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![α 1 F45K mutation may impair the GABA binding. A, binding site in the WT receptor (experimental structure from [6]). GABA-free (a) and GABA-bound (b) experimental structures. Distance between α 1 R66 and β 2 F200 as well as angle between the former residue and loop D is displayed. B, S201 scenario; α 1 F45K (in blue) polar interaction with β 2 S201 (in orange) from loop C. C, D44 scenario; electrostatic interaction of α 1 F45K with α 1 D44 (in orange) from loop G D, T47 scenario; polar interaction of α 1 F45K with α 1 T44 (in orange) from loop G.](publication/339976957/figure/fig3/AS:878139710377985@1586376230991/a-1-F45K-mutation-may-impair-the-GABA-binding-A-binding-site-in-the-WT-receptor.png)
α 1 F45K mutation may impair the GABA binding. A, binding site in the WT receptor (experimental structure from [6]). GABA-free (a) and GABA-bound (b) experimental structures. Distance between α 1 R66 and β 2 F200 as well as angle between the former residue and loop D is displayed. B, S201 scenario; α 1 F45K (in blue) polar interaction with β 2 S201 (in orange) from loop C. C, D44 scenario; electrostatic interaction of α 1 F45K with α 1 D44 (in orange) from loop G D, T47 scenario; polar interaction of α 1 F45K with α 1 T44 (in orange) from loop G.
Source publication
GABAA receptors (GABAARs) mediate inhibitory neurotransmission in the mammalian brain. Recently, numerous GABAAR static structures have been published, but the molecular mechanisms of receptor activation remain elusive. Loop G is a rigid β-strand belonging to an extensive β-sheet that spans the regions involved in GABA binding and the interdomain i...
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GABA A receptors mediate most inhibitory synaptic transmission in the brain of vertebrates. Following GABA binding and fast activation, these receptors undergo a slower desensitiza-tion, the conformational pathway of which remains largely elusive. To explore the mechanism of desensitization, we used concatemeric α1β2γ2 GABA A receptors to selective...
Citations
... Considering that models employed in single-channel analysis had two open states, for macroscopic modeling (one open state), the average values of α and β rate constants from single-channel modeling were taken as initial values. As explained in detail in our previous reports, 9,24 estimations for d and r rate constants from stationary singlechannel analysis yield markedly different values of these rate constants because of distinct experimental conditions. The resulting rate constants for the α 1 P277H mutant, estimated with ChannelLab for these initial values, are presented in Figure 7C. ...
... Surprisingly, in the steady-state single-channel recordings, all the rate constants for the considered substitutions (except only for d for α 1 P277E) describing desensitization (d and r) were significantly affected by the mutations (Table 3), whereas the relative effect on d and r rate constants appeared more moderate in the case of macroscopic recordings (only d for α 1 P277H was significantly altered, Figure 7B). This is intriguing because, as discussed in our previous reports, 8,9,23,24,51 macroscopic recordings of responses to rapid applications of the saturating agonist offer optimal conditions to reveal the desensitization onset, whereas steadystate measurements are carried out in conditions in which the majority of receptors are desensitized. For this reason, stationary single-channel recordings typically do not reveal rapid desensitization rate constants, which also explains differences in their estimation in macroscopic and singlechannel recordings (see Table 3 and Figure 7B). ...
... The most remarkable in this context is the observation that microscopic desensitization of the GABA A receptor is highly sensitive to mutations of residues in most of localizations studied by our group thus far. 8,9,[23][24][25]50,51 This feature of desensitization led us to propose the concept of a "diffuse desensitization gate" 8,9 as opposed to the desensitization gate largely restricted to the receptor's transmembrane domains. 54 In general, the emerging picture is that structural determinants of various conformational transitions are not compartmentalized, but rather specific elements of the protein structure are being shared upon distinct phases of the receptor activation. ...
