces in heteromeric GlyRs (26,27). Interactions between the Ϫ26Ј and 24Ј side groups in both subunits were weaker, but not negligible in the ␣1 subunit. The especially weak interaction energy between ␣1 QϪ26Ј and ␤ K24Ј supports the notion that the TZ of the ␤ subunit is of lesser significance in receptor activation than the ␣1 subunit.

Structure and Energy in Glycine Receptors
eukaryotic (14,16,20,21) pLGICs. Given that these structures are static representations obtained under non-physiological conditions, it is vital to check them against functional data to ascertain if the crystal structures correspond to functionally relevant states. It is also useful to determine whether the putative interactions identified here are likely to have universal relevance across the pLGIC family. A structural alignment of two GLIC structures, in open (18) and closed (19) states (Fig. 6, A  and B) reveals relative movements of the residues at Ϫ26Ј, 19Ј, and 24Ј during channel opening.
To determine whether the interactions examined in this study represent a universal structural principal of pLGIC activation we measured the ␣-carbon-␣-carbon (␣C-␣C) distances between the Ϫ26Ј and 19Ј positions and the Ϫ26Ј and 24Ј positions (Fig. 6C) in crystal structures of pLGICs in putative conducting and non-conducting configurations (Fig. 6D). Inclusion of structures in either the open-like or closed-like groups was based on a consideration of several features of each structure, including the minimum pore diameter, M2 orientation, and the molecules in which each receptor was co-crystalized. In each case, our classifications are in accord with those proposed by the original authors.
The residues at positions Ϫ26Ј and 19Ј have a significantly smaller mean ␣C-␣C distance in conducting states (7.8 Ϯ 0.1 Å) compared with nonconducting ones (12.9 Ϯ 1.0 Å). The mean distance between positions Ϫ26Ј and 24Ј differs less between the two states, with a nonsignificant decrease from 12.9 Ϯ 2.2 (nonconducting) to 9.7 Ϯ 0.1 Å (conducting). The distances between Ϫ26Ј and 19Ј, and Ϫ26Ј and 24Ј from a recently published GluCl structure in apo (closed) and POPC-bound confirmations (open) were excluded from the mean distance calculations (14). The decision to exclude these data were made on the basis that the ␣C-␣C distances from Ϫ26Ј to 19Ј or 24Ј were clear outliers, especially for Ϫ26Ј to 19Ј. Furthermore, the POPC-bound (presumably open) structure differs from an earlier ivermectin-and glutamate-bound structure (16) in terms of the distances measured, and this earlier structure does conform to the general trends observed. This suggests that the apo and POPC-bound structures might not resemble physiological conformations, and so were excluded from further analysis.
Prediction of Synaptic Currents Mediated by ␣1 QϪ26ЈE -containing GlyRs-Finally, we investigated how the ␣1 QϪ26ЈE hyperekplexia mutation might affect glycinergic inhibitory synaptic currents. Applying a saturating (1 mM) glycine concentration for brief periods (Ͻ1 ms) to outside-out membrane patches containing many receptors elicited synaptic-like ensemble currents. Wild-type ␣1 homomers and ␣1␤ heteromers activated rapidly, with 10 -100% rise time constants of 0.24 Ϯ 0.06 and 0.24 Ϯ 0.02 ms, respectively. The decay time constants were also relatively brief, being 24.2 Ϯ 7.3 ms for homomers and 15.7 Ϯ 4.3 ms for heteromers (Fig. 7A). The ␤ subunit had no significant influence on either parameter. Our results are consistent with those measured previously via similar techniques (36) and with synaptic currents of native synapses expressing ␣1 homomeric and ␣1␤ heteromeric GlyRs (37). This similarity in kinetics suggests that they are dominated by the ␣1 subunit. This inference was borne out in our experiments on ␣1 QϪ26ЈE homomers and ␣1 QϪ26ЈE -␤ heteromers. Currents mediated by both receptors containing the ␣1 QϪ26ЈE mutation were activated with rise time constants that were similar to wild-type receptors. These were 0.26 Ϯ 0.03 and 0.31 Ϯ 0.09 ms for homo-and heteromeric receptors, respectively. The decay time constants measured from ␣1 QϪ26ЈE and ␣1 QϪ26ЈE -␤ receptors were an order of magnitude greater compared with their respective wild-type receptors (280 Ϯ 41 and 272 Ϯ 35 ms, respectively) and were not significantly different from each other (Fig. 7A).
