Qiang Shan

A single β subunit M2 domain residue controls the picrotoxin sensitivity of αβ heteromeric glycine receptor chloride channels

This study investigated the residues responsible for the reduced picrotoxin sensitivity of the αβ heteromeric glycine receptor relative to the α homomeric receptor. By analogy with structurally related receptors, the β subunit M2 domain residues P278 and F282 were considered the most likely candidates for mediating this effect. These residues align with G254 and T258 of the α subunit. The T258A, T258C and T258F mutations dramatically reduced the picrotoxin sensitivity of the α homomeric receptor. Furthermore, the converse F282T mutation in the β subunit increased the picrotoxin sensitivity of the αβ heteromeric receptor. The P278G mutation in the β subunit did not affect the picrotoxin sensitivity of the αβ heteromer. Thus, a ring of five threonines at the M2 domain depth corresponding to α subunit T258 is specifically required for picrotoxin sensitivity. Mutations to α subunit T258 also profoundly influenced the apparent glycine affinity. A substituted cysteine accessibility analysis revealed that the T258C sidechain increases its pore exposure in the channel open state. This provides further evidence for an allosteric mechanism of picrotoxin inhibition, but renders it unlikely that picrotoxin (as an allosterically acting ‘competitive’ antagonist) binds to this residue.

Type 1 Adenylyl Cyclase Is Essential for Maintenance of Remote Contextual Fear Memory

Although molecular mechanisms for hippocampus-dependent memory have been extensively studied, much less is known about signaling events important for remote memory. Here we report that mice lacking type 1 adenylyl cyclase (AC1) are able to establish and retrieve remote contextual memory but unable to sustain it as long as wild-type mice. Interestingly, mice overexpressing AC1 show superior remote contextual memory even though they exhibit normal hippocampus-dependent contextual memory. These data illustrate that calcium coupling to cAMP contributes to the stability of remote memory and identifies AC1 as a potential drug target site to improve long-term remote memory.

Asymmetric contribution of α and β subunits to the activation of αβ heteromeric glycine receptors

This study investigated the role of β subunits in the activation of αβ heteromeric glycine receptor (GlyR) chloride channels recombinantly expressed in HEK293 cells. The approach involved incorporating mutations into corresponding positions in α and β subunits and comparing their effects on receptor function. Although cysteine‐substitution mutations to residues in the N‐terminal half of the α subunit M2–M3 loop dramatically impaired the gating efficacy, the same mutations exerted little effect when incorporated into corresponding positions of the β subunit. Furthermore, although the α subunit M2–M3 loop cysteines were modified by a cysteine‐specific reagent, the corresponding β subunit cysteines showed no evidence of reactivity. These observations suggest structural or functional differences between α and β subunit M2–M3 loops. In addition, a threonine→leucine mutation at the 9′ position in the β subunit M2 pore‐lining domain dramatically increased the glycine sensitivity. By analogy with the effects of the same mutation in other ligand‐gated ion channels, it was concluded that the mutation affected the GlyR activation mechanism. This supports the idea that the GlyR β subunit is involved in receptor gating. In conclusion, this study demonstrates that β subunits contribute to the activation of the GlyR, but that their involvement in this process is significantly different to that of the α subunit.

Phosphorylation of α3 glycine receptors induces a conformational change in the glycine-binding site.

Inflammatory pain sensitization is initiated by prostaglandin-induced phosphorylation of α3 glycine receptors (GlyRs) that are specifically located in inhibitory synapses on spinal pain sensory neurons. Phosphorylation reduces the magnitude of glycinergic synaptic currents, thereby disinhibiting nociceptive neurons. Although α1 and α3 subunits are both expressed on spinal nociceptive neurons, α3 is a more promising therapeutic target as its sparse expression elsewhere implies a reduced risk of side-effects. Here we compared glycine-mediated conformational changes in α1 and α3 GlyRs to identify structural differences that might be exploited in designing α3-specific analgesics. Using voltage-clamp fluorometry, we show that glycine-mediated conformational changes in the extracellular M2-M3 domain were significantly different between the two GlyR isoforms. Using a chimeric approach, we found that structural variations in the intracellular M3-M4 domain were responsible for this difference. This prompted us to test the hypothesis that phosphorylation of S346 in α3 GlyR might also induce extracellular conformation changes. We show using both voltage-clamp fluorometry and pharmacology that Ser346 phosphorylation elicits structural changes in the α3 glycine-binding site. These results provide the first direct evidence for phosphorylation-mediated extracellular conformational changes in pentameric ligand-gated ion channels, and thus suggest new loci for investigating how phosphorylation modulates structure and function in this receptor family. More importantly, by demonstrating that phosphorylation alters α3 GlyR glycine-binding site structure, they raise the possibility of developing analgesics that selectively target inflammation-modulated GlyRs.

β Subunit M2-M3 loop conformational changes are uncoupled from α1 β glycine receptor channel gating: implications for human hereditary hyperekplexia.

