The ?7 Nicotinic Acetylcholine Receptor Subunit Exists in Two
Isoforms that Contribute to Functional Ligand-Gated Ion
Emily G. Severance, Hongling Zhang, Yolmari Cruz, Sergei Pakhlevaniants,
Stephen H. Hadley, Jahanshah Amin, Lynn Wecker, Crystal Reed, and Javier Cuevas
Department of Pharmacology and Therapeutics, University of South Florida College of Medicine, Tampa, Florida
Received March 4, 2004; accepted June 17, 2004
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 ?7 subunit (?7-nAChRs). Unlike ?7-
nAChRs expressed in other cells, the predominant ?7-nAChR
subtype found in rat intracardiac and superior cervical ganglion
neurons exhibits a slow rate of desensitization and is reversibly
blocked by ?-bungarotoxin (?Bgt). We report here the identifi-
cation of an ?7 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 ?7 isoform was detected using reverse
transcriptase-polymerase chain reaction techniques in neona-
tal rat brain and intracardiac and superior cervical ganglion
neurons. Immunoblot experiments using a polyclonal antibody
directed against the deduced amino acid sequence of the ?7-2
insert showed a pattern of expression consistent with ?7-2
subunit mRNA distribution. Moreover, the ?7-2 subunit could
be immunodepleted from protein extracts by solid-phase im-
munoprecipitation techniques using the anti-?7 monoclonal
antibody 319. The ?7-2 subunit was shown to form functional
homomeric ion channels that were activated by acetylcholine
and blocked by ?-bungarotoxin when expressed in Xenopus
laevis oocytes. This ?7 isoform exhibited a slow rate of desen-
sitization, and inhibition of these channels by ?Bgt reversed
rapidly after washout. Taken together, these data indicate that
the ?7-2 subunit is capable of forming functional ?Bgt-sensitive
acetylcholine receptors that resemble the ?7-nAChRs previ-
ously identified in rat autonomic neurons. Furthermore, the
distribution of the ?7-2 isoform is not limited to peripheral
Nicotinic acetylcholine receptor channels (nAChRs) that
contain the ?7 gene product (?7-nAChRs) are one of the most
abundant types of nicotinic receptors in the vertebrate ner-
vous system. The ?7-nAChRs differ from most neuronal nic-
otinic receptors in that they bind ?-bungarotoxin (?Bgt) with
high affinity and have calcium permeability comparable with
glutamatergic N-methyl-D-aspartate receptors (Seguela et
al., 1993). The ?7-nAChRs modulate various cell processes
ranging from synaptic transmission (Zhang et al., 1996) to
apoptosis (Berger et al., 1998). In the central nervous system,
these receptors seem to be involved in learning and memory
(Radcliffe and Dani, 1998) and have been linked to patho-
physiological conditions such as schizophrenia (Freedman et
al., 1997). Experiments have also shown that the ?-amyloid
peptide is a ligand for ?7-nAChRs (Liu et al., 2001; Dineley et
al., 2002), suggesting that ?7-nAChR function may be altered
during Alzheimer’s disease. Furthermore, the ?7-nAChRs
participate in important physiological processes in the vis-
cera, such as the regulation of inflammation (Wang et al.,
2003) and nicotine-induced nitrergic neurogenic vasodilation
(Si and Lee, 2002).
The ?7 nicotinic acetylcholine receptor subunit was first
cloned from chick brain (Schoepfer et al., 1990). This subunit
was later found to form functional homomeric AChRs when
expressed in Xenopus laevis oocytes (Seguela et al., 1993) and
to contribute to functional nAChRs in native cells (Alkondon
and Albuquerque, 1993; Zhang et al., 1994). Although there
is strong evidence that native ?7-nAChRs are homopentam-
ers (Chen and Patrick, 1997; Drisdel and Green, 2000), the
composition and stoichiometry of native ?7-nAChRs remains
This work was supported by National Institutes of Health grant HL63247
Article, publication date, and citation information can be found at
ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; ?7-nAChR, nicotinic acetylcholine receptor containing the ?7 gene product; ?Bgt,
?-bungarotoxin; bp, base pair; RT-PCR, reverse transcriptase-polymerase chain reaction; ACh, acetylcholine; SCG, superior cervical ganglia;
PCR, polymerase chain reaction; ICG, intracardiac ganglia; Ab, antibody; mAb, monoclonal antibody.
Copyright © 2004 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 66:420–429, 2004
Vol. 66, No. 3
Printed in U.S.A.
at ASPET Journals on December 30, 2015
to be confirmed. For example, the ?7 subunit can combine
with the ?2 subunit to form functional heteropentamers in X.
laevis oocytes (Khiroug et al., 2002), and the widespread
coexpression of these two subunits in the central nervous
system has led investigators to suggest that they may form
such heteropentamers in vivo (Azam et al., 2003). In addi-
tion, differences exist in the pharmacological and biophysical
properties of ?7-nAChRs from a variety of cell types, an
indication of variability in the structure or subunit composi-
tion of ?7-nAChRs. For example, whereas ?7-nAChRs in rat
hippocampal neurons desensitize rapidly and bind ?Bgt in an
irreversible manner (Alkondon and Albuquerque, 1993), ?7-
nAChRs in mammalian autonomic neurons desensitize
slowly and recover rapidly from ?Bgt blockade (Cuevas and
Berg, 1998; Cuevas et al., 2000). More recently, denervated
mouse muscle was shown to express a subtype of ?7-AChRs
that desensitizes slowly and is not blocked by the classic
?7-AChR–selective antagonist methyllycaconitine (Tsuneki
et al., 2003).