It has been demonstrated that the time constant for deactivation of ensemble currents is the same as the burst durations at very low concentrations of ligand (38). However, it is difficult to predict what a sufficiently low concentration of glycine would be that might elicit burst durations that correspond to the measured decay times. We thus employed 2 M glycine to activate bursts of activity from wild-type and ␣1 QϪ26ЈE -containing homomers and heteromers (Fig. 7B). 2 M glycine induced bursts of activity in ␣1 QϪ26ЈE -containing receptors that were of longer duration compared with their wild-type counterparts. Wild-type ␣1 homomers were activated for a mean of 37 Ϯ 4 ms, whereas the mean burst duration of ␣1 QϪ26ЈE homomeric receptors was 947 Ϯ 141 ms. Wild-type heteromers had a mean burst duration of 40 Ϯ 3 ms and heteromers incorporating the ␣1 QϪ26ЈE mutation had a mean burst duration of 344 Ϯ 87 ms. The mean burst durations and ensemble current decay times were plotted on the same set of axes to investigate any correlation between them (Fig. 7C). A closer correspondence between burst duration and decay times was observed for wild-type receptors, the former quantity being ϳ1.5-2.5-fold greater than the latter. For receptors containing the ␣1 QϪ26ЈE mutation there was greater deviation between the burst duration and current decay times, especially for ␣1 QϪ26ЈE homomers. We make the following inferences from these data. First, that current kinetics in homo-and heteromeric receptors are dominated by the ␣1 subunit. Second, that there is a strong correlation between the burst duration and the decay rate of ensemble currents. Third, that 2 M glycine is likely too high a concentration to elicit bursts that are sufficiently brief to account for the current decay times, especially for mutant-containing receptors. Fourth, that synaptic currents in vivo mediated by GlyRs expressing the ␣1 QϪ26ЈE mutation will decay dramatically more slowly than wild-type.

DISCUSSION
Our aim was to investigate the mechanism by which the ␣1 QϪ26ЈE mutation induces spontaneous activity in homo-and heteromeric GlyRs on the level of single receptors. We reasoned that insights thus obtained may be used in conjunction with crystal structures of several, newly published pLCIC mem-bers to provide fundamental insights into the activation mechanisms of pLGICs in general. We investigated these receptors via single-channel analysis for two reasons. First, single channel kinetic analysis permits a quantitative understanding of the channel gating process and its underlying energetics (39,40). Second, single channel conductance analysis allows us to deter-  FEBRUARY 27, 2015 • VOLUME 290 • NUMBER 9 mine whether mutations to residues near the pore lumen alter side group charge (24,41). This would help us to resolve whether electrostatic interactions among the mutated residues affect receptor activation. The correlation between conductance and cluster length was critical for the interpretation of pairwise interactions because if a mutation did not affect conductance it also had no effect on cluster duration on receptors containing the ␣1 QϪ26ЈE as a background. The importance of connecting cluster duration and conductance becomes clearer when comparing the ␣1 QϪ26ЈE/R19ЈA receptors with ␣1 QϪ26ЈE/K24ЈA , ␣1 QϪ26ЈE -␤ K24ЈA , or ␣1 QϪ26ЈE -␤ A19ЈR receptors. The ␣1 R19ЈA mutation decreases cluster duration and conductance on a wild-type background, and on an ␣1 QϪ26ЈE background. In contrast, the ␣1 K24ЈA decreases cluster duration but not conductance. The ␣1 QϪ26ЈE/K24ЈA double mutations had no additional effect on either parameter. The ␤ K24ЈA or ␤ A19ЈR mutations had no significant effects on cluster duration or conductance when co-expressed with either ␣1 wild-type or ␣1 QϪ26ЈE . These observations support the notion that ␣1 K24Ј is a salient gating element, but not as part of the ␣1 QϪ26Ј -␣1 R19Ј pathway, whereas ␤ 24Ј or ␤ 19Ј positions seem not to support charge nor do they feature prominently in receptor activation.
