Modulation of gain-of-function α6*-nicotinic acetylcholine receptor by β3 subunits

We previously have shown that β3 subunits either eliminate (e.g. for all-human (h) or all-mouse (m) α6β4β3-nAChR) or potentiate (e.g. for hybrid mα6hβ4hβ3- or mα6mβ4hβ3-nAChR containing subunits from different species) function of α6*-nAChR expressed in Xenopus oocytes, and that nAChR hα6 subunit residues Asn-143 and Met-145 in N-terminal domain loop E are important for dominant-negative effects of nAChR hβ3 subunits on hα6*-nAChR function. Here, we tested the hypothesis that these effects of β3 subunits would be preserved even if nAChR α6 subunits harbored gain-of-function, leucine- or valine-to-serine mutations at 9' or 13' positions (L9'S or V13'S) in their second transmembrane domains, yielding receptors with heightened functional activity and more amenable to assessment of effects of β3 subunit incorporation. However, coexpression with β3 subunits potentiates rather than suppresses function of all-human, all-mouse, or hybrid α6((L9'S or V13'S))β4*- or α6(N143D+M145V)(L9'S)β2*-nAChR. This contrasts with the lack of consistent function when α6((L9'S or V13'S)) and β2 subunits are expressed alone or in the presence of wild-type β3 subunits. These results provide evidence that gain-of-function hα6hβ2*-nAChR (i.e. hα6(N143D+M145V)(L9'S)hβ2hβ3 nAChR) could be produced in vitro. These studies also indicate that nAChR β3 subunits can be assembly partners in functional α6*-nAChR and that 9' or 13' mutations in the nAChR α6 subunit second transmembrane domain can act as gain-of-function and/or reporter mutations. Moreover, our findings suggest that β3 subunit coexpression promotes function of α6*-nAChR.     

Creating an α7 nicotinic acetylcholine recognition domain from the acetylcholine-binding protein: crystallographic and ligand selectivity analyses

Determining the structure of the ligand-binding domain of the nicotinic acetylcholine receptor (nAChR) has been a long standing goal in the design of selective drugs useful in implicated diseases for this prevalent receptor family. Acetylcholine-binding proteins have proven to be valuable surrogates with structural similarity and sequence identity to the extracellular domain of the nicotinic receptor, yet these soluble proteins have their unique features and do not serve as exact replicates of the nAChRs of interest. Here we systematically modify the sequence of these proteins toward the homomeric human α7 nAChR. These chimeric proteins exhibit a shift in affinities to reflect α7 binding characteristics yet maintain expression levels and stability conducive for crystallization. We also present a pentameric humanoid nAChR extracellular domain with the structural determination of the α7 nAChR glycosylation site.     

Structural link between γ-aminobutyric acid type A (GABAA) receptor agonist binding site and inner β-sheet governs channel activation and allosteric drug modulation

Rapid opening and closing of pentameric ligand-gated ion channels (pLGICs) regulate information flow throughout the brain. For pLGICs, it is postulated that neurotransmitter-induced movements in the extracellular inner β-sheet trigger channel activation. Homology modeling reveals that the β4-β5 linker physically connects the neurotransmitter binding site to the inner β-sheet. Inserting 1, 2, 4, and 8 glycines in this region of the GABA(A) receptor β-subunit progressively decreases GABA activation and converts the competitive antagonist SR-95531 into a partial agonist, demonstrating that this linker is a key element whose length and flexibility are optimized for efficient signal propagation. Insertions in the α- and γ-subunits have little effect on GABA or SR-95531 actions, suggesting that asymmetric motions in the extracellular domain power pLGIC gating. The effects of insertions on allosteric modulator actions, pentobarbital, and benzodiazepines, have different subunit dependences, indicating that modulator-induced signaling is distinct from agonist gating.     

