Manuel Criado

Conservation within the RIC-3 Gene Family EFFECTORS OF MAMMALIAN NICOTINIC ACETYLCHOLINE RECEPTOR EXPRESSION

In Caenorhabditis elegans, the ric-3 gene is required for the maturation of multiple nicotinic acetylcholine receptors (nAChRs), whereas other neurotransmittergated channels expressed within the same cells are unaffected by the presence of RIC-3. Here we show that RIC-3 is a member of a conserved gene family with representatives in both vertebrates and invertebrates. All members of this family have two transmembrane domains followed by a coiled-coil domain. Expression of the human ric-3 homolog, hric3, like the C. elegans ric-3, enhances C. elegans DEG-3/DES-2, rat α7, and human α7 nAChR-dependent whole-cell current amplitudes in Xenopus leavis oocytes, thus demonstrating functional conservation. However, hric3 also reduces human α4β2 and α3β4 nAChR-dependent whole-cell current amplitudes. Thus, hric3 shows differential effects on human nAChRs unlike the observed uniform effect of ric-3 on C. elegans nAChRs. Moreover, hric3 totally abolished currents evoked by 5-HT3 serotonin receptors, whereas it barely modified α1 glycine receptor currents. With this caveat, RIC-3 belongs to a conserved family of genes likely to regulate nAChR-mediated transmission throughout evolution. Analysis of transcripts encoded by the hric3 locus shows that it encodes for multiple transcripts, likely to produce multiple hric3 isoforms, and that hric3 is expressed in neurons and muscles, thus enabling its interactions with nAChRs in vivo.

Primary structure of an agonist binding subunit of the nicotinic acetylcholine receptor from bovine adrenal chromaffin cells.

Activation by acetylcholine of a nicotinic acetylcholine receptor on the membrane of bovine chromaffin cells leads to membrane depolarization and to the subsequent triggering of catecholamine secretion. It is evident that acetylcholine receptors play a central role in the initial phase of the secretion process and, therefore, an extensive characterization of their molecular components and properties is of fundamental interest. With this intention, we have screened bovine adrenal medullary cDNA libraries with a probe coding for a fragment of the rat muscle acetylcholine receptor α subunit. Several cDNA clones were isolated. The longest cDNA had an open reading frame encoding a 495-amino acid protein with a molecular weight of 56,911. The deduced primary structure contains features that indicate that the encoded protein is an α or acetylcholine binding subunit, and, in fact, it manifests significant sequence similarity to previously cloned α subunits. Sequence identity is particularly high with the α3 subunit, which is expressed in the rat pheochromocytoma PC12 cell line and in several brain areas, and consequently, it is considered a component of a neuronal acetylcholine receptor. Accordingly, the present results suggest that the agonist binding subunit of the nicotinic acetylcholine receptor from bovine chromaffin cells is an α3-type subunit, corroborating previous immunological and pharmacological evidence for the presence of a neuronal nicotinic receptor in chromaffin cells.

Intragranular pH rapidly modulates exocytosis in adrenal chromaffin cells.

Several drugs produce rapid changes in the kinetics of exocytosis of catecholamines, as measured at the single event level with amperometry. This study is intended to unveil whether the mechanism(s) responsible for these effects involve changes in the intravesicular pH. Cell incubation with bafilomycin A1, a blocker of the vesicular proton pump, caused both a deceleration in the kinetics of exocytosis and a reduction in the catecholamine content of vesicle. These effects were also observed upon reduction of proton gradient by nigericin or NH4Cl. pH measurements using fluorescent probes (acridine orange, quinacrine or enhanced green fluorescent protein–synaptobrevin) showed a strong correlation between vesicular pH and the kinetics of exocytosis. Hence, all maneuvers tested that decelerated exocytosis also alkalinized secretory vesicles and vice versa. On the other hand, calcium entry caused a transient acidification of granules. We therefore propose that the regulation of vesicular pH is, at least partially, a necessary step in the modulation of the kinetics of exocytosis and quantal size operated by some cell signals.

