The alpha7 subunit of the neuronal nicotinic acetylcholine receptor (nAChR) is abundantly expressed in hippocampus and is implicated in modulating neurotransmitter release and in binding alpha-bungarotoxin (alpha-BGT). A null mutation for the alpha7 subunit was prepared by deleting the last three exons of the gene. Mice homozygous for the null mutation lack detectable mRNA, but the mice are viable and anatomically normal. Neuropathological examination of the brain revealed normal structure and cell layering, including normal cortical barrel fields; histochemical assessment of the hippocampus was also normal. Autoradiography with [3H]nicotine revealed no detectable abnormalities of high-affinity nicotine binding sites, but there was an absence of high-affinity [125I]alpha-BGT sites. Null mice also lack rapidly desensitizing, methyllycaconitine-sensitive, nicotinic currents that are present in hippocampal neurons. The results of this study indicate that the alpha-BGT binding sites are equivalent to the alpha7-containing nAChRs that mediate fast, desensitizing nicotinic currents in the hippocampus. These mice demonstrate that the alpha7 subunit is not essential for normal development or for apparently normal neurological function, but the mice may prove to have subtle phenotypic abnormalities and will be valuable in defining the functional role of this gene product in vivo.
A family of genes has been identified that encodes subunits of nicotinic acetylcholine receptors (nAChRs) and is expressed in the nervous system. Functional neuronal nAChRs can be expressed in Xenopus oocytes by injection of RNA encoding 1 of 2 different beta-subunits (beta 2, beta 4) in pairwise combination with RNA encoding 1 of 3 different alpha-subunits (alpha 2, alpha 3, alpha 4). We examined the sensitivity of these 6 different alpha- beta-subunit combinations to the nicotinic agonists ACh, nicotine, cytisine, and 1,1-dimethyl-4- phenylpiperazinium (DMPP). Each subunit combination displayed a distinct pattern of sensitivity to these 4 agonists. The alpha 2 beta 2 combination was 5-fold more sensitive to nicotine than to acetylcholine, while the alpha 3 beta 2 combination was 17-fold less sensitive to nicotine than to ACh, and the alpha 3 beta 4 combination was equally sensitive to both nicotine and ACh. nAChRs composed of alpha 2, alpha 3, or alpha 4 in combination with beta 2 were 14–100- fold less sensitive to cytisine than to ACh. In contrast, nAChRs composed of alpha 2, alpha 3, or alpha 4 in combination with beta 4 were 3–17-fold more sensitive to cytisine than to ACh. The alpha 2 beta 2, alpha 3 beta 2, and alpha 3 beta 4 combinations were each equally sensitive to DMPP and ACh, while the alpha 2 beta 4, alpha 4 beta 2, and alpha 4 beta 4 combinations were 4–24-fold less sensitive to DMPP than to ACh. We also demonstrated that these differences are neither a consequence of variation in the relative amounts of RNA injected nor an artifact of oocyte expression. The oocyte system can accurately express ligand-gated ion channels because mouse muscle nAChRs expressed in oocytes display pharmacological properties similar to those reported for these receptors expressed on BC3H-1 cells. We conclude that both the alpha- and the beta-subunits contribute to the pharmacological characteristics of neuronal nAChRs.