Hereditary hyperekplexia, or startle disease, is a neuromotor disorder caused mainly by mutations that either prevent the surface expression of, or modify the function of, the human heteromeric α1 β glycine receptor (GlyR) chloride channel. There is as yet no explanation as to why hyperekplexia mutations that modify channel function are almost exclusively located in the α1 to the exclusion of β subunit. The majority of these mutations are identified in the M2–M3 loop of the α1 subunit. Here we demonstrate that α1 β GlyR channel function is less sensitive to hyperekplexia-mimicking mutations introduced into the M2–M3 loop of the β than into the α1 subunit. This suggests that the M2–M3 loop of the α subunit dominates the β subunit in gating the α1 β GlyR channel. A further attempt to determine the possible mechanism underlying this phenomenon by using the voltage-clamp fluorometry technique revealed that agonist-induced conformational changes in the β subunit M2–M3 loop were uncoupled from α1 β GlyR channel gating. This is in contrast to the α subunit, where the M2–M3 loop conformational changes were shown to be directly coupled to α1 β GlyR channel gating. Finally, based on analysis of α1 β chimeric receptors, we demonstrate that the structural components responsible for this are distributed throughout the β subunit, implying that the β subunit has evolved without the functional constraint of a normal gating pathway within it. Our study provides a possible explanation of why hereditary hyperekplexia-causing mutations that modify α1 β GlyR channel function are almost exclusively located in the α1 to the exclusion of the β subunit.

Optimization of a cAMP response element signal pathway reporter system

A sensitive cAMP response element (CRE) reporter system is essential for studying the cAMP/protein kinase A/cAMP response element binding protein signal pathway. Here we have tested a few CRE promoters and found one with high sensitivity to external stimuli. Using this optimal CRE promoter and the enhanced green fluorescent protein as the reporter, we have established a CRE reporter cell line. This cell line can be used to study the signal pathway by fluorescent microscope, fluorescence-activated cell analysis and luciferase assay. This cell lines sensitivity to forskolin, using the technique of fluorescence-activated cell sorting, was increased to approximately seven times that of its parental HEK 293 cell line, which is currently the most commonly used cell line in the field for the signal pathway study. Therefore, this newly created cell line is potentially useful for studying the signal pathways modulators, which generally have weaker effect than its mediators. Our research has also established a general procedure for optimizing transcription-based reporter cell lines, which might be useful in performing the same task when studying many other transcription-based signal pathways.

Incompatibility between a pair of residues from the pre-M1 linker and Cys-loop blocks surface expression of the glycine receptor.

Background: Structural basis determining glycine receptor surface expression is barely known. Results: A pair of positively charged residues from the pre-M1 linker and Cys-loop blocks glycine receptor surface expression. Conclusion: Compatibility of residues, in close proximity to each other, is essential for glycine receptor surface expression. Significance: We provide a novel mechanism, i.e. residue incompatibility, for explaining mutation-induced reduction in channel surface expression. Regulation of cell membrane excitability can be achieved either by modulating the functional properties of cell membrane-expressed single channels or by varying the number of expressed channels. Whereas the structural basis underlying single channel properties has been intensively studied, the structural basis contributing to surface expression is less well characterized. Here we demonstrate that homologous substitution of the pre-M1 linker from the β subunit prevents surface expression of the α1 glycine receptor chloride channel. By investigating a series of chimeras comprising α1 and β subunits, we hypothesized that this effect was due to incompatibility between a pair of positively charged residues, which lie in close proximity to each other in the tertiary structure, from the pre-M1 linker and Cys-loop. Abolishing either positive charge restored surface expression. We propose that incompatibility (electrostatic repulsion) between this pair of residues misfolds the glycine receptor, and in consequence, the protein is retained in the cytoplasm and prevented from surface expression by the quality control machinery. This hypothesis suggests a novel mechanism, i.e. residue incompatibility, for explaining the mutation-induced reduction in channel surface expression, often present in the cases of hereditary hyperekplexia.

Distinct Properties of Glycine Receptor β+/α− Interface UNAMBIGUOUSLY CHARACTERIZING HETEROMERIC INTERFACE RECONSTITUTED IN HOMOMERIC PROTEIN

Background: Heteromeric αβ glycine receptor β+/α− interfaces have never been characterized unambiguously. Results: This interface, compared with the α+/α− interface, is highly sensitive to agonist and experiences distinct conformational changes upon agonist binding. Conclusion: The β+/α− interface exhibits distinct properties. Significance: Our investigation directs the β+/α− interface-specific drug design and provides a general methodology for unambiguously characterizing heteromeric proteins interfaces. The glycine receptor (GlyR) exists either in homomeric α or heteromeric αβ forms. Its agonists bind at extracellular subunit interfaces. Unlike subunit interfaces from the homomeric α GlyR, subunit interfaces from the heteromeric αβ GlyR have not been characterized unambiguously because of the existence of multiple types of interface within single receptors. Here, we report that, by reconstituting β+/α− interfaces in a homomeric GlyR (αChb+a− GlyR), we were able to functionally characterize the αβ GlyR β+/α− interfaces. We found that the β+/α− interface had a higher agonist sensitivity than that of the α+/α− interface. This high sensitivity was contributed primarily by loop A. We also found that the β+/α− interface differentially modulates the agonist properties of glycine and taurine. Using voltage clamp fluorometry, we found that the conformational changes induced by glycine binding to the β+/α− interface were different from those induced by glycine binding to the α+/α− interface in the α GlyR. Moreover, the distinct conformational changes found at the β+/α− interface in the αChb+a− GlyR were also found in the heteromeric αβ GlyR, which suggests that the αChb+a− GlyR reconstitutes structural components and recapitulates functional properties, of the β+/α− interface in the heteromeric αβ GlyR. Our investigation not only provides structural and functional information about the GlyR β+/α− interface, which could direct GlyR β+/α− interface-specific drug design, but also provides a general methodology for unambiguously characterizing properties of specific protein interfaces from heteromeric proteins.