Single Expressed Glycine Receptor Domains Reconstitute Functional Ion Channels without Subunit-specific Desensitization Behavior

Cys loop receptors are pentameric arrangements of independent subunits that assemble into functional ion channels. Each subunit shows a domain architecture. Functional ion channels can be reconstituted even from independent, nonfunctional subunit domains, as shown previously for GlyRα1 receptors. Here, we demonstrate that this reconstitution is not restricted to α1 but can be transferred to other members of the Cys loop receptor family. A nonfunctional GlyR subunit, truncated at the intracellular TM3–4 loop by a premature stop codon, can be complemented by co-expression of the missing tail portion of the receptor. Compared with α1 subunits, rescue by domain complementation was less efficient when GlyRα3 or the GABA_A/C subunit ρ1 was used. If truncation disrupted an alternative splicing cassette within the intracellular TM3–4 loop of α3 subunits, which also regulates receptor desensitization, functional rescue was not possible. When α3 receptors were restored by complementation using domains with and without the spliced insert, no difference in desensitization was found. In contrast, desensitization properties could even be transferred between α1/α3 receptor chimeras harboring or lacking the α3 splice cassette proving that functional rescue depends on the integrity of the alternative splicing cassette in α3. Thus, an intact α3 splicing cassette in the TM3–4 loop environment is indispensable for functional rescue, and the quality of receptor restoration can be assessed from desensitization properties.     

Potent and selective inhibition of a single alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit by an RNA aptamer

Inhibitors of AMPA-type glutamate ion channels are useful as biochemical probes for structure-function studies and as drug candidates for a number of neurological disorders and diseases. Here, we describe the identification of an RNA inhibitor or aptamer by an in vitro evolution approach and a characterization of its mechanism of inhibition on the sites of interaction by equilibrium binding and on the receptor channel opening rate by a laser-pulse photolysis technique. Our results show that the aptamer is a noncompetitive inhibitor that selectively inhibits the GluA2Q(flip) AMPA receptor subunit without any effect on other AMPA receptor subunits or kainate or NMDA receptors. On the GluA2 subunit, this aptamer preferentially inhibits the flip variant. Furthermore, the aptamer preferentially inhibits the closed-channel state of GluA2Q(flip) with a K(I) = 1.5 μM or by ～15-fold over the open-channel state. The potency and selectivity of this aptamer rival those of small molecule inhibitors. Together, these properties make this aptamer a promising candidate for the development of water-soluble, highly potent, and GluA2 subunit-selective drugs.     

Elucidation of Molecular Impediments in the α6 Subunit for in Vitro Expression of Functional α6β4* Nicotinic Acetylcholine Receptors

Explorations into the α6-containing nicotinic acetylcholine receptors (α6* nAChRs) as putative drug targets have been severely hampered by the inefficient functional expression of the receptors in heterologous expression systems. In this study, the molecular basis for the problem was investigated through the construction of chimeric α6/α3 and mutant α3 and α6 subunits and functional characterization of these co-expressed with β4 or β4β3 subunits in tsA201 cells in a fluorescence-based assay and in Xenopus oocytes using two-electrode voltage clamp electrophysiology. Substitution of a small C-terminal segment in the second intracellular loop or the Phe²²³ residue in transmembrane helix 1 of α6 with the corresponding α3 segment or residue was found to enhance α6β4 functionality in tsA201 cells significantly, in part due to increased cell surface expression of the receptors. The gain-of-function effects of these substitutions appeared to be additive since incorporation of both α3 elements into α6 resulted in assembly of α6β4* receptors exhibiting robust functional responses to acetylcholine. The pharmacological properties exhibited by α6β4β3 receptors comprising one of these novel α6/α3 chimeras in oocytes were found to be in good agreement with those from previous studies of α6* nAChRs formed from other surrogate α6 subunits or concatenated subunits and studies of other heteromeric nAChRs. In contrast, co-expression of this α6/α3 chimera with β2 or β2β3 subunits in oocytes did not result in efficient formation of functional receptors, indicating that the identified molecular elements in α6 could be specific impediments for the expression of functional α6β4* nAChRs.     