Cys-loop receptors are a superfamily of transmembrane, pentameric receptors that play a crucial role in mammalian CNS signaling. Physiological activation of these receptors is typically initiated by neurotransmitter binding to the orthosteric binding site, located at the extracellular domain (ECD), which leads to the opening of the channel pore (gate) at the transmembrane domain (TMD). Whereas considerable knowledge on molecular mechanisms of Cys-loop receptor activation was gathered for the acetylcholine receptor, little is known with this respect about the GABAA receptor (GABAAR), which mediates cellular inhibition. Importantly, several static structures of GABAAR were recently described, paving the way to more in-depth molecular functional studies. Moreover, it has been pointed out that the TMD-ECD interface region plays a crucial role in transduction of conformational changes from the ligand binding site to the channel gate. One of the interface structures implicated in this transduction process is the M2-M3 loop with a highly conserved proline (P277) residue. To address this issue specifically for α1β2γ2L GABAAR, we choose to substitute proline α1P277 with amino acids with different physicochemical features such as electrostatic charge or their ability to change the loop flexibility. To address the functional impact of these mutations, we performed macroscopic and single-channel patch-clamp analyses together with modeling. Our findings revealed that mutation of α1P277 weakly affected agonist binding but was critical for all transitions of GABAAR gating: opening/closing, preactivation, and desensitization. In conclusion, we provide evidence that conservative α1P277 at the interface is strongly involved in regulating the receptor gating.
... The allosteric BZD binding site is located at the interface between the principal α1 subunit and the complementary γ2 subunit (23). Many studies have reported the importance of NTD in the function of GABA A Rs. F45 at NTD of α1 subunit is involved in agonist binding and late channel gating transitions (24). N104 and N173 of β2 subunits, which are located around Cys-loop, exhibit the essential role in N-glycosylation (25). ...
ABSTRACT Epilepsy is one of the most common episodic neurological disorders, affecting 1% population worldwide. The genetic variations of γ-aminobutyric acid type A (GABAA) receptor, including missense, nonsense, splice site and frameshift variants in GABRA1-6, GABRB1-3, GABRG1-3, and GABRD, have been identified as some of the primary genetic causes of epilepsy. However, the lack of a complete understanding of the association between epilepsy syndromes and GABAA receptor variants makes it challenging to develop effective therapeutics. Here, we summarize a comprehensive list of over 150 epilepsy-associated variants in the major α1, β2, β3, and γ2 subunits of GABAA receptors and their functional defects. In addition, their spatial distribution is visualized in the cryo-EM structures of GABAA receptors. Many of the variants lead to reduced receptor surface expression and thus loss of function due to protein conformational defects and impaired trafficking. This knowledge aids the development of precision medicine-based therapeutic strategies to treat epilepsy.
... We reported that a benzodiazepine, flurazepam, affected desensitization, 24,28 and when we consider that the binding site for this modulator is located at ECD, very distantly from transmembrane segments indicated by Gielen et al., 27 it seems that desensitization might be also controlled by structures within ECD. Moreover, in our recent study, 22 we provided evidence that mutation of the F45 residue, located close to the agonist binding site at the loop G of the α 1 subunit, profoundly altered desensitization kinetics. Unexpectedly, we also found that mutation of the α 1 F14 and β 2 F31 residues, located "above" the orthosteric binding site in ECD, also had a strong impact on the desensitization transitions. ...
... There is no doubt that the M2 and M3 transmembrane segments play an important role in determining GABA A R desensitization, but several reports also indicate other structural determinants, practically in all major regions of this receptor. As already mentioned in the Introduction, mutation of the α 1 F45 residue (loop G) 22 affected practically all gating features of the receptor including preactivation, opening/closing, and desensitization providing another example that structural determinants for these gating transitions are shared. Similar conclusions were drawn in our recent study on the role of the loop C (β 2 F200) 24 in which the impact on all gating characteristics, including desensitization, was reported. ...
... Typically, traces exhibited 1−3 modes of activity. 22,23,50 Event detection analysis in Clampfit 10.7 software (Molecular Devices, Sunnyvale, CA, US) was used to assess open probability (P open ) for distinct clusters, three modes for β 2 G254V = 0.66 ± 0.08, 61% (predominant); 0.90 ± 0.05, 26%; and 0.16 ± 0.12, 13%; and 3 modes for β 2 L296V = 0.20 ± 0.05, 60% (predominant); 0.41 ± 0.04, 19%; and 0.05 ± 0.01, 21%. For α 1 G258V and α 1 L300V, only one mode of activity was present, P open , for α 1 G258V = 0.33 ± 0.08 and for α 1 L300V = 0.19 ± 0.01. ...