Assuming that the coupling energies calculated here reflect relative degrees of local conformational movements necessary for the receptor to transition between stable functional states, we can deduce the following about GlyR (and other pLGIC) activation. The TZ of the ␣1 subunit is the most mobile, especially the extracellular portions of M1, M2, and the M2-M3 linker, compared with the TZ of the ␤ subunit. The mean increase in proximity between ␣1 Ϫ26Ј and ␣1 19Ј , between putative non-conducting and conducting states derived from crystal structures is greatest (Ͼ5 Å, Fig. 6). So too, is the ⌬⌬G int for this interaction in the ␣1 subunit (Fig. 5). The ␣1 Ϫ26Ј -␣1 19Ј coupling energy is well within the range for a significant interaction between residues in pLGICs during activation (39,40,44) and between ligand binding residues and ligand (45). We propose that this interaction represents one point of energy transfer along what is likely to be a broad reaction coordinate (broad corrugated activation barrier) as proposed by Auerbach for the related nAChR (40,46).
The cross-linking evidence suggests that the ␣1 M2-M3 linker is also highly mobile. This mobility is evidently necessary to produce activations that exhibit the type of complexity (multiple dwell components, Fig. 4) characteristic of wild-type receptors (12,47). However, the mean change in separation between Ϫ26Ј and 24Ј between conducting and non-conducting states is less than that for Ϫ26Ј and 19Ј (ϳ3 Å), and the absolute distance between these two positions is greater (Fig. 6). The relatively low coupling energy associated with ␣1 Ϫ26Ј and ␣1 24Ј , along with evidence that ␣1 24Ј mutations lead to profound perturbations in receptor activation suggests that this residue (and likely the M2-M3 linker) is involved in a different route of energy transfer to that of ␣1 Ϫ26Ј and ␣1 19Ј . The ␣1 K24Ј may interact with aromatic residues in the ␤10-M1 linker (Fig.  1A, inset) to form cation-contacts, as predicted by the crystal structure of the ␤3 homomeric GABA A receptor (21). The coupling energies calculated for ␣1 Ϫ26Ј with ␤ 19Ј and ␤ 24Ј parallel those for the ␣1 subunit alone. However, the corresponding values suggest either weak or no interactions. This is reconcilable with the notion that the ␤ subunit has a subordinate role in receptor activation (34). Moreover, adding a basic residue at ␤ 19Ј or removing one from ␤ 24Ј had little or no effect on channel conductance, respectively. These data suggest that the microenvironment around these positions is such that basic residues are weakly protonated (24), or otherwise not felt by permeating ions. ␤ 24Ј -␤ 24Ј cysteine cross-linking is not amenable to electrophysiology, but an ␣1 Ϫ26Ј -␤ 24Ј coupling energy that is comparable with the thermal energy of the receptor (40) would imply the ␤M2-M3 linker is relatively static with respect to ␣1 Ϫ26Ј during receptor activation. Together, these findings imply that many parts of the receptor change energy during activation.
We have demonstrated that the increased duration of unliganded (spontaneous) activity was paralleled by an increase in the durations of fully liganded activity (clusters) in receptors containing the ␣1 QϪ26ЈE mutation. This is also the case for wildtype receptors. From this observation we infer that the ␣1 QϪ26ЈE mutation has not uncoupled the activation mechanism as it occurs in wild-type receptors, allowing the transmembrane domains to adopt open, conducting conformation(s) by autonomously interacting with ␣1 R19Ј . The indistinguishable conductance levels between unliganded and fully liganded receptors lend support to this inference (48,49). Furthermore, we infer that by enhancing the ␣1 Ϫ26Ј -␣1 19Ј interaction, the ␣1 QϪ26ЈE mutation has exposed a salient component of the activation mechanism of the wild-type receptor. If we assume that the receptors are in thermodynamic equilibrium, and that the ligand binding properties of receptors mutated at the TZ are little changed relative to wild-type (12,50), then it follows that for an increase in the activation equilibrium constant for unliganded activation (an index of the efficacy with which the receptor transitions from non-conducting to conducting configurations) there will be a corresponding increase in the fully liganded activation equilibrium constant. This result has been well documented for constitutively active muscle nAChRs that bear mutations within the TZ (49,51).
In summary, we sought to quantitate the strength of interactions between the 19Ј, 24Ј, and Ϫ26Ј positions in contributing to the activation mechanism of both homo-and heteromeric GlyRs. We conclude that the ␣1 Ϫ26Ј -␣1 19Ј interaction is likely part of a significant pathway that distributes the energy of ligand binding to receptor activation. The significance of the interaction was made evident by the discovery that a single electrostatic interaction per ␣1 QϪ26ЈE -containing pentamer is sufficient to enhance receptor activation. The 24Ј position, although situated on a segment that is also highly mobile, is likely involved in a different energetic pathway. The local movements associated with these energetic interactions are evident in published crystal structures, indicating that they may pertain widely across the pLGIC family.