Potential state-selective hydrogen bond formation can modulate activation and desensitization of the α7 nicotinic acetylcholine receptor

A series of arylidene anabaseines were synthesized to probe the functional impact of hydrogen bonding on human α7 nicotinic acetylcholine receptor (nAChR) activation and desensitization. The aryl groups were either hydrogen bond acceptors (furans), donors (pyrroles), or neither (thiophenes). These compounds were tested against a series of point mutants of the ligand-binding domain residue Gln-57, a residue hypothesized to be proximate to the aryl group of the bound agonist and a putative hydrogen bonding partner. Q57K, Q57D, Q57E, and Q57L were chosen to remove the dual hydrogen bonding donor/acceptor ability of Gln-57 and replace it with hydrogen bond donating, hydrogen bond accepting, or nonhydrogen bonding ability. Activation of the receptor was compromised with hydrogen bonding mismatches, for example, pairing a pyrrole with Q57K or Q57L, or a furan anabaseine with Q57D or Q57E. Ligand co-applications with the positive allosteric modulator PNU-120596 produced significantly enhanced currents whose degree of enhancement was greater for 2-furans or -pyrroles than for their 3-substituted isomers, whereas the nonhydrogen bonding thiophenes failed to show this correlation. Interestingly, the PNU-120596 agonist co-application data revealed that for wild-type α7 nAChR, the 3-furan desensitized state was relatively stabilized compared with that of 2-furan, a reversal of the relationship observed with respect to the barrier for entry into the desensitized state. These data highlight the importance of hydrogen bonding on the receptor-ligand state, and suggest that it may be possible to fine-tune features of agonists that mediate state selection in the nAChR.     

Intersubunit bridge formation governs agonist efficacy at nicotinic acetylcholine α4β2 receptors: unique role of halogen bonding revealed

The α4β2 subtype of the nicotinic acetylcholine receptor has been pursued as a drug target for treatment of psychiatric and neurodegenerative disorders and smoking cessation aids for decades. Still, a thorough understanding of structure-function relationships of α4β2 agonists is lacking. Using binding experiments, electrophysiology and x-ray crystallography we have investigated a consecutive series of five prototypical pyridine-containing agonists derived from 1-(pyridin-3-yl)-1,4-diazepane. A correlation between binding affinities at α4β2 and the acetylcholine-binding protein from Lymnaea stagnalis (Ls-AChBP) confirms Ls-AChBP as structural surrogate for α4β2 receptors. Crystal structures of five agonists with efficacies at α4β2 from 21-76% were determined in complex with Ls-AChBP. No variation in closure of loop C is observed despite large efficacy variations. Instead, the efficacy of a compound appears tightly coupled to its ability to form a strong intersubunit bridge linking the primary and complementary binding interfaces. For the tested agonists, a specific halogen bond was observed to play a large role in establishing such strong intersubunit anchoring.     

The interaction between two extracellular linker regions controls sustained opening of acid-sensing ion channel 1

Activation of acid-sensing ion channels (ASICs) contributes to neuronal death during stroke, to axonal degeneration during neuroinflammation, and to pain during inflammation. Although understanding ASIC gating may help to modulate ASIC activity during these pathologic situations, at present it is poorly understood. The ligand, H(+), probably binds to several sites, among them amino acids within the large extracellular domain. The extracellular domain is linked to the two transmembrane domains by the wrist region that is connected to two anti-parallel β-strands, β1 and β12. Thus, the wrist region together with those β-strands may have a crucial role in transmitting ligand binding to pore opening and closing. Here we show that amino acids in the β1-β2 linker determine constitutive opening of ASIC1b from shark. The most crucial residue within the β1-β2 linker (Asp(110)), when mutated from aspartate to cysteine, can be altered by cysteine-modifying reagents much more readily when channels are closed than when they are desensitized. Finally, engineering of a cysteine at position 110 and at an adjacent position in the β11-β12 linker leads to spontaneous formation of a disulfide bond that traps the channel in the desensitized conformation. Collectively, our results suggest that the β1-β2 and β11-β12 linkers are dynamic during gating and tightly appose to each other during desensitization gating. Hindrance of this tight apposition leads to reopening of the channel. It follows that the β1-β2 and β11-β12 linkers modulate gating movements of ASIC1 and may thus be drug targets to modulate ASIC activity.     

