The 7 Nicotinic Acetylcholine Receptor Subunit Exists in Two Isoforms that Contribute to Functional Ligand-Gated Ion Channels

University of South Florida, Tampa, Florida, United States
Molecular Pharmacology (Impact Factor: 4.13). 10/2004; 66(3):420-9. DOI: 10.1124/mol.104.000059
Source: PubMed


Fast synaptic transmission in mammalian autonomic ganglia is mediated primarily by nicotinic receptors, and one of the most abundant nicotinic acetylcholine receptor subtypes in these neurons contains the alpha7 subunit (alpha7-nAChRs). Unlike alpha7-nAChRs expressed in other cells, the predominant alpha7-nAChR subtype found in rat intracardiac and superior cervical ganglion neurons exhibits a slow rate of desensitization and is reversibly blocked by alpha-bungarotoxin (alphaBgt). We report here the identification of an alpha7 subunit sequence variant in rat autonomic neurons that incorporates a novel 87-base pair cassette exon in the N terminus of the receptor and preserves the reading frame of the transcript. This alpha7 isoform was detected using reverse transcriptase-polymerase chain reaction techniques in neonatal rat brain and intracardiac and superior cervical ganglion neurons. Immunoblot experiments using a polyclonal antibody directed against the deduced amino acid sequence of the alpha7-2 insert showed a pattern of expression consistent with alpha7-2 subunit mRNA distribution. Moreover, the alpha7-2 subunit could be immunodepleted from protein extracts by solid-phase immunoprecipitation techniques using the anti-alpha7 monoclonal antibody 319. The alpha7-2 subunit was shown to form functional homomeric ion channels that were activated by acetylcholine and blocked by alpha-bungarotoxin when expressed in Xenopus laevis oocytes. This alpha7 isoform exhibited a slow rate of desensitization, and inhibition of these channels by alphaBgt reversed rapidly after washout. Taken together, these data indicate that the alpha7-2 subunit is capable of forming functional alphaBgt-sensitive acetylcholine receptors that resemble the alpha7-nAChRs previously identified in rat autonomic neurons. Furthermore, the distribution of the alpha7-2 isoform is not limited to peripheral neurons.

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    • "The alternative splicing has been also reported for various mammalian nAChR genes, and can be associated with human diseases. For example, alternative splicing in the neuronal α4 and α7 nAChR subunits is associated with Tourette syndrome [32] and schizophrenia [33], respectively, and that of the α1 and ε subunits of the muscle-type nAChR—with congenital myasthenic syndromes [34], [35], [36]. Indeed, the presence of non-functional α1 nAChR resulting from RNA splicing was found in muscle cells [37]. "
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    • "These mutations exist within different regions of the receptor, such as within the second transmembrane domain lining the pore of the channel (Revah et al. 1991), as well as in the D-loop near the ligand-binding domain (Gay et al. 2008). Other studies have shown that α7 nAChR desensitization kinetics can be affected by co-assembly with non-α7 subunits such as β2 (Khiroug et al. 2002), or by alternative splicing of the α7 receptor (Severance et al. 2004). In this study, we have identified a critical proline residue near the middle of the β9 strand which, when mutated to specific amino acids, confers markedly slower desensitization to the rat α7 receptor. "
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    • "With the tip of the superfusion placed 120 μm above our cells we reach 75 % of the final concentration of solutions within 35 msec. This is considerably slower than the rapid application system used by others (full concentration reached within 5 msec) to record the currents carried by the fast desensitizing splice variant α7-1 of the α7 nAChR (Zhang et al., 1994; Severance et al., 2004). By comparing their rapid application with a conventional but slower puffer pipette, these authors concluded that the high speed of the superfusion is a prerequisite for the detection of currents in response to α7-1 activation in chick ciliary ganglion neurons (Zhang et al., 1994). "
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