GABA type A receptors (GABAARs) belong to the pentameric ligand-gated ion channel (pLGIC) family and play a crucial role in mediating inhibition in the adult mammalian brain. Recently, a major progress in determining the static structure of GABAARs was achieved, although precise molecular scenarios underlying conformational transitions remain unclear. The ligand binding sites (LBSs) are located at the extracellular domain (ECD), very distant from the receptor gate at the channel pore. GABAAR gating is complex, comprising three major categories of transitions: openings/closings, preactivation, and desensitization. Interestingly, mutations at, e.g., the ligand binding site affect not only binding but often also more than one gating category, suggesting that structural determinants for distinct conformational transitions are shared. Gielen and co-workers (2015) proposed that the GABAAR desensitization gate is located at the second and third transmembrane segment. However, studies of our and others’ groups indicated that other parts of the GABAAR macromolecule might be involved in this process. In the present study, we asked how selected point mutations (β2G254V, α1G258V, α1L300V, and β2L296V) at the M2 and M3 transmembrane segments affect gating transitions of the α1β2γ2 GABAAR. Using high resolution macroscopic and single-channel recordings and analysis, we report that these substitutions, besides affecting desensitization, also profoundly altered openings/closings, having some minor effect on preactivation and agonist binding. Thus, the M2 and M3 segments primarily control late gating transitions of the receptor (desensitization, opening/closing), providing a further support for the concept of diffuse gating mechanisms for conformational transitions of GABAAR.
... In particular, single-channel analysis and modeling revealed only minor and nonsignificant changes in desensitization rate constants (table shown in Figure 6), whereas macroscopic investigations strongly underscored the impact of the H55 mutation on these transitions. A similar discrepancy was observed in our previous papers, 18,20 and it was attributed primarily to profoundly different experimental conditions: nonequilibrium (macroscopic) vs steady-state (single-channel). Indeed, whereas in rapid application experiments, the vast majority of receptors desensitizes within a few milliseconds, in the single-channel recordings, the rapid desensitization may have a contribution to some components of shut times distributions, while longer sojourns in the desensitized states are not included in the clusters. ...
... We may speculate that in the case of these mutants, in highly dynamic conditions, the receptor might operate in a different mode than in the steady-state. However, when we analyzed other mutants such as α 1 F64, 18,33 β 2 E155, 21 or α 1 F45, 20 there was typically a good qualitative correspondence between gating parameters (except for desensitization) estimated from macroscopic and single-channel activity. We may speculate that in these different situations, the mutation at this critical interface domain might differentiate the modus operandi of this receptor in steady-state and dynamic conditions. ...
... Considering that the benzodiazepine binding site is located at the ECD, very distantly from transmembrane segments indicated by Gielen and co-workers, 45 it seems likely that desensitization might be additionally controlled by some structures at the ECD. Moreover, in our recent study, 20 we reported that desensitization is strongly affected by the mutation of the F45 residue, located at the loop G of the α 1 subunit, close to the agonist binding site. Even more surprisingly, in our most recent paper, 47 we report that the mutation of α 1 F14 and β 2 F31 residues, located at the "top" of the ECD, also influences the desensitization transitions. ...