Effects of ginsenosides, active components of ginseng, on nicotinic acetylcholine receptors expressed in Xenopus oocytes.

We investigated the effects of ginsenosides, the active ingredient of ginseng, on neuronal or muscle-type nicotinic acetylcholine receptor channel activity expressed in Xenopus oocytes after injection of cRNA encoding bovine neuronal alpha3beta4, alpha7 or human muscle alphabetadeltavarepsilon subunits. Treatment with acetylcholine elicited an inward peak current (I(ACh)) in oocytes expressing nicotinic acetylcholine receptor subtypes. Cotreatment with ginsenoside Rg2 and acetylcholine inhibited I(ACh) in oocytes expressing with alpha3beta4 or alphabetadeltavarepsilon but not in oocytes expressing alpha7 nicotinic acetylcholine receptors. The inhibition of I(ACh) by ginsenoside Rg2 was reversible and dose-dependent. The half-inhibitory concentrations (IC50) of ginsenoside Rg2 were 60.2+/-14.1 and 15.7+/-3.5 microM in oocytes expressing alpha3beta4 and alphabetadeltavarepsilon nicotinic acetylcholine receptors, respectively. The inhibition of I(ACh) by ginsenoside Rg2 was voltage-independent and noncompetitive. Other ginsenosides besides ginsenoside Rg2 also inhibited I(ACh) in oocytes expressing alpha3beta4 or alphabetadeltavarepsilon nicotinic acetylcholine receptors. The order of potency for the inhibition of I(ACh) was ginsenoside Rg2>Rf>Re>Rg1>Rc>Rb2>Rb1 in oocytes expressing alpha3beta4 nicotinic acetylcholine receptors and was ginsenoside Rg2>Rf>Rg1>Re>Rb1>Rc>Rb2 in oocytes expressing alphabetadeltavarepsilon nicotinic acetylcholine receptors. These results indicate that ginsenosides might regulate nicotinic acetylcholine receptors in a differential manner and this regulation might be one of the pharmacological actions of Panax ginseng.

Modifications in the C terminus of the synaptosome-associated protein of 25 kDa (SNAP-25) and in the complementary region of synaptobrevin affect the final steps of exocytosis

Fusion proteins made of green fluorescent protein coupled to SNAP-25 or synaptobrevin were overexpressed in bovine chromaffin cells in order to study the role of critical protein domains in exocytosis. Point mutations in the C-terminal domain of SNAP-25 (K201E and L203E) produced a marked inhibition of secretion, whereas single (Q174K, Q53K) and double mutants (Q174K/Q53K) of amino acids from the so-called zero layer only produced a moderate alteration in secretion. The importance of the SNAP-25 C-terminal domain in exocytosis was also confirmed by the similar effect on secretion of mutations in analogous residues of synaptobrevin (A82D, L84E). The effects on the initial rate and magnitude of secretion correlated with the alteration of single vesicle fusion kinetics since the amperometric spikes from cells expressing SNAP-25 L203E and K201E and synaptobrevin A82D and L84E mutants had lower amplitudes and larger half-width values than the ones from controls, suggesting slower neurotransmitter release kinetics than that found in cells expressing the wild-type proteins or zero layer mutants of SNAP-25. We conclude that a small domain of the SNAP-25 C terminus and its counterpart in synaptobrevin play an essential role in the final membrane fusion step of exocytosis.

