Pharmacological Chaperoning of Nicotinic Acetylcholine Receptors Reduces the Endoplasmic Reticulum Stress Response

We report the first observation that endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) can decrease when a central nervous system drug acts as an intracellular pharmacological chaperone for its classic receptor. Transient expression of α4β2 nicotinic receptors (nAChRs) in Neuro-2a cells induced the nuclear translocation of activating transcription factor 6 (ATF6), which is part of the UPR. Cells were exposed for 48 h to the full agonist nicotine, the partial agonist cytisine, or the competitive antagonist dihydro-β-erythroidine; we also tested mutant nAChRs that readily exit the ER. Each of these four manipulations increased Sec24D-enhanced green fluorescent protein fluorescence of condensed ER exit sites and attenuated translocation of ATF6-enhanced green fluorescent protein to the nucleus. However, we found no correlation among the manipulations regarding other tested parameters [i.e., changes in nAChR stoichiometry (α4_2β2_3 versus α4_3β2_2), changes in ER and trans-Golgi structures, or the degree of nAChR up-regulation at the plasma membrane]. The four manipulations activated 0 to 0.4% of nAChRs, which shows that activation of the nAChR channel did not underlie the reduced ER stress. Nicotine also attenuated endogenously expressed ATF6 translocation and phosphorylation of eukaryotic initiation factor 2α in mouse cortical neurons transfected with α4β2 nAChRs. We conclude that, when nicotine accelerates ER export of α4β2 nAChRs, this suppresses ER stress and the UPR. Suppression of a sustained UPR may explain the apparent neuroprotective effect that causes the inverse correlation between a person's history of tobacco use and susceptibility to developing Parkinson's disease. This suggests a novel mechanism for neuroprotection by nicotine

Single-Molecule Imaging of a Fluorescent Unnatural Amino Acid Incorporated Into Nicotinic Receptors

We report on the first, to our knowledge, successful detection of a fluorescent unnatural amino acid (fUAA), Lys(BODIPYFL), incorporated into a membrane protein (the muscle nicotinic acetylcholine receptor, nAChR) in a living cell. Xenopus oocytes were injected with a frameshift-suppressor tRNA, amino-acylated with Lys(BODIPYFL) and nAChR (α/β19′GGGU/γ/δ) mRNAs. We measured fluorescence from oocytes expressing nAChR β19′Lys(BODIPYFL), using time-resolved total internal reflection fluorescence microscopy. Under conditions of relatively low receptor density (<0.1 receptors/μm^2), we observed puncta with diffraction-limited profiles that were consistent with the point-spread function of our microscope. Furthermore, diffraction-limited puncta displayed step decreases in fluorescence intensity, consistent with single-molecule photobleaching. The puncta densities agreed with macroscopic ACh-induced current densities, showing that the fUAA was incorporated, and that receptors were functional. Dose-response relations for the nAChR β19′Lys(BODIPYFL) receptors were similar to those for wild-type receptors. We also studied nAChR β19′Lys(BODIPYFL) receptors labeled with α-bungarotoxin monoconjugated with Alexa488 (αBtxAlexa488). The nAChR has two αBtx binding sites, and puncta containing the Lys(BODIPYFL) labeled with αBtxAlexa488 yielded the expected three discrete photobleaching steps. We also performed positive control experiments with a nAChR containing enhanced green fluorescent protein in the γ-subunit M3-M4 loop, which confirmed our nAChR β19′Lys(BODIPYFL) measurements. Thus, we report on the cell-based single-molecule detection of nAChR β19′Lys(BODIPYFL)

α7β2 Nicotinic Acetylcholine Receptors Assemble, Function, and Are Activated Primarily via Their α7-α7 Interfaces

We investigated assembly and function of nicotinic acetylcholine receptors (nAChRs) composed of α7 and β2 subunits. We measured optical and electrophysiological properties of wild-type and mutant subunits expressed in cell lines and Xenopus laevis oocytes. Laser scanning confocal microscopy indicated that fluorescently tagged α7 and β2 subunits colocalize. Förster resonance energy transfer between fluorescently tagged subunits strongly suggested that α7 and β2 subunits coassemble. Total internal reflection fluorescence microscopy revealed that assemblies localized to filopodia-like processes of SH-EP1 cells. Gain-of-function α7 and β2 subunits confirmed that these subunits coassemble within functional receptors. Moreover, α7β2 nAChRs composed of wild-type subunits or fluorescently tagged subunits had pharmacological properties similar to those of α7 nAChRs, although amplitudes of α7β2 nAChR-mediated, agonist-evoked currents were generally ∼2-fold lower than those for α7 nAChRs. It is noteworthy that α7β2 nAChRs displayed sensitivity to low concentrations of the antagonist dihydro-β-erythroidine that was not observed for α7 nAChRs at comparable concentrations. In addition, cysteine mutants revealed that the α7-β2 subunit interface does not bind ligand in a functionally productive manner, partly explaining lower α7β2 nAChR current amplitudes and challenges in identifying the function of native α7β2 nAChRs. On the basis of our findings, we have constructed a model predicting receptor function that is based on stoichiometry and position of β2 subunits within the α7β2 nAChRs