The GABAA receptor is a member of the Cys-loop family and plays a crucial role in the adult mammalian brain inhibition. Although the static structure of this receptor is emerging, the molecular mechanisms underlying its conformational transitions remain elusive. It is known that in the Cys-loop receptors, the interface between extracellular and transmembrane domains plays a key role in transmitting the “activation wave” down to the channel gate in the pore. It has been previously reported that histidine 55 (H55), located centrally at the interfacial β1−β2 loop of the α1 subunit, is important in the receptor activation, but it is unknown which specific gating steps it is affecting. In the present study, we addressed this issue by taking advantage of the state-of-the-art macroscopic and single-channel recordings together with extensive modeling. Considering that H55 is known to affect the local electrostatic landscape and because it is neighbored by two negatively charged aspartates, a well conserved feature in the α subunits, we considered substitution with negative (E) and positive (K) residues. We found that these mutations markedly affected the receptor gating, altering primarily preactivation and desensitization transitions. Importantly, opposite effects were observed for these two mutations strongly suggesting involvement of electrostatic interactions. Single-channel recordings suggested also a minor effect on opening/closing transitions which did not depend on the electric charge of the substituting amino acid. Altogether, we demonstrate that H55 mutations affect primarily preactivation and desensitization most likely by influencing local electrostatic interactions at the receptor interface.
Aims
GABAA receptors belong to Cys-loop ion channel family and mediate inhibition in the brain. Despite the abundance of structural data on receptor structure, the molecular scenarios of activation are unknown. In this study we investigated the role of a β2P273 residue in channel gating transitions. This residue is located in a central position of the M2-M3 linker of the interdomain interface, expected to be predisposed to interact with another interfacial element, the β1–β2 loop of the extracellular side. The interactions occurring on this interface have been reported to couple agonist binding to channel gating.
Main methods
We recorded micro- and macroscopic current responses of recombinant GABAA receptors mutated at the β2P273 residue (to A, K, E) to saturating GABA. Electrophysiological data served as basis to kinetic modeling, used to decipher which gating transition were affected by mutations.
Key findings
Mutations of this residue impaired macroscopic desensitization and accelerated current deactivation with P273E mutant showing greatest deviation from wild-type. Single-channel analysis revealed alterations mainly in short-lived shut times and shortening of openings, resulting in dramatic changes in intraburst open probability. Kinetic modeling indicated that β2P273 mutants show diminished entry into desensitized and open states as well as faster channel closing transitions.
Significance
In conclusion, we demonstrate that β2P273 of the M2-M3 linker is a crucial element of the ECD-TMD interface regulating the receptor's ability to undergo late gating transitions. Henceforth, this region could be an important target for new pharmacological tools affecting GABAAR-mediated inhibition.
GABA A receptors (GABA A Rs) mediate the majority of fast inhibitory transmission throughout the brain. Although it is widely known that pore-forming subunits critically determine receptor function, it is unclear whether their single-channel properties are modulated by GABA A R-associated transmembrane proteins. We previously identified Shisa7 as a GABA A R auxiliary subunit that modulates the trafficking, pharmacology, and kinetic properties of these receptors. In particular, Shisa7 accelerates receptor deactivation; however, the underlying mechanisms by which Shisa7 controls GABA A R kinetics have yet to be determined. Here we have performed single-channel recordings and find that while Shisa7 does not change channel slope conductance, it reduces the frequency of openings. Importantly, Shisa7 modulates GABA A R gating by decreasing the duration and open probability (Po) within bursts. Kinetic analysis of dwell times, activation modeling, and macroscopic simulations indicate that Shisa7 accelerates GABA A R deactivation by governing the time spent between close and open states during gating. Together, our data provide a mechanistic basis for how Shisa7 controls GABA A R gating kinetics and reveal for the first time that GABA A R single-channel properties can be modulated by an auxiliary subunit. These findings shed light on processes that shape the temporal dynamics of GABAergic transmission.
GABAA receptors (GABAARs) mediate the majority of fast inhibitory transmission throughout the brain. Although it is widely known that pore-forming subunits critically determine receptor function, it is unclear whether their single-channel properties are modulated by GABAAR-associated transmembrane proteins. We previously identified Shisa7 as a GABAAR auxiliary subunit that modulates the trafficking, pharmacology, and kinetic properties of these receptors. In particular, Shisa7 accelerates receptor deactivation; however, the underlying mechanisms by which Shisa7 controls GABAAR kinetics have yet to be determined. Here we have performed single-channel recordings and find that while Shisa7 does not change channel slope conductance, it reduces the frequency of openings. Importantly, Shisa7 modulates GABAAR gating by decreasing the duration and open probability (Po) within bursts. Kinetic analysis of dwell times, activation modeling, and macroscopic simulations indicate that Shisa7 accelerates GABAAR deactivation by governing the time spent between close and open states during gating. Together, our data provide a mechanistic basis for how Shisa7 controls GABAAR gating kinetics and reveal for the first time that GABAAR single-channel properties can be modulated by an auxiliary subunit. These findings shed light on processes that shape the temporal dynamics of GABAergic transmission.
