α4β2 nicotinic acetylcholine receptors in the early postnatal mouse superior cervical ganglion

Abstract

Heteropentameric nicotinic acetylcholine receptors (nAChR) mediate fast synaptic transmission in ganglia of the autonomic nervous system. It is undisputed that α3 and β4 are the predominant subunits in the superior cervical ganglion (SCG); however, reports on the presence of receptors that contain α4 have been controversial. Here, we have searched for the presence of α4-containing nAChRs in the postnatal rat and mouse SCG. We now show by immunoprecipitation combined with radioligand binding that α4-containing receptors constitute about 20% of hetero-oligomeric nAChRs in postnatal Day 3 (P3) mice. However, already by P9, the level of α4 approaches zero. In contrast, the number of α4-containing receptors is close to zero in the rat SCG at all times investigated. Deletion of the β2 subunit by using α5β2-double knockout (KO) mice removes all α4-containing receptors, suggesting that in the postnatal mouse SCG, α4 co-assembles only with β2 but not with β4. α4β2 receptors are, on the other hand, up-regulated in the SCG of P3 α5β4-double KO mice, where they make up about 50% of receptors that bind [(3) H]-epibatidine. Nonetheless, receptors on the surface of SCG neurons from α5β4-double KO mice maintained for one to two days in culture comprise <10% of α4β2 and >90% of α3β2, as determined by patch clamp recordings with α4β2- and α3β2-specific ligands. We propose that in the P3 SCG of wild type mice, α3β4 (±α5) represent about 62% of receptors, whereas 17% are α3β2β4, and 21% are α4β2 (±α5) receptors.

Figure 1. α4-containing nAChRs in the mouse SCG decline soon after birth and are barely detectable in the rat

nAChRs from SCG membranes of 2, 3, 5, 9 or 18 day old WT mice and 2, 5 or 18 day old rats were solubilized, labeled with 1 nM [³H]-epibatidine, and immunoprecipitated with our anti-α4 antibodies. Nonspecific binding was measured in the presence of 300 μM nicotine and subtracted from overall to obtain the specific binding shown. Data are the mean ± SEM of 3-6 independent experiments, each performed with duplicate (P3, P9) or triplicate (P2, P5, P18) measurements. *, **, ***: significantly different from zero (P < 0.05, 0.01, and 0.001, respectively; one sample Students t-test). The specificity and efficacy of our anti-α4 antibodies is documented in David et al., 2010 (for mouse nAChRs) and in the supplementary material (for rat nAChRs).

Figure 2. The nAChR subunit α4 is present in short-term SCG cell cultures from P3 mice

Indirect immunofluorescence of actin (A1-A3) and the α4 (B1-B2) and α3 (B3) nAChR subunits as detected by mouse anti-actin (1:1000), rabbit anti-α4, and rabbit anti-α3 (each 0.4μg/ml). Since the experiments were designed to parallel the patch clamp recordings shown in Fig. 5, cultures were prepared from α5α7β4-triple KO mice. A1 and B1: Cell culture 1 day in vitro prepared from P3 α5α7β4-triple KO mice. A2, A3, B2, and B3: Cell cultures 6 days in vitro prepared from P6 α5α7β4-triple KO mice. Note that the intensity of the fluorescent signal due to α4 is greatly attenuated in cells taken from P6 animals and cultured for 6 days (B2). Panel B3 shows the fluorescent signal due to α3 for comparison. Calibration: 25 μm.

Figure 3. Subunit composition of P3 wild-type mice nAChRs

nAChRs from SCG membranes of three day old wild-type mice were solubilized, labeled with 1 nM [³H]-epibatidine and immunoprecipitated with each of the subunit-specific antibodies indicated at the abscissa. Nonspecific binding was measured in the presence of 300 μM nicotine and subtracted from overall to obtain the specific binding shown in the figure. Data are the mean ± SEM of 3-5 independent experiments, each performed with duplicate measurements. The dashed line indicates the total number of receptors which are precipitated by the combined use of anti-β2 and anti-β4 antibodies. Though comparisons don't reach levels of significance, the results suggest that separate α3- (82 %) and α4-containing receptors (19 %) make up for 100 % of the receptors. Likewise, α4-containing receptors seem to be less frequent than β2-containing receptors, whereas the combined use of anti- α4 and anti-β2 antibodies does not precipitate more receptors than the single use of anti-β2 antibodies. Statistical significance between data points was calculated by one-way ANOVA, followed by Bonferroni's post-hoc multiple comparison test (n.s.: P > 0.05; **, ***: significantly different from β2 + β4 with P < 0.01 and P < 0.001, respectively).