The N-terminal Domain Modulates α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) Receptor Desensitization

AMPA receptors are tetrameric glutamate-gated ion channels that mediate fast synaptic neurotransmission in mammalian brain. Their subunits contain a two-lobed N-terminal domain (NTD) that comprises over 40% of the mature polypeptide. The NTD is not obligatory for the assembly of tetrameric receptors, and its functional role is still unclear. By analyzing full-length and NTD-deleted GluA1–4 AMPA receptors expressed in HEK 293 cells, we found that the removal of the NTD leads to a significant reduction in receptor transport to the plasma membrane, a higher steady state-to-peak current ratio of glutamate responses, and strongly increased sensitivity to glutamate toxicity in cell culture. Further analyses showed that NTD-deleted receptors display both a slower onset of desensitization and a faster recovery from desensitization of agonist responses. Our results indicate that the NTD promotes the biosynthetic maturation of AMPA receptors and, for membrane-expressed channels, enhances the stability of the desensitized state. Moreover, these findings suggest that interactions of the NTD with extracellular/synaptic ligands may be able to fine-tune AMPA receptor-mediated responses, in analogy with the allosteric regulatory role demonstrated for the NTD of NMDA receptors.     

Proton-mediated Conformational Changes in an Acid-sensing Ion Channel

Acid-sensing ion channels are cation channels activated by external protons and play roles in nociception, synaptic transmission, and the physiopathology of ischemic stroke. Using luminescence resonance energy transfer (LRET), we show that upon proton binding, there is a conformational change that increases LRET efficiency between the probes at the thumb and finger subdomains in the extracellular domain of acid-sensing ion channels. Additionally, we show that this conformational change is lost upon mutating Asp-238, Glu-239, and Asp-260, which line the finger domains, to alanines. Electrophysiological studies showed that the single mutant D260A shifted the EC₅₀ by 0.2 pH units, the double mutant D238A/E239A shifted the EC₅₀ by 2.5 pH units, and the triple mutant D238A/E239A/D260A exhibited no response to protons despite surface expression. The LRET experiments on D238A/E239A/D260A showed no changes in LRET efficiency upon reduction in pH from 8 to 6. The LRET and electrophysiological studies thus suggest that the three carboxylates, two of which are involved in carboxyl/carboxylate interactions, are essential for proton-induced conformational changes in the extracellular domain, which in turn are necessary for receptor activation.     

Mechanism of AMPA receptor activation by partial agonists: disulfide trapping of closed lobe conformations

The mechanism by which agonist binding to an ionotropic glutamate receptor leads to channel opening is a central issue in molecular neurobiology. Partial agonists are useful tools for studying the activation mechanism because they produce full channel activation with lower probability than full agonists. Structural transitions that determine the efficacy of partial agonists can provide information on the trigger that begins the channel-opening process. The ligand-binding domain of AMPA receptors is a bilobed structure, and the closure of the lobes is associated with channel activation. One possibility is that partial agonists sterically block full lobe closure but that partial degrees of closure trigger the channel with a lower probability. Alternatively, full lobe closure may be required for activation, and the stability of the fully closed state could determine efficacy with the fully closed state having a lower stability when bound to partial relative to full agonists. Disulfide-trapping experiments demonstrated that even extremely low efficacy ligands such as 6-cyano-7-nitroquinoxaline-2,3-dione can produce a full lobe closure, presumably with low probability. The results are consistent the hypothesis that the efficacy is determined at least in part by the stability of the state in which the lobes are fully closed.     

Opposite Effects of KCTD Subunit Domains on GABAB Receptor-mediated Desensitization

