Susana Gerber

Charged Amino Acids of the N-terminal Domain Are Involved in Coupling Binding and Gating in α7 Nicotinic Receptors

Binding of agonists to nicotinic acetylcholine receptors generates a sequence of conformational changes resulting in channel opening. Previously, we have shown that the aspartate residue Asp-266 at the M2-M3 linker of the α7 nicotinic receptor is involved in connecting binding and gating. High resolution structural data suggest that this region could interact with the so-called loops 2 and 7 of the extracellular N-terminal region. In this case, certain charged amino acids present in these loops could integrate together with Asp-266 and other amino acids, a mechanism involved in channel activation. To test this hypothesis, all charged residues in these loops, Asp-42, Asp-44, Glu-45, Lys-46, Asp-128, Arg-130, and Asp-135, were substituted with other amino acids, and expression levels and electrophysiological responses of mutant receptors were determined. Mutants at positions Glu-45, Lys-46, and Asp-135 exhibited poor or null functional responses to different nicotinic agonists regardless of significant membrane expression, whereas D128A showed a gain of function effect. Because the double reverse charge mutant K46D/D266K did not restore receptor function, a gating mechanism controlled by the pairwise electrostatic interaction between these residues is not likely. Rather, a network of interactions formed by residues Lys-46, Asp-128, Asp-135, Asp-266, and possibly others appears to link agonist binding to channel gating.

The cysteine-rich with EGF-Like domains 2 (CRELD2) protein interacts with the large cytoplasmic domain of human neuronal nicotinic acetylcholine receptor α4 and β2 subunits

Using a yeast two‐hybrid screening we report the isolation of a novel human protein, hCRELD2β, that interacts specifically with the large cytoplasmic regions of human nicotinic acetylcholine receptor (nAChR) α4 and β2 subunits, both in yeast cells and in vitro. This interaction is not detected with nAChR α7 and α3 subunits. The hCRELD2 gene encodes for multiple transcripts, likely to produce multiple protein isoforms. A previously reported one has been renamed as CRELD2α. Isoforms α and β are expressed in all tissues examined and have the same N‐terminal and central regions but alternative C‐terminal regions. Both isoforms interact with the α4 subunit. Within this subunit the interaction was localized to the N‐terminal region of the large cytoplasmic loop. The CRELD2β protein is present at the endoplasmic reticulum where colocalized with α4β2 nAChRs upon cell transfection. Immunohistochemistry experiments demonstrated the presence of CRELD2 in the rat brain at sites where α4β2 receptors have been previously detected. Labeling was restricted to neuronal perikarya. Finally, CRELD2 decreases the functional expression and impairs membrane transport of α4β2 nAChRs in Xenopus leavis oocytes, without affecting α3β4 and α7 nAChR expression. These results suggest that CRELD2 can act as a specific regulator of α4β2 nAChR expression.

Role of the N‐terminal α‐helix in biogenesis of α7 nicotinic receptors

We studied the role of the α‐helix present at the N‐terminus of nicotinic acetylcholine receptor (nAChR) subunits in the expression of functional channels. Deletion of this motif in α7 subunits abolished expression of nAChRs at the membrane of Xenopus oocytes. The same effect was observed upon substitution by homologous motifs of other ligand‐gated receptors. When residues from Gln4 to Tyr15 were individually mutated to proline, receptor expression strongly decreased or was totally abolished. Equivalent substitutions to alanine were less harmful, suggesting that proline‐induced break of the α‐helix is responsible for the low expression. Steady‐state levels of wild‐type and mutant subunits were similar but the formation of pentameric receptors was impaired in the latter. In addition, those mutants that reached the membrane showed a slightly increased internalization rate. Expression of α7 nAChRs in neuroblastoma cells confirmed that mutant subunits, although stable, were unable to reach the cell membrane. Analogous mutations in heteromeric nAChRs (α3β4 and α4β2) and 5‐HT3A receptors also abolished their expression at the membrane. We conclude that the N‐terminal α‐helix of nAChRs is an important requirement for receptor assembly and, therefore, for membrane expression.

