Attenuation of nicotine-evoked Ca 2 + influx by antibody to the nicotinic acetylcholine receptor α 3 subunits in human embryonic kidney cells

Autoantibody against neuronal nicotinic acetylcholine receptor (nAChR) 3 subunit is implicated in severe autonomic dysfunction in the patients with autoimmune autonomic ganglionopathy (AAG). Although this autoantibody has been revealed to impair fast excitatory synaptic transmission in autonomic ganglia, its precise mechanism remains unknown. Here, we show that antibody-induced reduction of cell-surface 3 subunits result in impairment of nicotine-evoked Ca influx in stably transfected human embryonic kidney cells. These effects of the antibody were remarkably inhibited by interfering with the endocytic machinery at low-temperature. We conclude that reduction of nAChR in autonomic ganglia can be mediated by the endocytosis of 3 subunits, and resulted in autonomic failure in AAG patients.

Previous electrophysiological studies have revealed pathogenic roles of anti-α3 subunit autoantibody.Autonomic dysfunction was induced in rabbits immunized with recombinant α3 subunit protein [5] and in mice given anti-α3 subunit antibodies [6].Vernino et al. [7] demonstrated that IgG from the seropositive patients specifically reduced ganglionic nAChR current in human embryonic kidney (HEK) cells.In this study, we further examined whether antibody-induced reduction in the protein levels of cell-surface α3 subunits could cause ganglionic nAChR dysfunction in HEK293 cells.

Cell Culture
HEK293 cells stably co-expressing α3 and 4 subunits (HEK293-α34 cells) or mock-transfected HEK293 cells were established in our laboratory [8] and cultured in a humidified incubator with 5% CO 2 at 37˚C in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 100 g/ml G418 (Roche Applied Science, Germany).

Subcellular Fractionation
The cells on culture dishes were scraped, pelleted by centrifugation, and homogenized in a solution containing 0.32 M sucrose, 2 mM EDTA, 2 mM EGTA, 20 mM HEPES and protease inhibitor cocktail (BioVision Research Products, USA) at pH 7.2.Subcellular fractionation was performed as summarized in Figure 1(a).At first, we centrifuged out the nuclear pellet (P1 fraction) at 1000 ×g for 5 min.The resulting supernatant was then centrifuged at 170,000 ×g for 60 min, and cytosolic supernatant was transferred to a new tube (S2 fraction).The resulting pellet of membrane fraction was resuspended in the homogenization solution (P2 fraction).Protein concentration of each fraction was determined using DC protein assay (BIO-RAD, USA).

Assay for Antibody-Induced Internalization of α3 Subunits
HEK293-α34 cells were seeded at 5.0 × 10 4 cells/cm 2 on glass coverslips.On the second day, the cells were pre-incubated in Locke's buffer (154 mM NaCl, 5.6 mM KCl, 2.3 mM CaCl 2 , 1.0 mM MgCl 2 , 3.6 mM NaHCO 3 , 5 mM glucose and 5 mM HEPES, at pH 7.2) for 30 min at 4˚C or 37˚C, and then incubated with 1 g/mL rat anti-α3 subunit antibody (Covance, USA) in the Locke's buffer at 4˚C or 37˚C.In some cases, Locke's buffer containing 0.45 M sucrose was used as hypertonic condition.

Immunofluorescence Staining
HEK293-α34 and mock-transfected HEK293 cells were fixed in ice-cold 4% paraformaldehyde (PFA) for 20 min, washed in phosphate-buffered saline (PBS), blocked in PBS containing 1% BSA and 0.3% Triton X-100 for 30 min, and incubated with primary antibodies (Table 1) overnight at 4˚C.The cells were washed in PBS containing 0.3% Triton X-100 (PBS-T), incubated with secondary antibodies (Table 1) for 1 hr at RT, and again washed in PBS-T.For cell-surface staining, Triton X-100 was omitted from all solutions.The coverslips were mounted on glass slides with Dapi-Fluoromount-G (Southern Biotech, USA).Images were captured by a BX51 fluorescence microscope (Olympus, Japan).

Cell-Surface Enzyme-Linked
Immunosorbent Assay (ELISA) α3 subunits expressed on cell-surface membrane were quantitated by cell-surface ELISA.HEK293-α34 cells  were seeded into each well of a 24-well culture plate at 2.5 × 10 5 cells/cm 2 .On the second day, the cells were incubated with 1 g/mL rat anti-α3 subunit antibody in isotonic or hypertonic Locke's buffer for 5 -30 min at 37˚C.The cells were washed in Locke's buffer at 4˚C and α3 subunits on the cell-surface were labeled with 1 g/mL rat anti-α3 subunit antibody in the Locke's buffer for 30 min at 4˚C.The cells were then fixed in ice-cold 4% PFA for 20 min.After fixation, the cells were washed in PBS and blocked in PBS containing 1% BSA for 30 min.And then, the cells were incubated with HRP-conjugated anti-rat IgG antibody (Table 1) for 1 hr at RT and washed in PBS.Signals were generated using TMB microwell peroxidase substrate system (KPL, USA).After stopping the reaction with 2 M sulfuric acid, absorbance of the dye was measured at a wavelength of 450 nm using Multiskan MS-UV (Thermo Fisher Scientific, Finland).