Potentiation of human α4β2 neuronal nicotinic receptors by a Flustra foliacea metabolite

Abstract The effects of various Flustra foliacea metabolites on different types of human neuronal nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes were investigated. Whereas most of the compounds tested had a small blocking effect, one of them, deformylflustrabromine, selectively increased the current obtained in α4β2 receptors when co-applied with acetylcholine (ACh). The current increase was reversible and concentration-dependent. This potentiating effect was still present at saturating concentrations of acetylcholine, and no changes in single-channel conductance or reversal potential were observed, thus suggesting a modification in the gating of α4β2 receptors. Dwell time analysis of single channel records indicates that the mechanism of action of deformylflustrabromine could be both an increase of the opening rate constant and a decrease of the closing rate constant on α4β2 receptors. Thus, deformylflustrabromine may constitute an excellent starting point for the future development of related agents able to potentiate human neuronal nicotinic receptor function.

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.

Multiple Functional Sp1 Domains in the Minimal Promoter Region of the Neuronal Nicotinic Receptor α5 Subunit Gene

The α5 subunit is a component of the neuronal nicotinic acetylcholine receptors, which are probably involved in the activation step of the catecholamine secretion process in bovine adrenomedullary chromaffin cells. The promoter of the gene coding for this subunit was isolated, and its proximal region was characterized, revealing several GC boxes located close to the site of transcription initiation (from −111 to −40). Deletion analysis and transient transfections showed that a 266-base pair region (−111 to +155) gave rise to ∼77 and 100% of the maximal transcriptional activity observed in chromaffin and SHSY-5Y neuroblastoma cells, respectively. Site-directed mutagenesis of five different GC motifs indicated that all of them contribute to the activity of the α5 gene, but in a different way, depending on the type of transfected cell. Thus, in SHSY-5Y cells, alteration of the most promoter-proximal of the GC boxes decreased α5 promoter activity by ∼50%, whereas single mutations of the other GC boxes had no effect. In chromaffin cells, by contrast, modification of any of the GC boxes produced a similar decrease in promoter activity (50–69%). In both cell types, however, activity was almost abolished when four GC boxes were suppressed simultaneously. Electrophoretic mobility shift assays using nuclear extracts from either chromaffin or SHSY-5Y cells showed the specific binding of Sp1 protein to fragment −111 to −27. Binding of Sp1 to the GC boxes was also demonstrated by DNase I footprint analysis. This study suggests that the general transcription factor Sp1 plays a dominant role in α5 subunit expression, as has also been demonstrated previously for α3 and β4 subunits. Since these three subunits have their genes tightly clustered and are expressed in chromaffin cells, probably as components of the same receptor subtype, we propose that Sp1 constitutes the key factor of a regulatory mechanism common to the three subunits.

GC- and E-box Motifs as Regulatory Elements in the Proximal Promoter Region of the Neuronal Nicotinic Receptor α7 Subunit Gene

The α7 subunit is a component of α-bungarotoxin-sensitive nicotinic acetylcholine receptors expressed in bovine adrenomedullary chromaffin cells. The proximal promoter of the gene coding for this subunit contains several GC-boxes and one E-box. Deletion analysis and transient transfections showed that a 120-base pair region (−77 to +43) including all of these elements gave rise to ∼70 and 95% of the maximal transcriptional activity observed in chromaffin and SHSY-5Y neuroblastoma cells, respectively. Site-directed mutagenesis of the different elements indicated that both GC and E motifs contribute to the activity of the α7 gene in a very prominent way. Using electrophoretic mobility shift assays, the upstream stimulatory factor (USF) was shown to be a component of the complexes that interacted with the E-box when nuclear extracts from chromaffin and SHSY-5Y cells were used. Binding of the early growth response gene transcription factor (Egr-1) to three different GC-boxes was also demonstrated by shift assays and DNase I footprint analysis. Likewise, α7 promoter activity increased by up to 5-fold when α7 constructs and an Egr-1 expression vector were cotransfected into chromaffin cell cultures. Mutagenesis of individual GC-boxes had little effect on Egr-1 activation. By contrast, pairwise suppression of GC-boxes abolished activation, especially when the most promoter-proximal of the Egr-1 sites was removed. Taken together, these studies indicate that the α7 gene is likely to be a target for multiple signaling pathways, in which various regulatory elements are involved.

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.