Role of the RIC-3 protein in trafficking of serotonin and nicotinic acetylcholine receptors.

Neurotransmitter-gated receptors are assembled in the endoplasmic reticulum and transported to the cell surface through a process that might be of central importance to regulate the efficacy of synaptic transmission (Kneussel and Betz, 2000; Kittler and Moss, 2003). This process is relatively inefficient- what may be the consequence of tight quality controls that guarantee the functional competence of the final product. For this purpose, specific proteins involved in assembly and trafficking of receptors might be required (Keller and Taylor, 1999; Millar, 2003; Wanamaker et al., 2003). The RIC-3 protein could be one of them, as mutations in the ric-3 gene affect maturation of nicotinic acetylcholine receptors (nAChRs) in Caenorhabditis elegans (Halevi et al., 2002). Moreover, the human homolog hRIC-3 showed differential effects when coexpressed with several ligand-gated receptors (Halevi et al., 2003). Thus, it enhanced alpha7 nAChR expression while inhibiting expression of other nAChR subtypes (alpha4beta2 and alpha3beta4) and 5-HT3 serotonin receptors (5-HT3Rs). These opposite effects suggested that the RIC-3 protein might play a key role in the biogenesis of some ligand-gated receptors and prompted us to investigate how it performs its action. Here, we show that the RIC-3 protein acts as a barrier for some receptors like alpha4beta2 nAChRs and 5-HT3Rs, stopping the traffic of mature receptors to the membrane. In contrast, the inefficient transport of alpha7 nAChRs is enhanced by RIC-3 in a process in which certain amino acids at the amphipathic helix located at the C-terminal region of the large cytoplasmic domain are involved.

Expression and functional properties of α7 acetylcholine nicotinic receptors are modified in the presence of other receptor subunits

Although α7 nicotinic receptors are predominantly homopentamers, previous reports have indicated that α7 and β2 subunits are able to form heteromers. We have studied whether other nicotinic receptor subunits can also assemble with α7 subunits and the effect of this potential association. Coexpression of α7 with α2, α3, or β4 subunits reduced to about half, surface α‐bungarotoxin binding sites and acetylcholine‐gated currents. This is probably because of inhibition of membrane trafficking, as the total amount of α7 subunits was similar in all cases and a significant proportion of mature α7 receptors was present inside the cell. Only β4 subunits appeared to directly associate with α7 receptors at the membrane and these heteromeric receptors showed some kinetic and pharmacological differences when compared with homomeric α7 receptors. Finally, we emulated the situation of bovine chromaffin cells in Xenopus laevis oocytes by using the same proportion of α3, β4, α5, and α7 mRNAs, finding that α‐bungarotoxin binding was similarly reduced in spite of increased currents, apparently mediated by α3β4(α5) receptors.

The loop between β-strands β2 and β3 and its interaction with the N-terminal α-helix is essential for biogenesis of α7 nicotinic receptors

J. Neurochem. (2010) 112, 103–111.

A small cytoplasmic region adjacent to the fourth transmembrane segment of the α7 nicotinic receptor is essential for its biogenesis

Deletion of a small cytoplasmic fragment close to the fourth transmembrane segment of the nicotinic α7 receptor (Glu437 to Arg447) abolished membrane expression. Different single mutants showed moderate to strong decreases in expression whereas the latter was totally abolished upon proline substitutions. We hypothesize that preservation of an α‐helix formed by the fourth transmembrane segment and the adjacent cytoplasmic region is essential for membrane receptor expression. Moreover, in selected mutants with low or null membrane expression, a significant proportion of mature receptors was present inside the cell. Hence, elements in this cytoplasmic fragment might influence receptor transport to the membrane.

Mutants of β-strand β3 and the loop B in the interface between α7 subunits of a homomeric acetylcholine receptor show functional and pharmacological alterations

J. Neurochem. (2011) 118, 968–978.