Transcripts for the beta2 and the beta4 nicotinic acetylcholine receptor (nAChR) subunits are found throughout the CNS and the peripheral nervous system. These two beta subunits can form heteromultimeric channels with any of the alpha2, alpha3, alpha4, or alpha5 subunits in heterologous expression systems. Nonetheless, the subunit composition of native nAChRs and the role of different nAChR subtypes in vivo remain unclear. We prepared null mutations for the beta2 and the beta4 genes and bred beta2-/-beta4-/- mice by mating mice of identical beta2-/-beta4+/- or beta2+/-beta4-/- genotype. The beta2-/- and the beta4-/- single-mutant mice grow to adulthood with no visible phenotypic abnormalities. The beta2-/-beta4-/- double mutants survive to birth but have impaired growth and increased perinatal mortality. They also present enlarged bladders with dribbling urination and develop urinary infection and bladder stones. The ocular pupils are widely dilated and do not constrict in response to light. Histological studies revealed no significant abnormalities of brain or peripheral tissues except for hyperplasia in the bladder mucosa of beta4-/- and beta2-/-beta4-/- mutants. Bladder strips from beta2-/-beta4-/- mice did not respond to nicotine but contracted when stimulated with a muscarinic agonist or electric field stimulation. Bladder strips from beta4 mutants did not respond to nicotine despite the absence of major bladder dysfunction in vivo. Acetylcholine-activated whole-cell currents were absent in superior cervical ganglion neurons from beta2-/-beta4-/- mice and reduced in neurons from beta4-/- mice. Although there is apparent redundancy and a superficially normal phenotype in beta2-/- and beta4-/- mice, physiological studies indicate major deficits in the beta4-/- mice. Our previous description of a similar phenotype in alpha3-/- mice and the current data suggest that the alpha3 and the beta4 subunits are major components in autonomic nAChRs. The phenotype of the beta2-/-beta4-/- and alpha3-/- mice resembles the autosomal recessive megacystis-microcolon-hypoperistalsis syndrome in humans.
The ␣3 subunit of the neuronal nicotinic acetylcholine receptor is widely expressed in autonomic ganglia and in some parts of the brain. The ␣3 subunit can form heteromultimeric ion channels with other ␣ subunits and with 2 and 4 subunits, but its function in vivo is poorly understood. We prepared a null mutation for the ␣3 gene by deletion of exon 5 and found that homozygous (؊͞؊) mice lacked detectable mRNA on Northern blotting. The ؊͞؊ mice survive to birth but have impaired growth and increased mortality before and after weaning. The ؊͞؊ mice have extreme bladder enlargement, dribbling urination, bladder infection, urinary stones, and widely dilated ocular pupils that do not contract in response to light. Detailed histological studies of ؊͞؊ mice revealed no significant abnormalities in brain or peripheral tissues except urinary bladder, where inf lammation was prominent. Ganglion cells and axons were present in bladder and bowel. Bladder strips from ؊͞؊ mice failed to contract in response to 0.1 mM nicotine, but did contract in response to electrical field stimulation or carbamoylcholine. The number of acetylcholine-activated single-channel currents was severely reduced in the neurons of superior cervical ganglia in ؊͞؊ mice with five physiologically distinguishable nicotinic acetylcholine receptor subtypes with different conductance and kinetic properties in wild-type mice, all of which were reduced in ؊͞؊ mice. The findings in the ␣3-null mice suggest that this subunit is an essential component of the nicotinic receptors mediating normal function of the autonomic nervous system. The phenotype in ؊͞؊ mice may be similar to the rare human genetic disorder of megacystismicrocolon-intestinal hypoperistalsis syndrome.The neuronal nicotinic acetylcholine (ACh) receptor (nAChR) gene family consists of eight ␣ subunits (␣2-␣9) and three  subunits (2-4), each containing four membranespanning domains (1-4). Expression studies in Xenopus oocytes have shown that any one of the ␣2, ␣3, or ␣4 subunits in combination with either 2 or 4 can produce functional receptors (2, 4-6). Diverse combinations of subunits occur, and even a single population of neurons can express multiple classes of nAChRs (7-9). The ␣3 subunit is widely expressed in autonomic ganglia and in some parts of the brain in multiple organisms (1, 2, 10-13). Although the role of ␣3-containing nAChRs in synaptic transmission in the central nervous system is not known, presynaptic receptors have been implicated in the modulation of the release of dopamine, norepinephrine, and glutamate (13-16).In the peripheral autonomic nervous system, efferent signals are relayed by both sympathetic and parasympathetic ganglia. The ␣3 subunit is the predominant ␣ gene expressed in these ganglia in the chicken (17). Functional deletion of the ␣3 subunit by antisense oligomers eliminated specific subtypes of channels expressed by chicken sympathetic neurons (8). In rat trigeminal sensory neurons, ␣34 is the principal subtype (18). The ␣3 subunit is expressed...
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