GABA_B receptors assemble from principle and auxiliary subunits. The principle subunits GABA_B1 and GABA_B2 form functional heteromeric GABA_B(1,2) receptors that associate with homotetramers of auxiliary KCTD8, -12, -12b, or -16 (named after their K⁺ channel tetramerization domain) subunits. These auxiliary subunits constitute receptor subtypes with distinct functional properties. KCTD12 and -12b generate desensitizing receptor responses while KCTD8 and -16 generate largely non-desensitizing receptor responses. The structural elements of the KCTDs underlying these differences in desensitization are unknown. KCTDs are modular proteins comprising a T1 tetramerization domain, which binds to GABA_B2, and a H1 homology domain. KCTD8 and -16 contain an additional C-terminal H2 homology domain that is not sequence-related to the H1 domains. No functions are known for the H1 and H2 domains. Here we addressed which domains and sequence motifs in KCTD proteins regulate desensitization of the receptor response. We found that the H1 domains in KCTD12 and -12b mediate desensitization through a particular sequence motif, T/NFLEQ, which is not present in the H1 domains of KCTD8 and -16. In addition, the H2 domains in KCTD8 and -16 inhibit desensitization when expressed C-terminal to the H1 domains but not when expressed as a separate protein in trans. Intriguingly, the inhibitory effect of the H2 domain is sequence-independent, suggesting that the H2 domain sterically hinders desensitization by the H1 domain. Evolutionary analysis supports that KCTD12 and -12b evolved desensitizing properties by liberating their H1 domains from antagonistic H2 domains and acquisition of the T/NFLEQ motif.     

A Conserved Salt Bridge between Transmembrane Segments 1 and 10 Constitutes an Extracellular Gate in the Dopamine Transporter

Neurotransmitter transporters play an important role in termination of synaptic transmission by mediating reuptake of neurotransmitter, but the molecular processes behind translocation are still unclear. The crystal structures of the bacterial homologue, LeuT, provided valuable insight into the structural and dynamic requirements for substrate transport. These structures support the existence of gating domains controlling access to a central binding site. On the extracellular side, access is controlled by the “thin gate” formed by an interaction between Arg-30 and Asp-404. In the human dopamine transporter (DAT), the corresponding residues are Arg-85 and Asp-476. Here, we present results supporting the existence of a similar interaction in DAT. The DAT R85D mutant has a complete loss of function, but the additional insertion of an arginine in opposite position (R85D/D476R), causing a charge reversal, results in a rescue of binding sites for the cocaine analogue [³H]CFT. Also, the coordination of Zn²⁺ between introduced histidines (R85H/D476H) caused a ∼2.5-fold increase in [³H]CFT binding (B_max). Importantly, Zn²⁺ also inhibited [³H]dopamine transport in R85H/D476H, suggesting that a dynamic interaction is required for the transport process. Furthermore, cysteine-reactive chemistry shows that mutation of the gating residues causes a higher proportion of transporters to reside in the outward facing conformation. Finally, we show that charge reversal of the corresponding residues (R104E/E493R) in the serotonin transporter also rescues [³H](S)-citalopram binding, suggesting a conserved feature. Taken together, these data suggest that the extracellular thin gate is present in monoamine transporters and that a dynamic interaction is required for substrate transport.     

Role of Conformational Dynamics in α-Amino-3-hydroxy-5-methylisoxazole-4-propionic Acid (AMPA) Receptor Partial Agonism

We have investigated the range of cleft closure conformational states that the agonist-binding domains of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors occupy when bound to a series of willardiine derivatives using single-molecule FRET. These studies show that the agonist-binding domain exhibits varying degrees of dynamics when bound to the different willardiines with differing efficacies. The chlorowillardiine- and nitrowillardiine-bound form of the agonist-binding domain probes a narrower range of cleft closure states relative to the iodowillardiine bound form of the protein, with the antagonist (αS)-α-amino-3-[(4-carboxyphenyl)methyl]-3,4-dihydro-2,4-dioxo-1(2H)-pyrimidinepropanoic acid (UBP-282)-bound form exhibiting the widest range of cleft closure states. Additionally, the average cleft closure follows the order UBP-282 > iodowillardiine > chlorowillardiine > nitrowillardiine-bound forms of agonist-binding domain. These single-molecule FRET data, along with our previously reported data for the glutamate-bound forms of wild type and T686S mutant proteins, show that the mean currents under nondesensitizing conditions can be directly correlated to the fraction of the agonist-binding domains in the “closed” cleft conformation. These results indicate that channel opening in the AMPA receptors is controlled by both the ability of the agonist to induce cleft closure and the dynamics of the agonist-binding domain when bound to the agonist.     