GABAA receptors (GABAARs) play a crucial role in mediating inhibition in adult mammalian brains. In the recent years, an impressive progress in revealing the static structure of GABAARs was achieved but the molecular mechanisms underlying their conformational transitions remain elusive. Phenylalanine 64 (α1F64) is located at the loop D of the orthosteric binding site of GABAAR and was found to directly interact with GABA molecule. Mutations of α1F64 were demonstrated to affect not only binding but also some gating properties. Loop D is a rigid β strand which seems to be particularly suitable to convey activatory signaling from the ligand binding site (LBS) to the gate at the channel pore. To test this scenario, we have investigated the substitution of α1F64 with glycine, the smallest amino acid, widely recognized as a rigidity "reducer" of protein structures. To this end, we assessed the impact of the α1F64G mutation in the α1β2γ2L type of GABAARs on gating properties by analyzing both macroscopic responses to rapid agonist applications and single-channel currents. We found that this substitution dramatically altered all gating features of the receptor (opening/closing, preactivation and desensitization) which contrasts with markedly weaker effects of previously considered substitutions (α1F64L and α1F64A). In particular, α1F64G mutation practically abolished the desensitization process. At the same time, the α1F64G mutant maintained gating integrity manifested as single-channel activity in the form of clusters. We conclude that rigidity of the loop D plays a crucial role in conveying the activation signal from the LBS to the channel gate.
GABA type A receptor plays a key role in inhibitory signaling in the adult central nervous system. This receptor can be modulated by protons but the underlying molecular mechanisms have not been fully explored. To find possible pH-sensor residues, a comparative study for proton-activated GLIC channel and α1β2γ2 GABA receptor was performed and pK 's of respective residues were estimated by numerical algorithms which consider local interactions. β E155, located at the GABA binding site, showed pKa values close to physiological values and dependence on the receptor state and ligation, suggesting a role in modulation by pH. To validate this prediction, pH sensitivity of current responses to GABA was investigated using patch-clamp technique for WT and mutated (β2E155[C, S, Q, L]) GABA receptors. Cysteine mutation preserved pH sensitivity. However, for remaining mutants, the sensitivity to acidification (pH = 6.0) was reduced becoming not statistically significant. The effect of alkaline pH (8.0) was maintained for all mutants with exception for β2E155L for which it was nearly abolished. To further explore the impact of considered mutations, molecular docking was performed which indicated that pH modulation is probably affected by interplay between binding site residues, zwitterion GABA and protons. These data, altogether, indicate that mutation of β2E155 to hydrophobic residue (L) maximally impaired pH modulation while for polar substitutions the effect was smaller. In conclusion, our data provide evidence that a key binding site residue β2E155 plays an important role in proton sensitivity of GABA receptor.
Pentameric ligand gated ion channels (pLGICs) are crucial in electrochemical signaling but exact molecular mechanisms of their activation remain elusive. So far, major effort focused on the top-down molecular pathway between the ligand binding site and the channel gate. However, recent studies revealed that pLGIC activation is associated with coordinated subunit twisting in the membrane plane. This suggests a key role of intersubunit interactions but the underlying mechanisms remain largely unknown. Herein, we investigated a “peripheral” subunit interface region of GABAA receptor where structural modeling indicated interaction between N-terminal α1F14 and β2F31 residues. Our experiments underscored a crucial role of this interaction in ligand binding and gating, especially preactivation and opening, showing that the intersubunit cross-talk taking place outside (above) the top-down pathway can be strongly involved in receptor activation. Thus, described here intersubunit interaction appears to operate across a particularly long distance, affecting vast portions of the macromolecule.