Figure 4. β subunits markedly affect the expression of both α3- and α4-containing receptors

nAChRs from SCG membranes of WT (black bars), α5β2 (white bars), and α5β4 (grey bars) KO mice were solubilized, labeled with 1 nM [³H]-epibatidine, and immunoprecipitated with either anti-α3 or anti-α4 antibody. Nonspecific binding was measured in the presence of 300 μM nicotine and subtracted from overall to obtain the specific binding shown in the figure. Data are the mean specific binding relative to immunoprecipitation with anti-β2 + anti-β4 (for WT), anti-β2 (for α5β4 KO), and anti-β4 (for α5β2 KO) antibodies, respectively (dashed line, 100 %) ± SEM of 3-6 independent experiments, each performed with duplicate measurements. Note that α4 is lost (n.s.: not significantly different from zero, P > 0.05, one sample Students t-test) in the SCG of α5β2 KO and significantly upregulated in α5β4 KO mice. α3-containing receptors are, on the other hand, significantly reduced in α5β4 KO mice. Statistical significance between data points was calculated by one-way ANOVA, followed by Bonferroni's post-hoc multiple comparison test (n.s.: P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001).

Fig. 5. Few α4-containing nAChR reach the cell surface

A. Patch clamp recordings (perforated patch) from cultured postnatal day 3 SCG neurons (α5β4α7-triple KO), kept for 2 days in culture. Agonists and α-conotoxin MII were always applied in the indicated order: (1) 100 μM nicotine, the 2 sec application indicated by bar. (2) 300 μM RJR 2403, the 2 sec application indicated by bar. (3) 100 μM nicotine after 10 sec of application of 100 nM α-conotoxin MII. (4) 100 μM nicotine after 130 sec of application of 100 nM α-conotoxin MII. Calibration bars: 100 pA; 1 sec. B. Pooled observations from 38 cells. Data are mean peak currents ± SEM. Figures on top of the bars indicate current densities relative to the currents induced by nicotine (in the absence of α-conotoxin MII). C. Comparison of effects obtained in α5β4α7-triple KO (filled bars; n = 32) and α5β4-double KO mice (open bars; n = 6). Peak amplitudes by indicated protocols relative to 100 μM nicotine did not differ between the two genotypes (P > 0.05, Student's t-test).

Fig. 6. Proposed nAChR subtypes occurring in the SCG of P3 WT, α5β2-, and α5β4 KO mice

α5β2-double KO (white bars) mice also lack the α4 subunit (see Fig. 4), which leaves only α3β4 hetero-oligomeric receptors in this genotype. Compared to WT mice, the overall number of receptors remains unaffected by the KO. α5β4-double KO (grey bars) mice contain equal numbers of α3- and α4-containing receptors (see Fig. 4). In the absence of β4, the two subunits share the β2 subunit at about identical rates of recurrence (Fig. 4). Compared to WT mice, the overall number of receptors is significantly reduced in the KO. WT (black bars): About 80 % of receptors contain the subunit α3, versus 20 % of receptors with α4 (Fig. 3). β2 subunits (which occur in about 40 % of the receptors, Fig. 3) that do not co-assemble with α4 (about 50 % according to experiments shown in Fig. 2) assemble with α3. The model infers from our observations in P19 WT animals (David et al., 2010) that α3β2 receptors always contain the β4 subunit, whereas β2 and α5 never occur in the same receptor. It also infers that α4 only co-assembles with β2 but not with β4 (Fig. 4). We did not probe α5-containing receptors which may concur with α3 and β4 as in P19 animals (David et al., 2010). The α5 subunit could, in principle, also co-assemble with α4β2 receptors (Gotti et al., 2006). According to this model, 62 % of receptors in P3 WT animals consist of α3β4 with and without α5, 17 % are of the α3β2β4 type, and 21 % are α4β2 receptors with our without α5.