Structural characterization of binding mode of smoking cessation drugs to nicotinic acetylcholine receptors through study of ligand complexes with acetylcholine-binding protein

Smoking cessation is an important aim in public health worldwide as tobacco smoking causes many preventable deaths. Addiction to tobacco smoking results from the binding of nicotine to nicotinic acetylcholine receptors (nAChRs) in the brain, in particular the α4β2 receptor. One way to aid smoking cessation is by the use of nicotine replacement therapies or partial nAChR agonists like cytisine or varenicline. Here we present the co-crystal structures of cytisine and varenicline in complex with Aplysia californica acetylcholine-binding protein and use these as models to investigate binding of these ligands binding to nAChRs. This analysis of the binding properties of these two partial agonists provides insight into differences with nicotine binding to nAChRs. A mutational analysis reveals that the residues conveying subtype selectivity in nAChRs reside on the binding site complementary face and include features extending beyond the first shell of contacting residues.     

Gating-induced Conformational Rearrangement of the γ-Aminobutyric Acid Type A Receptor β-α Subunit Interface in the Membrane-spanning Domain

GABA(A) receptors mediate fast inhibitory synaptic transmission. The transmembrane ion channel is lined by a ring of five α helices, M2 segments, one from each subunit. An outer ring of helices comprising the alternating M1, M3, and M4 segments from each subunit surrounds the inner ring and forms the interface with the lipid bilayer. The structural rearrangements that follow agonist binding and culminate in opening of the ion pore remain incompletely characterized. Propofol and other intravenous general anesthetics bind at the βM3-αM1 subunit interface. We sought to determine whether this region undergoes conformational changes during GABA activation. We measured the reaction rate of p-chloromercuribenzenesulfonate (pCMBS) with cysteines substituted in the GABA(A) receptor α1M1 and β2M3 segments. In the presence of GABA, the pCMBS reaction rate increased significantly in a cluster of residues in the extracellular third of the α1M1 segment facing the β2M3 segment. Mutation of the β2M2 segment 19' position, R269Q, altered the pCMBS reaction rate with several α1M1 Cys, some only in the resting state and others only in the GABA-activated state. Thus, β2R269 is charged in both states. GABA activation induced disulfide bond formation between β2R269C and α1I228C. The experiments demonstrate that α1M1 moves in relationship to β2M2R269 during gating. Thus, channel gating does not involve rigid body movements of the entire transmembrane domain. Channel gating causes changes in the relative position of transmembrane segments both within a single subunit and relative to the neighboring subunits.     

Interactions among Positions in the Third and Fourth Membrane-associated Domains at the Intersubunit Interface of the N-Methyl-D-aspartate Receptor Forming Sites of Alcohol Action

The N-methyl-d-aspartate (NMDA) glutamate receptor is a major target of ethanol in the brain. Previous studies have identified positions in the third and fourth membrane-associated (M) domains of the NMDA receptor GluN1 and GluN2A subunits that influence alcohol sensitivity. The predicted structure of the NMDA receptor, based on that of the related GluA2 subunit, indicates a close apposition of the alcohol-sensitive positions in M3 and M4 between the two subunit types. We tested the hypothesis that these positions interact to regulate receptor kinetics and ethanol sensitivity by using dual substitution mutants. In single-substitution mutants, we found that a position in both subunits adjacent to one previously identified, GluN1(Gly-638) and GluN2A(Phe-636), can strongly regulate ethanol sensitivity. Significant interactions affecting ethanol inhibition and receptor deactivation were observed at four pairs of positions in GluN1/GluN2A: Gly-638/Met-823, Phe-639/Leu-824, Met-818/Phe-636, and Leu-819/Phe-637; the latter pair also interacted with respect to desensitization. Two interactions involved a position in M4 of both subunits, GluN1(Met-818) and GluN2A(Leu-824), that does not by itself alter ethanol sensitivity, whereas a previously identified ethanol-sensitive position, GluN2A(Ala-825), did not unequivocally interact with any other position tested. These results also indicate a shift by one position of the predicted alignment of the GluN1 M4 domain. These findings have allowed for the refinement of the NMDA receptor M domain structure, demonstrate that this region can influence apparent agonist affinity, and support the existence of four sites of alcohol action on the NMDA receptor, each consisting of five amino acids at the M3-M4 domain intersubunit interfaces.