nAChRs also underlie the behavioral and addictive properties of nicotine
Numerous studies have shown that chronic nicotine exposure enhances surface expression of nAChRs, especially for the alpha4beta2 subtype (Schwartz and Kellar, 1983)
We found that nicotine exposure does not affect alpha7 in absence and presence of NACHO (Figures S1C and S1D), and nicotine markedly enhanced alpha4beta2 surface in a manner additive with NACHO (Figures 3G and 3H)
At the cellular level, nAChRs can underlie synaptic responses, neuronal excitability, and neurotransmitter release (Dajas-Bailador and Wonnacott, 2004; Gotti and Clementi, 2004; Hogg et al., 2003)
This NACHO-mediated [3H]epibatidine binding to alpha6beta2 transfectants was also displaced by conotoxin MII (Figure 4E)
Homomeric alpha7 receptors are also abundant and are of particular interest as they show very high calcium permeability and are linked to both physiological and disease processes (Gotti and Clementi, 2004; Hogg et al., 2003; Le Nove` re et al., 2002; Lindstrom, 1997; Picciotto, 2003; Role and Berg, 1996
Strikingly, we found that alpha-bungarotoxin did not completely displace [3H]epibatidine binding to alpha7 receptors with NACHO (Figure 3B)
General characterization showed that the knockout mice as compared to their wild-type littermates show no changes in body weight, have slight increases in body temperature (Figures S3A and S3B), and exhibit significantly enhanced locomotor activity (Figures S2A and S2B)
As ACh-gated cation channels, these broadly distributed receptors participate in arousal, motivation, and numerous aspects of cognition (Gotti and Clementi, 2004; Hogg et al., 2003; Le Nove` re et al., 2002; Lindstrom, 1997; Picciotto, 2003; Role and Berg, 1996).
Because nAChRs play important roles in cognition (Gotti and Clementi, 2004; Hogg et al., 2003; Le Nove` re et al., 2002; Lindstrom, 1997; Picciotto, 2003; Role and Berg, 1996), we focused attention on this behavi
Indeed, presynaptic effects of nAChRs in regulating dopamine release in basal ganglia and prefrontal cortex likely participate in nicotine’s addictive and cognitive effects, respectively (Jasinska et al., 2014)
In substantia nigra, postsynaptic nicotinic receptors induce inward currents to excite dopaminergic neurons that project to corpus striatum (Matsubayashi et al., 2003)
Through these postsynaptic effects and by influencing neural excitability, nAChRs can modulate or induce synaptic plasticity (Yakel, 2014)
Resistance to inhibitor of cholinesterase-3 (RIC-3) is required for nAChR function in C. elegans (Nguyen et al., 1995)
Whereas RIC-3 can enhance function of certain mammalian nAChRs, RIC-3 is not essential (Koperniak et al., 2013)
In C. elegans, RIC-3 protein is essential for nAChR function (Nguyen et al., 1995), and mammalian RIC-3 has modest and mixed effects on nAChRs (Halevi et al., 2003; Millar, 2008)
For alpha7 and alpha3beta2, RIC-3 did not enable detectable [3H]epibatidine binding, yet RIC-3 profoundly augmented the effects of NACHO on [3H]epibatidine binding to either receptor subtype (Figures 3A and S1A)
As previously reported using fluorescently labeled alpha-bungarotoxin (Gu et al., 2016), NACHO enabled formation of assembled surface alpha7 receptors, and RIC-3 further enhanced this (Figures 3E and 3F)
Using a genomic cDNA screening strategy, we recently identified NACHO (Gu et al., 2016), a small multi-pass transmembrane protein enriched in neuronal endoplasmic reticulum (ER) that can mediate functional reconstitution of alpha7 receptors in non-neuronal cell lines
NACHO serves as a molecular chaperone to mediate folding, assembly, and surface expression of alpha7 receptors (Gu et al., 2016)
As published previously (Gu et al., 2016), ACh evoked currents from alpha7 require NACHO, and currents from alpha4beta2 were augmented ~3-fold by NACHO, which did not alter the desensitization kinetics of alpha4beta2 receptors (Figures 1A and 1B)
As previously published (Gu et al., 2016), robust surface alpha7 and alpha4beta2 expression required co-transfection with NACHO (Figures 1C and D), and we find that alpha3beta2 is also NACHO-dependent (Figures 1C and D)
Our previous studies demonstrated that NACHO promotes assembly of alpha7 receptors as evidenced by alpha-bungarotoxin labeling, (Gu et al., 2016) which in brain only binds with high affinity to properly folded pentameric alpha7 receptors (Couturier et al., 1990; Schoepfer et al., 1990)
