Functional Nicotinic Acetylcholine Receptors That Mediate Ganglionic Transmission in Cardiac Parasympathetic Neurons

Abstract

Nicotinic acetylcholine receptors (nAChRs) mediate ganglionic transmission in the peripheral autonomic nervous system in mammals. Functional neuronal nAChRs have been shown to assemble from a combination of alpha and beta subunits, including alpha3, alpha5, alpha7, beta2, and beta4 in RNA-injected oocytes, but the subunit composition of functional neuronal nAChRs in vivo in mammals remains unknown. We examined the subunit composition of functional nAChRs in the intracardiac parasympathetic ganglion in a physiologically intact system in vivo. We report here that localized perfusion of the canine intracardiac ganglion in situ with an antagonist specific for nAChRs containing an alpha3/beta2 subunit interface (alpha-conotoxin MII 100-200 nm) resulted in reversible attenuation of the sinus cycle length (SCL) response by approximately 70% to electrical stimulation of the preganglionic vagus nerve. Perfusion with antagonist specific for receptors containing an alpha3/beta4 subunit interface (alpha-conotoxin AuIB 1 micrometer) resulted in attenuation in SCL responses (approximately 20%) compared with baseline when applied by itself, but not in animals pretreated with alpha-conotoxin MII. Perfusion of the ganglion with alpha-bungarotoxin (1 micrometer, which blocks alpha7 receptors) caused a reduction in SCL response by approximately 30% compared with baseline when perfused on its own and when added after blockade with MII and AuIB. Perfusion with hexamethonium bromide resulted in complete blockade of ganglionic transmission, confirming total perfusion of the ganglion and the nicotinic nature of ganglionic transmission at this synapse. Immunohistochemistry using monoclonal antibodies against specific nicotinic subunits confirmed the presence of alpha3, alpha7, beta2, and beta4 subunits. We conclude that functional ganglionic transmission in the canine intracardiac ganglion is mediated primarily by receptors containing an alpha3/beta2 subunit interface, with a smaller contribution by receptors containing alpha7 nAChRs. Despite the presence of beta4 subunits in functional channels, a contribution of a distinct alpha3/beta4 receptor population that does not include an alpha3/beta2 subunit interface was less clear.

Selective Modulation of Neuronal Nicotinic Acetylcholine Receptor Channel Subunits by Go-Protein Subunits

“…The high degree of subunit specificity of G-protein modulation of neuronal nAChRs is intriguing, given that neuronal nAChRs of rat intrinsic cardiac ganglia have been shown to be composed mainly of ␣3, ␣5, ␣7, ␤2, and ␤4 subunits (Poth et al, 1997;Bibevski et al, 2000) and that a large component of cholinergic (vagal) transmission in intracardiac neurons is mediated by the ␣3␤2 subunit (Bibevski et al, 2000). G-protein activation did not modify currents evoked by the cholinergic agonist, cytisine, which has been shown to be most potent for ␣3␤4 and ␣3␤4␣5 subunit combinations (Covernton et al, 1994).…”

“…The lack of G-protein binding and potentiation of ␣7 nAChRmediated currents by G-protein activation supports the hypothesis that at least one ␤ subunit of the nAChR is required for G-protein activation. The high probability of the occurrence of ␣3␤2-containing nAChRs in mammalian intracardiac neurons (Poth et al, 1997;Bibevski et al, 2000) explains the robust potentiation of ACh-and nicotine-evoked currents by G-protein subunits. The coimmunoprecipitation of native nAChRs with G o ␣ and G␤␥ subunits clearly demonstrates direct protein interaction.…”

“…To date, eight ␣ (␣2-␣7, ␣9, and ␣10) and three ␤ (␤2-␤4) subunits have been identified in the mammalian nervous system. Neuronal nAChRs on intrinsic cardiac ganglion (ICG) neurons mediate ganglionic transmission and are composed of ␣3, ␣5, ␣7, ␤2, and ␤4 subunits (Poth et al, 1997;Bibevski et al, 2000). In contrast to the metabotropic muscarinic AChR (mAChR), the nAChR does not require the activation of guanine nucleotide-binding proteins (G-proteins) to open its associated ion channel.…”

Nicotinic mechanisms in the autonomic control of organ systems

“…While in mice and rats ␤2* nAChRs seem to have a minor role in the autonomic control of cardiac function, in dogs ␣3␤2* nAChRs have been proposed as the main mediators of ganglionic transmission in intracardiac ganglia. In these animals, the contribution of ␣3␤4* receptors that do not contain ␤2 at the same time (i.e., ␣3␤4 vs. ␣3␤2␤4 combinations) is not clear (Bibevski et al, 2000).…”

“…The ␣3 nAChR subunit is expressed at high levels in all autonomic ganglia, including the sympathetic ganglia innervating the heart and the cardiac ganglionated plexuses (Cimino et al, 1992;Mandelzys et al, 1994;Rust et al, 1994;Poth et al, 1997). ␣3 is likely to participate in the majority of nAChR subtypes expressed by autonomic neurons (Listerud et al, 1991;Poth et al, 1997;Xu et al, 1999a;Bibevski et al, 2000) because its absence produces a severe in vivo phenotype with high perinatal mortality and autonomic dysfunction at the level of several organ systems (Xu et al, 1999a;De Biasi et al, 2000). Acutely isolated superior cervical ganglion (SCG) neurons from ␣3 null (Ϫ/Ϫ) mice express only two out of the five nAChR currents recorded from wild-type (ϩ/ϩ) neurons.…”