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Cys-loop receptors are important drug targets that are involved in signaling in the nervous system. The binding of neurotransmitters in the extracellular region of these receptors triggers an allosteric activation mechanism, the full details of which remain elusive, although structurally flexible loops in the interface between the extracellular region of Cys-loop receptors and the pore-forming transmembrane domain are known to play an important role. Here we explore the roles of three largely conserved Pro residues in two of these loops, the Cys-loop and M2-M3 loop, in 5-HT3A and α7 nACh receptors. The data from natural and noncanonical amino acid mutagenesis suggest that in both proteins a Pro is essential in the Cys-loop, probably because of its enhanced ability to form a cis peptide bond, although other factors are also involved. The important characteristics of Pros in the M2-M3 loop, however, differ in these two receptors: in the 5-HT3 receptor, the Pros can be replaced by some charged amino acids resulting in EC50s similar to those of wild-type receptors, while such substitutions in the nACh receptor ablate function. Ala substitution at one of these Pros also has different effects in the two receptors. Thus, our data show that even highly conserved residues can have distinct behaviors in related Cys-loop receptors.

Cys-loop receptors are pentameric ligand-gated ion channels that facilitate communication within the nervous system. Upon neurotransmitter binding, these receptors undergo an allosteric activation mechanism connecting the binding event to the membrane-spanning channel pore, which expands to conduct ions. Some of the earliest steps in this activation mechanism are carried out by residues proximal to the binding site, the relative positioning of which may reflect functional differences among members of the Cys-loop family of receptors. Herein, we investigated key side-chain interactions near the binding site via mutagenesis and two-electrode voltage-clamp electrophysiology in serotonin-gated 5-HT_(3A) receptors (5-HT_(3A)Rs) and nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus laevis oocytes. We found that a triad of residues aligning to Thr-152, Glu-209, and Lys-211 in the 5-HT_(3A)R can be exchanged between the homomeric 5-HT_(3A)R and the muscle-type nAChR α-subunit with small functional consequences. Via triple mutant cycle analysis, we demonstrated that this triad forms an interdependent network in the muscle-type nAChR. Furthermore, nAChR-type mutations of the 5-HT_(3A)R affect the affinity of nicotine, a competitive antagonist of 5-HT_(3A)Rs, in a cooperative manner. Using mutant cycle analyses between the 5-HT_(3A) triad, loop A residues Asn-101 and Glu-102, β9 residue Lys-197, and the channel gate at Thr-257, we observed that residues in this region are energetically linked to the channel gate and are particularly sensitive to mutations that introduce a net positive charge. This study expands our understanding of the differences and similarities in the activation mechanisms of Cys-loop receptors.

Erwinia ligand-gated ion channel (ELIC) is a bacterial homologue of vertebrate pentameric ligand-gated ion channels (pLGICs) and has proven to be a valuable model for understanding the structure and function of this important protein family. There is nevertheless still a question about whether molecular details can be accurately extrapolated from this protein to those found in eukaryotes. Here we explore the role of proline residues (Pros) in ELIC by creating mutant receptors, expressing them in Xenopus laevis oocytes, and using whole-cell voltage-clamp electrophysiology to monitor channel activity. In contrast to eukaryotic pLGICs, proline-to-alanine (Pro-to-Ala) substitution in ELIC mostly resulted in gain of function, and even altering highly conserved Pro residues in M1 and the M2-M3 loop did not ablate function. These substitutions also mostly resulted in ablation of the modulation by Ca2+ observed in wild-type receptors. Substitution of the Pro in the "Cys loop", however, did result in nonfunctional receptors. Probing this residue with noncanonical amino acids revealed a requirement for a substituted amine at this position, as well as a general preference for Pro analogues with greater intrinsic cis biases. We propose there is likely a cis bond at the apex of the Cys loop in this protein, which is consistent with some, but not all, findings from other pLGICs. Overall, the data show that the roles of proline residues are less critical in ELIC than in other pLGICs, supporting other studies that suggest caution must be applied in using data from this prokaryotic receptor to understand molecular details of eukaryotic pLGIC receptor function., This work was supported by National Institutes of Health Grant NS-34407 (D.A.D.) and MRC Grant MR L021676 (S.C.R.L.).

Cys-loop receptors are pentameric ligand-gated ion channels that facilitate communication within the nervous system. Upon neurotransmitter binding, these receptors undergo an allosteric activation mechanism connecting the binding event to the membrane-spanning channel pore, which expands to conduct ions. Some of the earliest steps in this activation mechanism are carried out by residues proximal to the binding site, the relative positioning of which may reflect functional differences among members of the Cys-loop family of receptors. Herein, we investigated key side-chain interactions near the binding site via mutagenesis and two-electrode voltage-clamp electrophysiology in serotonin-gated 5-HT(3A) receptors (5-HT(3A)Rs) and nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus laevis oocytes. We found that a triad of residues aligning to Thr-152, Glu-209, and Lys-211 in the 5-HT(3A)R can be exchanged between the homomeric 5-HT(3A)R and the muscle-type nAChR α-subunit with small functional consequences. Via triple mutant cycle analysis, we demonstrated that this triad forms an interdependent network in the muscle-type nAChR. Furthermore, nAChR-type mutations of the 5-HT(3A)R affect the affinity of nicotine, a competitive antagonist of 5-HT(3A)Rs, in a cooperative manner. Using mutant cycle analyses between the 5-HT(3A) triad, loop A residues Asn-101 and Glu-102, β9 residue Lys-197, and the channel gate at Thr-257, we observed that residues in this region are energetically linked to the channel gate and are particularly sensitive to mutations that introduce a net positive charge. This study expands our understanding of the differences and similarities in the activation mechanisms of Cys-loop receptors.

Pentameric ligand gated ion channels (pLGIC) mediate fast synaptic transmission in animals, where they are known as Cys-loop receptors. Prokaryotic homologues of these proteins have been found in bacteria, although these do not possess the eponymous Cys-loop. Each receptor is constituted of five subunits which surround a central pore. A detailed analysis of sequences of all these subunits reveals that only the Pro in the Cys-loop is absolutely conserved; this has led to the proposal (which has not been widely adopted) that these receptors should be renamed ‘Pro-loop’ receptors. In addition to this Pro, there are 3 other Pros that are largely conserved and have been shown to play important but different roles. Here we combine old and new data to compare the characteristics of these Pro residues that allow them to have these distinct roles. Pro has a unique chemistry in proteins because it lacks a backbone hydrogen bond, and has a stronger cis-bias, and greater steric bulk at the amine than other amino acids. Determining which of these properties is essential at which location in Cys-loop receptors can only be accomplished using proline analogues, i.e non-conventional amino acids. These were inserted using nonsense suppression into nACh and 5-HT_3 receptors and receptor characteristics were determined using two electrode voltage clamp in Xenopus oocytes. The data reveal some similarities but also some differences in the roles of Pro at equivalent positions, and identify differences in the specific Pro properties that allow these two related Cys-loop receptors to function.