In membranes from alpha7-transfected cells, [3H]epibatidine binding absolutely required NACHO (Figure 2A), which fits with an essential role for NACHO in alpha7 assembly
NACHO does not affect function of ion channels gated by numerous other ligands including glutamate, GABA, serotonin, and capsaicin, suggesting that NACHO represents the only essential client-specific chaperone yet identified for any mammalian neurotransmitter receptor (Gu et al., 2016)
Cells transfected with alpha4beta2 or alpha3beta4 alone showed [3H]epibatidine binding, and this was markedly increased (5- to 10-fold) in presence of NACHO (Figures 2B and 2D)
We now also find that alpha3beta2 receptor function in HEK cells requires NACHO, and these channels showed desensitization kinetics generally similar to alpha4beta2 (Figures 1A and 1B)
Similarly, quantifiable [3H]epibatidine binding to alpha3beta2 required cotransfection with NACHO (Figure 2C)
For alpha3beta4 alone, ACh evoked large slowly-desensitizing responses, and NACHO amplified their magnitudes ~5-fold (Figures 1A and 1B)
For alpha3beta4 alone, we found abundant surface labeling and this was enhanced by NACHO (Figures 1C and 1D)
However, we did find that NACHO enhances [3H]epibatidine binding to cells transfected with alpha6beta2beta3 (Figure 4C) indicating that NACHO can enhance intracellular receptor assembly
Whereas this subunit combination is not competent to form a functional channel (Champtiaux et al., 2002; Dash et al., 2014; Kuryatov et al., 2000), NACHO still mediated partial receptor assembly as reflected by [3H]epibatidine binding (Figure 4D)
These experiments showed that NACHO is most concentrated in the heavy (P3) and light (P4) microsomal fractions (Figures 3C and 3D), which contain resident endoplasmic reticulum (ER) proteins including calnexin
Importantly, NACHO knockout mice show complete loss of alpha7 ligand binding and channel function indicating that NACHO is required for formation of alpha7 receptors (Gu et al., 2016)
We previously showed that [125I]alpha-bungarotoxin binding is absent in NACHO knockout mice brain (Gu et al., 2016), which express normal levels of RIC-3 mRNA (Figure S1B)
[125I]epibatidine binding was decreased in all brain regions evaluated—it was reduced by 66% in cortex, 39% in striatum, and 51% in medial vestibular nucleus (MVN) (Figures 5A and 5D)
This partial reduction fits with our data showing that NACHO enhances [3H]epibatidine binding to alpha4beta2 and other heteromeric nAChRs but that some [3H]epibatidine binding occurs to certain heteromeric nAChRs in the absence of NACHO (Figure 2B)
Labeling of alpha6-containing nAChRs in striatum by [125I]conotoxin MII is virtually abolished in NACHO knockout mice (Figures 5B and 5D), indicating a vital role for NACHO in assembly of these presynaptic receptors
Consistent with our previous autoradiographic results (Gu et al., 2016), we found no detectable [3H]alpha-bungarotoxin binding to brain membranes from NACHO knockouts
In membranes from cerebral cortex, hippocampus, and striatum, we found that levels of [3H]epibatidine binding sites are decreased by 50%–75%
A very recent study also reported memory defects in mice lacking NACHO/TMEM35 done by scientists unaware of NACHO’s role in controlling nAChRs (Kennedy et al., 2016)
NACHO knockouts did not show changes in levels of [3H]epibatidine binding to membranes from superior cervical ganglia (wild-type [WT] 329.0 ± 17.2, knockout [KO] 322.8 ± 4.0 fmol/mg protein), which primarily express receptors containing alpha3beta4-containing receptors (David et al., 2010)
The NACHO knockouts showed increased total number of arm entries in the Y-maze, which fits with their enhanced locomotor activity (Figure S2F)
In the Morris water maze, NACHO knockout mice were delayed in learning the task during acquisition days 1–4 and showed fewer target crossings in the probe test (Figures S2Cand S2D), despite showing no significant difference for genotype on average speed through each acquisition day (ANOVA: F[1,28] = 0.004; p = 0.9492; data not shown)
NACHO knockout mice also showed deficits of spontaneous alternation in the Y-maze compared to wild-type littermates (Figure S2E)
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If you find BEL Commons useful in your work, please consider citing: Hoyt, C. T., Domingo-Fernández, D., & Hofmann-Apitius, M. (2018). BEL Commons: an environment for exploration and analysis of networks encoded in Biological Expression Language. Database, 2018(3), 1–11.