The role of the nAChR subunits α 5, β 2, and β 4 on synaptic transmission in the mouse superior cervical ganglion

Abstract

Our previous immunoprecipitation analysis of nicotinic acetylcholine receptors ( nAChR s) in the mouse superior cervical ganglion ( SCG ) revealed that approximately 55%, 24%, and 21% of receptors are comprised of α 3 β 4, α 3 β 4 α 5, and α 3 β 4 β 2 subunits, respectively. Moreover, mice lacking β 4 subunits do not express α 5‐containing receptors but still express a small number of α 3 β 2 receptors. Here, we investigated how synaptic transmission is affected in the SCG of α 5 β 4‐ KO and α 5 β 2‐ KO mice. Using an ex vivo SCG preparation, we stimulated the preganglionic cervical sympathetic trunk and measured compound action potentials ( CAP s) in the postganglionic internal carotid nerve. We found that CAP amplitude was unaffected in α 5 β 4‐ KO and α 5 β 2‐ KO ganglia, whereas the stimulation threshold for eliciting CAP s was significantly higher in α 5 β 4‐ KO ganglia. Moreover, intracellular recordings in SCG neurons revealed no difference in EPSP amplitude. We also found that the ganglionic blocking agent hexamethonium was the most potent in α 5 β 4‐ KO ganglia ( IC 50 : 22.1 μ mol/L), followed by α 5 β 2‐ KO ( IC 50 : 126.7 μ mol/L) and WT ganglia ( IC 50 : 389.2 μ mol/L). Based on these data, we estimated an IC 50 of 568.6 μ mol/L for a receptor population consisting solely of α 3 β 4 α 5 receptors; and we estimated that α 3 β 4 α 5 receptors comprise 72% of nAChR s expressed in the mouse SCG . Similarly, by measuring the effects of hexamethonium on AC h‐induced currents in cultured SCG neurons, we found that α 3 β ...

The α5 Nicotinic Acetylcholine Receptor Subunit Differentially Modulates α4β2* and α3β4* Receptors

“…In β2 KO mice, the SCG neurons express ~75% α3β4 * receptors and ~25% (α3β4) 2 α5 receptors, whereas α5β2 double-KO mice express exclusively α3β4 * hetero-pentameric receptors, making these ideal models for investigating the role of the α5 subunit in endogenous α3β4 * receptors (David et al, 2010 ). Moreover, as discussed below, experiments showed that the percentage of (α3β4) 2 α5 receptors in the plasma membrane is significantly higher than the percentage of the overall receptor pool determined by immunoprecipitation (Simeone et al, 2019 ). With respect to ligand potency, David and colleagues found no difference in either the potency or efficacy of cytisine or DMPP between cultured neurons prepared from either α5β2 double-KO mice or β2 KO mice, and the total number of nACh receptors was not reduced in either α5β2 double-KO mice or β2 KO mice (David et al, 2010 ).…”

“…These KO mouse models were also used to measure receptor desensitization during prolonged exposure to nicotine in an intact SCG preparation in which compound action potentials (CAPs) were recorded from the postganglionic nerve in response to supramaximal stimulation of the preganglionic nerve. Nicotine added to the superfusion buffer at increasing concentrations yielded IC 50 values of 3.01 μM for α3β4 * receptors and 3.67 μM for (α3β4) 2 α5 receptors (Simeone et al, 2019 ). This small, yet statistically significant difference indicates that the presence of the α5 subunit provides a slight degree of protection from receptor desensitization, although these levels of nicotine are not achieved, even with heavy smoking (Moreyra et al, 1992 ).…”

“…Varying the stimulation frequency of the preganglionic nerve also revealed differences in CAP amplitude between ganglia expressing α3β4 * receptors and ganglia expressing (α3β4) 2 α5 receptors (Simeone et al, 2019 ). With a stimulation frequency of 5 Hz, CAP amplitude increased significantly in WT ganglia but not in α5β2 KO ganglia; with 10-Hz stimulation, however, CAP amplitude decreased in α5β2 KO ganglia but not in WT ganglia, suggesting differences in activation and/or desensitization properties between these two receptors (Simeone et al, 2019 ).…”

“…As noted above, only 25% of all receptors in the SCG of β2 KO mice contain the α5 subunit, and this relatively small contribution to the total receptor pool may not be sufficient to reveal differences at the whole-cell level. However, the non-competitive nAChR antagonist hexamethonium was significantly less potent at blocking (α3β4) 2 α5 receptors compared to the α3β4 * hetero-pentameric receptors expressed in α5β2 double-KO mice (see also Wang et al, 2002 ; Simeone et al, 2019 ). Using the difference in the right-shift in the concentration-response curve allowed Simeone and colleagues to calculate the potency of hexamethonium at blocking a hypothetical “pure” population of (α3β4) 2 α5 receptors, as well as the percentage of these receptors present at the cell surface.…”

“…Using the difference in the right-shift in the concentration-response curve allowed Simeone and colleagues to calculate the potency of hexamethonium at blocking a hypothetical “pure” population of (α3β4) 2 α5 receptors, as well as the percentage of these receptors present at the cell surface. Interestingly, the authors found that hexamethonium inhibited transganglionic transmission in intact ganglia to the same extent as cultured SCG neurons stimulated with 100 μM ACh, suggesting that α5-containing receptors are dispersed along the surface of SCG neurons and are not specifically targeted to synaptic sites (Simeone et al, 2019 ). The finding that 72 and 63% of surface receptors are (α3β4) 2 α5 receptors in intact ganglia and cultured SCG neurons, respectively, indicates that α5-containing receptors are enriched at the plasma membrane (Simeone et al, 2019 ).…”