Thus, the sequences of siRNA primers and location of the target sequences (in parentheses) in the dup7 mRNA sequence (NM-139320.1) were the following: siRNA-1, sense: 5-CAACAUUAAGAUUACAAGUTT-3 and reverse: 5-ACUUGUAAUCUUAAUGUUGCG-3 (junction region between exons D and C); siRNA-2, sense: 5-CGGUGGAGUCGGUUAUAAATT-3 and reverse: 5-UUUAUAACCGACUCCACCGAC-3 (exon D); and siRNA-3, sense: 5-CCAUUAUUGACAAUCCAAATT-3 and reverse: 5-UUUGGAUUGUCAAUAAUGGGG-3 (exon C). by combining immunoblotting, quantitative RT-PCR and FRET techniques with functional assays of 7-nAChR activity using human neuroblastoma HO-3867 SH-SY5Y cell variants with different dup7 expression levels. Our findings reveal a physical conversation between dup7 and 7 subunits in fluorescent protein-tagged dup7/7 transfected cells that negatively affects normal 7-nAChR activity. Specifically, in both single cells and cell populations, the [Ca2+]i transmission and the exocytotic response induced by selective activation of 7-nAChR were either significantly inhibited by stable dup7 overexpression or augmented after silencing gene expression with specific siRNAs. These findings identify a new role for the dup7 subunit as a negative regulator of 7-nAChR-mediated control of exocytotic neurotransmitter release. If this effect is usually excessive, it would result in an impaired synaptic transmission that could underlie the neurocognitive and neuropsychiatric disorders associated with 7-nAChR dysfunction. chimeric gene, which is usually evolutionarily a relatively recent gene since it appears in the human genome after its divergence from other higher primates (30). The new hybrid gene results from partial duplication (exons 5C10) of the parent gene, coding the 7 subunit that forms the 7-nAChR, fused to the genetic element (31, 32). Although expression has been associated with neurocognitive disorders such as schizophrenia, psychosis, bipolar disorder, autism, and dementia (33, 34, 35), the functional role of the chimeric gene was long unidentified until we reported that its product, the dup7 subunit, acted as a dominant unfavorable regulator of 7-nAChR-induced currents in a pioneering electrophysiological study conducted in oocytes (36). Our obtaining was corroborated shortly afterward by others, also in oocytes (37) and, a few years later, by HO-3867 our own group in diverse mammalian cell types. Thus, we reported that dup7 overexpression inhibits, both and and and overlaid around the bar graphs represent individual data points obtained in impartial cultures for each condition. The error bars show mean? SD. ??shows the concentrationCresponse curves to the agonist in nontransfected cells (Control), in both the absence and presence of the PAM; it can be observed that this last agent greatly enhanced the agonist-induced response at all tested concentrations. Figure?2shows the original fluorescence traces induced by different concentrations of PNU 282987 (+PAM) in control cells (black traces; left panel) or in cells expanded from your N1 clone (reddish traces; right panel). Physique?2, and show pooled results from indie cultures (n?= 4C12) of normalized [Ca2+]i responses ([Ca2+]i) evoked by increasing concentrations of PNU 282987 in the four cell variants tested [control cells, and cells overexpressing dup7 (clones L1 and N1), or vacant vector]. The [Ca2+]i signals were normalized as a percentage of the maximum response induced by PNU 282987 (3?M; 100%) in control cells (Fig.?2showed that while the [Ca2+]i signal induced by PNU 282987 in cells overexpressing the vacant Rabbit polyclonal to PBX3 vector was indistinguishable from that found in control cells, the overexpression of dup7 significantly reduced the 7-nAChR-mediated signal, particularly at the highest agonist screening concentrations (from 30?nM to 10?M), but not the transmission generated by a depolarizing stimulus of high K+ (70?mM, 1?s). The table inserted in Physique?2shows the EC50 and slope values obtained from the concentrationCresponse curves of PNU 282987 in the four cell variants tested; the application of the above statistical analysis to both parameters did not show significant differences between the [Ca2+]i signals generated in the four variants. Open in a separate window Physique?2 Effect of dup7 overexpression around the 7-nAChR-mediated [Ca2+]isignal in populations of SH-SY5Y cells.and shows original traces of the fluorescence increase (F) induced by the above two pulses applied successively in two Fura 2-loaded cells representative of the Control (upper HO-3867 panel) and the L1 clone (lower panel) groups. Note that overexpression of dup7 markedly reduces the 7-nAChR-mediated transmission but not that induced by the depolarizing stimulus. In fact, the high K+-evoked response is usually higher in the dup7 overexpressing cell than in.