Mar Castillo

Molecular characterization and localization of the RIC‐3 protein, an effector of nicotinic acetylcholine receptor expression

The RIC‐3 protein acts as a regulator of acetylcholine nicotinic receptor (nAChR) expression. In Xenopus laevis oocytes the human RIC‐3 (hRIC‐3) protein enhances expression of α7 receptors and abolishes expression of α4β2 receptors. In vitro translation of hRIC‐3 evidenced its membrane insertion but not the role as signal peptide of its first transmembrane domain (TMD). When the TMDs of hRIC‐3 were substituted, its effects on nAChR expression were attenuated. A certain linker length between the TMDs was also needed for α7 expression enhancement but not for α4β2 inhibition. A combination of increased α7 receptor steady state levels, facilitated transport and reduced receptor internalization appears to be responsible for the increase in α7 membrane expression induced by hRIC‐3. Antibodies against hRIC‐3 showed its expression in SH‐SY5Y and PC12 cells and its induction upon differentiation. Immunohistochemistry demonstrated the presence of RIC‐3 in rat brain localized, in general, in places where α7 nAChRs were found.

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.

Improved gating of a chimeric α7‐5HT3A receptor upon mutations at the M2–M3 extracellular loop

Acetylcholine‐evoked currents of the receptor chimera α7‐5HT3A V201 expressed in Xenopus oocytes are strikingly small when compared to the amount of α‐bungarotoxin binding sites detected at the oocyte membrane. Since the chimeric receptor is made of the extracellular N‐terminal region of the rat α7 nicotinic acetylcholine receptor and the C‐terminal region of the mouse 5‐HT3A receptor, which includes the ion channel, we hypothesized that communication between these two regions was not optimal. Here, we show that mutating to aspartate several adjacent positions in the M2–M3 extracellular linker increases current amplitudes to different extents, thus confirming the important role of this region on receptor gating.

Transcriptional Regulation by Activation and Repression Elements Located at the 5′-Noncoding Region of the Human α9 Nicotinic Receptor Subunit Gene

The α9 subunit is a component of the neuronal nicotinic acetylcholine receptor gene superfamily that is expressed in very restricted locations. The promoter of the human gene has been analyzed in the human neuroblastoma SH-SY5Y, where α9 subunit expression was detected, and in C2C12 cells that do not express α9. A proximal promoter region (from –322 to +113) showed maximal transcriptional activity in SH-SY5Y cells, whereas its activity in C1C12 cells was much lower. Two elements unusually located at the 5′-noncoding region exhibited opposite roles. A negative element located between +15 and +48 appears to be cell-specific because it was effective in C2C12 but not in SH-SY5Y cells, where it was counterbalanced by the presence of the promoter region 5′ to the initiation site. An activating element located between +66 and +79 and formed by two adjacent Sox boxes increased the activity of the α9 promoter about 4-fold and was even able to activate other promoters. This element interacts with Sox proteins, probably through a cooperative mechanism in which the two Sox boxes are necessary. We propose that the Sox complex provides an initial scaffold that facilitates the recruiting of the transcriptional machinery responsible for α9 subunit expression.

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.

Role of the extracellular transmembrane domain interface in gating and pharmacology of a heteromeric neuronal nicotinic receptor

J. Neurochem. (2010) 113, 1036–1045.

Interactions between loop 5 and β-strand β6′ are involved in α7 nicotinic acetylcholine receptors channel gating

Binding of agonists to nicotinic acetylcholine receptors (nAChR) is coupled to channel opening through local rearrangements of different domains of the protein. Recent structural data suggest that two of these regions could be the loop 5 (L5) and the β‐strand β6′, both forming the inner part of the N‐terminal domain. Amino acids in these domains were mutated in α7 nAChRs, and expression levels and functional responses of mutant receptors were measured. Mutations located at the putative apex of L5, Asp97 and Glu98, and also at Phe100, gave receptors with smaller currents, showing qualitative differences with respect to muscle nAChRs. In contrast, mutations in the β‐strand β6′ (at Phe124 and Lys125) showed increased functional responses. Mutations affected equally the responses to acetylcholine and dimethylphenylpiperazinium, except in Phe100 where the latter was sevenfold less effective than in wild‐type. Currents in mutants decayed with almost the same kinetics, ruling out large effects on desensitization. Analysis of double mutants demonstrated a functional coupling among the three electrically charged amino acids Asp97, Glu98, and Lys125, and also between Phe100 and Phe124. The results are compatible with the involvement of functional interactions between L5 and β‐strand β6′ during nAChR activation.

Role of loop 9 on the function of neuronal nicotinic receptors.

We have studied the role of loop 9 in the function of neuronal nicotinic receptors. By systematically mutating the residues in the loop we have determined that the most important amino acids determining the coupling of binding to gating are the ones closer to the transmembrane region. Single mutations at location E173 in homomeric alpha7 receptors destroyed their function by completely abolishing the current while preserving the expression at the membrane. In contrast, heteromeric receptor alpha3beta4 with the same mutations retained some function. We conclude that loop 9 has a different role in the function of homomeric and heteromeric receptors.