Intracellular Ca 2+ Assay
Intracellular Ca 2+ levels were measured using Fluo-3 AM, membrane-permeable Ca 2+ -sensitive fluorescent dye.HEK293-α34 and mock-transfected HEK293 cells were seeded into 35-mm glass bottom dish or each well of a 96-well culture plate at 2.0 × 10 5 cells/cm 2 .On the third day, we incubated the cells in Locke's buffer containing 10 M Fluo-3 AM (Dojindo Laboratories, Japan) for 60 min at 37˚C.The cells were washed in Locke's buffer, and incubated with 1 g/mL rat anti-α3 subunit antibody or 1 mM mecamylamine (Sigma-Aldrich, USA) in Locke's buffer for 5 -30 min at 4˚C or 37˚C.Nicotine (Wako Pure Chemical Industries, Japan) was diluted in Locke's buffer and added to the cells at a final concentration of 1 mM.Fluorescence images of the cells on the 35-mm glass bottom dish were recorded using BIO-REVO BZ-9000 fluorescence microscope (Keyence, Japan).Fluorescence intensity of the cells on the 96-well culture plate was measured using a fluorometric plate reader (Fluoroskan Ascent; Thermo Fisher Scientific) at 485 nm excitation and 538 nm emission.

RESULTS
To examine the effects of the antibody on cell-surface α3 subunits in vitro, we used HEK293-α34 cells [8].The cellular distribution of both subunits was examined by immunoblot analysis using subcellular fractions prepared from HEK293-α34 cells (Figure 1(a)).We used P1, P2 and S2 fractions as nuclear, membrane and cytosolic fractions, respectively.Predicted bands of α3 (50-kDa) and 4 (55-kDa) subunits were detected using each specific antibody in all subcellular fractions from HEK293-α34 cells, but not in homogenate from mock-transfected cells.Both subunits were mainly detected in P2 fraction of HEK293-α34 cells.The 42-kDa band recognized by anti-4 subunit antibody might represent a proteolytic form.The 70-kDa band, which was also present in the mock-transfected cells, might be due to non-specific reactions.Immunoreactivities for α3 and 4 subunits were detected by immunofluorescence staining under non-permeable condition, indicating sta-ble co-expression on the cell-surface (Figure 1

(b)).
To confirm whether exogenous α3 and 4 subunits formed functional nAChR, we examined nicotine-evoked Ca 2+ influx using Fluo-3 AM (Figure 1(c)).Nicotine elicited a transient elevation of fluorescence intensity (1.56-fold) in HEK293-α34 cells, but not in mock-transfected cells (<1.1-fold).Mecamylamine, an antagonist of neuronal nAChRs, inhibited the Ca 2+ influx in HEK293-α34 cells (Figure 1(c)).These data indicate the expression of functional nAChRs assembled from α3 and 4 subunits on the cell-surface.Using this cell line, we examined antibody-induced internalization of α3 subunits.After application of rat antibody recognizing extracellular NH 2 -terminal domain of α3 subunit (Anti-α3) for 30 min, internalized antibodies were detected by Alexa Fluor 488-conjugated anti-rat IgG antibody.At the same time, α3 subunits were stained with rabbit antibody against second intracellular loop of α3 subunit, followed by detection with Alexa Fluor 594-conjugated anti-rabbit IgG antibody.When the cells were incubated with Anti-α3 at 37˚C under isotonic condition, punctate rat IgG-immunoreactivity (IR) overlapped largely with punctateα3 subunit-IR (Figure 2(a)).In contrast, low-temperature and hypertonic condition inhibited the formation of punctate-IR for the rat IgG and α3 subunit.We next examined involvement of endocytic machinery in antibody internalization by double-labeled immunofluorescence staining using anti-early endosome antigen1 (EEA1) antibody (Figure 2(b)).When HEK293-α34 cells were incubated with Anti-α3 for 30 min at 37˚C in isotonic Locke's buffer, punctate rat IgG-IR were largely overlapped with punctate EEA1-IR.In contrast, no double-positive dots were formed under hypertonic condition.α3 subunits on the cell-surface were quantitated by cell-surface ELISA (Figure 2(c)).Application of Anti-α3 in isotonic Locke's buffer for 5, 15 and 30 min at 37˚C resulted in 37%, 60% and 67% decreases, respectively.Under hypertonic condition, the reduction at 30 min was significantly suppressed (21%, p < 0.01).
To examine whether the Anti-α3 inhibited nAChR function, we evaluated nicotine-evoked Ca 2+ influx using Fluo-3 AM.When HEK293-34 cells were incubated with Anti-3 for 30 min, nicotine-evoked elevation of fluorescence intensity was remarkably suppressed compared with the control cells (Figure 3(a)).We analyzed peak Fluo-3 fluorescence intensities obtained by addition of nicotine (Figures 3(b) and (c)).Application of Anti-3 for 5, 15 and 30 min resulted in 24%, 42% and 63% decreases of peak fluorescence intensity, respectively (Figure 3(b)).In contrast, when the cells were incubated with the antibody at 4˚C, no reduction was detected (Figure 3(c)).These results clearly indicate that the reduction in Ca 2+ influx is due to decrease of cell-surface nAChRs, and that antagonistic effect of the antibody is little, if any.

