To date, 11 members (␣2-␣9 and ␤2-␤4) of the neuronal nicotinic acetylcholine receptor gene family have been identified. These genes encode subunits that form distinct receptors with different pharmacological and physiological profiles in temporally and spatially restricted patterns within the nervous system. Distinct molecular mechanisms probably orchestrate the expression of various receptor subtypes, yet little is known of specific transcriptional regulatory elements and their associated factors that are responsible for this segregated pattern of expression. Here we report the identification of an element, in the 5-flanking region of the rat ␤4 subunit gene, containing a CA box that is necessary for ␤4 promoter activity in a transiently transfected cholinergic cell line, SN17. This element was shown to interact with a protein(s) in SN17 nuclear extracts that is antigenically related to the transcriptional activator Sp1. Furthermore, co-transfection experiments confirmed that Sp1 can transactivate a ␤4 promoter-reporter gene construct, indicating that Sp1 is necessary, at least in part, for transcriptional activation of the ␤4 subunit gene. Neuronal nicotinic acetylcholine (nACh)1 receptors belong to a large family of related neurotransmitter receptors that are expressed within the central and peripheral nervous systems (CNS and PNS). Little is known of their function within the CNS, although a recent report suggests that presynaptic nACh receptors may modify fast excitatory transmission in the CNS (1). Additionally, the loss of cholinergic neurons in pathological states involving alterations in cognition and memory (e.g. Alzheimer's disease) implicates nACh receptors in these processes (2). In support of this premise, transgenic mice lacking the nACh receptor ␤2 subunit performed poorly in passive avoidance testing, suggesting a defect in associative memory (3). Further underscoring the importance of nACh receptors within the CNS is a recent report describing the development of autosomal dominant, nocturnal, frontal lobe epilepsy in humans, resulting from a missense mutation in the ␣4 subunit gene (4). It is clear that a significant amount of work remains to be done in order to fully understand the function of nACh receptors, including the identification and characterization of individual subunit genes.Eleven members of the nACh receptor gene family have been identified to date, and they include ␣2-␣9 and ␤2-␤4 (5-20). These subunits have been demonstrated in either Xenopus oocytes or in vivo to form a variety of distinct heteromeric (␣2, ␣3, and ␣4 with either ␤2 or ␤4) and homomeric (␣7, ␣8, and ␣9) receptors with different pharmacological and physiological profiles (21-23). This functional heterogeneity likely results from incorporation of different ␣ and ␤ subunits into mature receptors (19,24). Further complicating the characterization of nACh receptors is the observation that individual nACh receptor subunit gene expression occurs in distinct yet overlapping temporally and spatially restricted patterns ...

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Fluorescence Resonance Energy Transfer (FRET) is a process by which a fluorophore (the donor) in the excited state transfers its energy to a neighboring fluorophore (the acceptor) non-radiatively through dipole-dipole interactions. Since the efficiency of energy transfer varies as the inverse of the sixth power of the distance separating the donor and acceptor chromophores, for FRET to occur the distance between the two molecules cannot exceed 10 to 100 angstroms (1 to l0nm). The combination of FRET and optical microscopy allows examination and quantitation of dynamic molecular interactions between cellular constituents at resolutions beyond the Abbe diffraction limit of light microscopy. Through the microscope one may detect FRET by an overall decrease in fluorescence emission of the donor with a concomitant increase in fluorescence emission of the acceptor.