Mutations at the M2 and M3 Transmembrane Helices of the GABA_ARs α₁ and β₂ Subunits Affect Primarily Late Gating Transitions Including Opening/Closing and Desensitization

Abstract

GABA type A receptors (GABA A Rs) 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 GABA A Rs 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. GABA A R 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 GABA A R desensitization gate is located at the second and third transmembrane segment. However, studies of our and others’ groups indicated that other parts of the GABA A R macromolecule might be involved in this process. In the present study, we asked how selected point mutations (β 2 G254V, α 1 G258V, α 1 L300V, and β 2 L296V) at the M2 and M3 transmembrane segments affect gating transitions of the α 1 β 2 γ 2 GABA A R. 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 GABA A R.

α1 Proline 277 Residues Regulate GABAAR Gating through M2-M3 Loop Interaction in the Interface Region

“…A minor involvement of α 1 P277 on the agonist binding is not surprising considering a large distance between this residue and the binding cassette. Similarly, our previous studies on substitutions at residues distant from the orthosteric binding site, in the ECD-TMD interface or in the transmembrane domain, also reported the weak impact of these mutations on the agonist binding. On the other hand, it is worth noting that dose–response relationships for point mutations of the H56 residue (numbering derived for the cDNA coding α 1 subunit for humans), which is in close proximity to P277, were characterized by shifts correlated with the side-chain charge: leftward for lysine and rightward for glutamate .…”

“…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, , estimations for d and r rate constants from stationary single-channel 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 C.…”

“…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 ), 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 B). This is intriguing because, as discussed in our previous reports, ,,,, macroscopic recordings of responses to rapid applications of the saturating agonist offer optimal conditions to reveal the desensitization onset, whereas steady-state 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 single-channel recordings (see Table and Figure B).…”

“…It is worth emphasizing that a similar modus operandi has been observed also for a number of other residues throughout the structure of the GABA A R macromolecule. Indeed, mutations of, e.g., binding site α 1 F64, β2F200, , peripheral α 1 F14, β2F31, interface-located α 1 H55, and transmembrane M2 and M3 helices β2G254V, α 1 G258V, α 1 L300V, and β2L296V affected most of the gating transitions. 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. ,,− ,, This feature of desensitization led us to propose the concept of a “diffuse desensitization gate” , as opposed to the desensitization gate largely restricted to the receptor’s transmembrane domains .…”

“…Cys-loop receptors have transmitter binding sites located on the extracellular domain (ECD), which are particularly distant (around 50 Å) from the channel gate situated roughly in the middle of the pore in the transmembrane domain (TMD) . Thus, activation of the Cys-loop receptors is believed to occur as a consequence of a complex “wave” of structural changes within the receptor macromolecule that starts upon ligand binding and eventually leads to channel pore opening. − The activation process of the Cys-loop receptor was best described for the nicotinic acetylcholine receptors. − Much less is known about anion-selective GABA A receptors, which in an adult mammalian brain mediate inhibition . Importantly, GABA A Rs are targets for many clinically important pharmacological agents such as benzodiazepines, barbiturates, or anesthetics. − Several studies dedicated to molecular mechanisms of GABA A R activation have shown that mutation of residues located near the GABA binding site affected not only the binding step but also channel gating. − These results suggest that the mechanism of the GABA A R activation involving energy transfer from the binding side to the channel gate might occur in a form of the widespread structural rearrangements as it was proposed for nAChRs .…”