DISCUSSION
In the present study, we showed that the antibody-induced reduction of cell-surface α3 subunits is the main cause of ganglionic nAChR dysfunction.Since these effects of the antibody were markedly inhibited under low-temperature or hypertonic condition, endocytic machinery could impair the fast excitatory synaptic transmission in autonomic ganglia of AAG patients.
The antibody-induced internalization of ganglionic nAChR has been suggested to be involved in the auto- nomic dysfunction.Interestingly, Wang et al. [9] reported that whole IgG from seropositive AAG patients decreased the nAChR current in IMR-32 neuroblastoma cells, but Fab fragments did not.In addition, nAChR current was not inhibited by the autoantibodies under low-temperature condition.Although their results suggest that the impairment of the ganglionic nAChR function requires the antibody-mediated cross-linking and the subsequent internalization of cell-surface 3 subunits, there is no direct experimental evidence.In this study, we demonstrated intracellular punctate accumulation of antibody-3 subunit complexes and localization of the antibodies in endocytic vesicles.In addition, our results from cell-surface ELISA indicated that antibody-induced reduction in cell-surface 3 subunits was rapidly progressed within 30 min as predicted by Wang et al. [9].The antibody simultaneously caused nAChR dysfunction within 30 min as shown by loss of nicotine-evoked Ca 2+ influx.When the endocytic machinery was inhibited by low-temperature or hypertonicity [10], the effects of the antibody described above were remarkably attenuated.
Although it remains to be seen whether these effects of anti-3 subunit antibody could be induced in vivo, Lennon et al. [5] reported that rabbit injected with NH 2 -terminal extracellular domain (ECD) of 3 subunit protein exhibited autonomic failure.Furthermore, IgG collected from this animal model produced a progressive decline of nAChR current in IMR-32 neuroblastoma cells [9].We previously demonstrated in HEK293-α34 cells that the antibody against ECD of 3 subunit induced the internalization and cytoplasmic accumulation of the 3 subunits [8].Our present study extends these previous observations about pathogenic mechanisms of anti-3 subunit autoantibody.
In conclusion, this study clearly demonstrates the involvement of endocytic machinery in the antibody-induced reduction in cell-surface 3 subunits and impairment of ganglionic nAChR function.Our data reinforce the idea that reduction of the ganglionic nAChR in autonomic ganglia plays an important pathogenic role in seropositive AAG patients.Our approaches described in this report may help to clarify the pathogenicity of the individual anti-3 subunit autoantibody.

Figure 2 .
Figure 2. Antibody-induced endocytosis of α3 subunits in HEK293-α3β4 cells.(a, b) HEK293-α3β4 cells were incubated with 1 μg/mL rat anti-α3 subunit antibody for 30 min at 37˚C or 4˚C in isotonic or hypertonic condition.The cells were then fixed, permeabilized and co-stained with rabbit anti-α3 subunit antibody (a) or rabbit anti-EEA1 antibody (b), followed by detection with Alexa Fluor 488-conjugated anti-rat IgG (green) and Alexa Fluor 594-conjugated anti-rabbit IgG (red) antibodies.Scale bars, 5 μm.(c) Quantification of α3 subunit expressed on the cell-surface membrane by cell-surface ELISA.HEK293-α3β4 cells were incubated with 1 μg/mL rat anti-α3 subunit antibody in isotonic or hypertonic condition for indicated time periods at 37˚C.Data are expressed as means ± S.D. from three independent experiments.Statistical comparisons were carried out by one-way ANOVA followed by Tukey test.* p < 0.01 vs. Control; # p < 0.01.

Figure 3 .
Figure 3. Antibody-induced dysfunction of nAChR.(a) Fluo-3 AM-loaded HEK293-α3β4 cells were incubated with or without 1 μg/mL rat anti-α3 subunit antibody (Anti-α3) for 30 min at 37˚C, and then stimulated by 1 mM nicotine.Changes in intracellular Ca 2+ concentration was monitored using a fluorescence microscope (left) and a fluorometric plate reader (right).Addition of nicotine is pointed by arrow.Data shown are representative responses from two independent experiments, and expressed as F/F 0 .(b, c) Peak Fluo-3 fluorescence intensity.The cells were incubated with or without 1 μg/mL rat anti-α3 subunit antibodies (Anti-α3) for indicated time periods at 37˚C (b) or for 30 min at 4˚C (c).Data are expressed as means ± S.D. of [(F max -F min )/F min ] from three independent experiments.F max is peak fluorescence intensity; F min is averaged fluorescence intensity of the first 1 to 20 counts before the addition of nicotine.Statistical comparisons were carried out by one-way ANOVA followed by Dunnett's test or two-tailed t-test.* p < 0.05, ** p < 0.01 vs. Control.NS, not significant.

Table 1 .
List of antibodies used in this study.