Cuevas and Berg (1998) proposed that the heterogeneity in
?7-nAChRs might be caused by cell-dependent expression of
?7 subunit isoforms. This theory was supported by the ob-
servation that splice variations of the ?7 subunit are detected
in human brain and leukocytes (Gault et al., 1998; Villiger et
al., 2002). However, most of these ?7 isoforms contain a
premature stop codon (Gault et al., 1998) or form a truncated
subunit that is not activated by acetylcholine (Villiger et al.,
2002). Although various other ligand-gated ion channels such
as 5-hydroxytryptamine-3 and GABA receptors express func-
tional splice variants with distinct properties (Bruss et al.,
2000), this type of diversity has been less forthcoming in
nicotinic acetylcholine receptors.
In the current study, we present the first evidence for a
functional splice variant of a nicotinic receptor subunit that
contributes to a distinctive channel. The ?7-2 isoform of the
?7 nicotinic receptor subunit contains an 87-base pair insert
that represents the incorporation of a novel exon (exon 4a)
between exons 4 and 5 of the conventional ?7 gene product
(?7-1). The ?7-2 splice variant is expressed in both peripheral
and central neurons, and when expressed in X. laevis oocytes,
it produces functional ACh-gated ion channels. The biophys-
ical and pharmacological properties of the homomeric chan-
nel formed by ?7-2 differ from those of homomeric ?7-1–
nAChRs and resemble those of the slowly desensitizing ?7-
nAChRs of rat autonomic neurons.
Materials and Methods
RT-PCR. The expression of ?7 nicotinic receptor subunit splice
variants in central and peripheral neurons was examined using
RT-PCR techniques similar to those reported previously (Zhang and
Cuevas, 2002). Total RNA was isolated from rat intracardiac ganglia
and associated tissue, superior cervical ganglia (SCG), nodosal gan-
glia and brain (RNeasy; QIAGEN GmbH, Hilden, Germany). Prim-
ers specific for the ?7 gene product (?7-P1) were designed to span an
intron to discriminate between genomic DNA and cDNA. Table 1
lists the sequences of the primers used in this study and the pre-
dicted size for the products. For single-cell RT-PCR experiments,
intracardiac neurons were dissociated from 4- to 7-day-old neonatal
rats, and cytoplasm was extracted from isolated neurons as de-
scribed previously (Zhang and Cuevas, 2002). All procedures were
done in accordance with the regulations of the University of South
Florida Institutional Animal Care and Use Committee. Negative
controls for these experiments included the use of water as template
for the RT-PCR and the suctioning of extracellular solution to control
for cytoplasmic contamination in the single-cell RT-PCR reactions.
PCR products were gel-purified using a QIAEX II Gel Purification kit
(QIAGEN) and sequenced by the Molecular Biology Core Facility at
the H. Lee Moffitt Cancer Center and Research Institute (University
of South Florida, Tampa, FL).
The relative abundance of ?7-1 and ?7-2 isoforms in central and
peripheral tissues was determined using the ?7-P5 primer combina-
tion (Table 1) containing two forward primers, one selective for both
isoforms and a second that specifically amplifies ?7-2 transcripts,
and a reverse primer that recognizes both ?7 splice variants. This
primer combination was used to overcome preferential amplification
of the smaller ?7-1 transcripts, which would occur if only a single
primer pair, such as ?7-P1 was used. The ability of this primer pair
to properly detect the relative abundance of ?7-1 and ?7-2 tran-
scripts was assessed by using various ratios of ?7-1- and ?7-2-pCIneo
plasmids (1:10 to 10:1) and was linear over the range tested.
Genomic DNA Extraction and PCR Amplification. Genomic
DNA was isolated from the livers of 7-day-old neonatal rats using the
Wizard Genomic DNA Purification Kit (Promega, Madison, WI).
Rats were killed by decapitation for these experiments. Primer pairs
Sequence of oligonucleotide primers used in this study, and the predicted size for the individual products
Primers specific for the splice insert are shown in bold italics. Nucleotide numbers correspond to the sequence of the RATNARAD (accession number L31619) in Fig. 2, except
for those that are specific for the ?7-2 (bold italics), which correspond to the nucleotide numbers used for the splice variant in Fig. 2. Product sizes shown in bold italics
represent the diagnostic bands used to determine relative abundance of the ?7-1 and ?7-2 isoforms.
Name Sequence Nucleotide NumbersProduct Size
Functional Isoforms of the ?7 Nicotinic ACh Receptor Subunit
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Address correspondence to: Dr. Javier Cuevas, Department of Pharmacol-
ogy and Therapeutics, University of South Florida College of Medicine, 12901
Bruce B. Downs Boulevard, MDC 9, Tampa, FL 33612-4799. E-mail:
Functional Isoforms of the ?7 Nicotinic ACh Receptor Subunit
at ASPET Journals on December 30, 2015