Nicotine up-regulates α4β2 nicotinic receptors and ER exit sites via stoichiometry-dependent chaperoning

The up-regulation of α4β2* nicotinic acetylcholine receptors (nAChRs) by chronic nicotine is a cell-delimited process and may be necessary and sufficient for the initial events of nicotine dependence. Clinical literature documents an inverse relationship between a person’s history of tobacco use and his or her susceptibility to Parkinson’s disease; this may also result from up-regulation. This study visualizes and quantifies the subcellular mechanisms involved in nicotine-induced nAChR up-regulation by using transfected fluorescent protein (FP)-tagged α4 nAChR subunits and an FP-tagged Sec24D endoplasmic reticulum (ER) exit site marker. Total internal reflection fluorescence microscopy shows that nicotine (0.1 µM for 48 h) up-regulates α4β2 nAChRs at the plasma membrane (PM), despite increasing the fraction of α4β2 nAChRs that remain in near-PM ER. Pixel-resolved normalized Förster resonance energy transfer microscopy between α4-FP subunits shows that nicotine stabilizes the (α4)2(β2)3 stoichiometry before the nAChRs reach the trans-Golgi apparatus. Nicotine also induces the formation of additional ER exit sites (ERES). To aid in the mechanistic analysis of these phenomena, we generated a β2enhanced-ER-export mutant subunit that mimics two regions of the β4 subunit sequence: the presence of an ER export motif and the absence of an ER retention/retrieval motif. The α4β2enhanced-ER-export nAChR resembles nicotine-exposed nAChRs with regard to stoichiometry, intracellular mobility, ERES enhancement, and PM localization. Nicotine produces only small additional PM up-regulation of α4β2enhanced-ER-export receptors. The experimental data are simulated with a model incorporating two mechanisms: (1) nicotine acts as a stabilizing pharmacological chaperone for nascent α4β2 nAChRs in the ER, eventually increasing PM receptors despite a bottleneck(s) in ER export; and (2) removal of the bottleneck (e.g., by expression of the β2enhanced-ER-export subunit) is sufficient to increase PM nAChR numbers, even without nicotine. The data also suggest that pharmacological chaperoning of nAChRs by nicotine can alter the physiology of ER processes

Pharmacological chaperoning of nicotinic receptors begins in the endoplasmic reticulum: Compartments and stoichiometries

[Poster abstract

Silicon chip-based patch-clamp electrodes integrated with PDMS microfluidics

We report on a silicon wafer-based device that can be used for recording macroscopic ion channel protein activities across a diverse group of cell-types. Gigaohm seals were achieved for CHO-K1 and RIN m5F cells, and both cell-attached and whole-cell mode configurations were also demonstrated. Two distinct intrinsic potassium ion channels were recorded in whole-cell mode for HIT-T15 and RAW 264.7 cells. Polydimethylsiloxane (PDMS) microfluidics were also coupled with the micromachined silicon chips in order to demonstrate that a single cell could be selectively directed to a micropore, and membrane protein currents could subsequently be recorded. These silicon chip-based devices have significant advantages over traditional micropipette approaches, and may serve as combinatorial tools for investigating membrane biophysics, pharmaceutical screening, and other bio-sensing tasks

Pharmacological chaperoning of nicotinic receptors begins in the endoplasmic reticulum: High-resolution imaging

[No abstract

Nicotine is a Selective Pharmacological Chaperone of Acetylcholine Receptor Number and Stoichiometry. Implications for Drug Discovery

The acronym SePhaChARNS, for “selective pharmacological chaperoning of acetylcholine receptor number and stoichiometry,” is introduced. We hypothesize that SePhaChARNS underlies classical observations that chronic exposure to nicotine causes “upregulation” of nicotinic receptors (nAChRs). If the hypothesis is proven, (1) SePhaChARNS is the molecular mechanism of the first step in neuroadaptation to chronic nicotine; and (2) nicotine addiction is partially a disease of excessive chaperoning. The chaperone is a pharmacological one, nicotine; and the chaperoned molecules are α4β2* nAChRs. SePhaChARNS may also underlie two inadvertent therapeutic effects of tobacco use: (1) the inverse correlation between tobacco use and Parkinson’s disease; and (2) the suppression of seizures by nicotine in autosomal dominant nocturnal frontal lobe epilepsy. SePhaChARNS arises from the thermodynamics of pharmacological chaperoning: ligand binding, especially at subunit interfaces, stabilizes AChRs during assembly and maturation, and this stabilization is most pronounced for the highest-affinity subunit compositions, stoichiometries, and functional states of receptors. Several chemical and pharmacokinetic characteristics render exogenous nicotine a more potent pharmacological chaperone than endogenous acetylcholine. SePhaChARNS is modified by desensitized states of nAChRs, by acid trapping of nicotine in organelles, and by other aspects of proteostasis. SePhaChARNS is selective at the cellular, and possibly subcellular, levels because of variations in the detailed nAChR subunit composition, as well as in expression of auxiliary proteins such as lynx. One important implication of the SePhaChARNS hypothesis is that therapeutically relevant nicotinic receptor drugs could be discovered by studying events in intracellular compartments rather than exclusively at the surface membrane