Background: Nicotine-induced changes in nAChRs are linked to nicotine addiction. Results: Cotinine, the primary metabolite of nicotine, alters the assembly and expression of some subtypes of nAChRs. Conclusion: Cotinine affects trafficking and assembly of a subset of nAChRs. Significance: Cotinine has a much longer half-life in the body than nicotine, and therefore may contribute to physiological effects attributed to nicotine.

A new approach is presented for the application of single-molecule imaging to membrane receptors through the use of vesicles derived from cells expressing fluorescently labeled receptors. During the isolation of vesicles, receptors remain embedded in the membrane of the resultant vesicles, thus allowing these vesicles to serve as nanocontainers for single-molecule measurements. Cell-derived vesicles maintain the structural integrity of transmembrane receptors by keeping them in their physiological membrane. It was demonstrated that receptors isolated in these vesicles can be studied with solution-based fluorescence correlation spectroscopy (FCS) and can be isolated on a solid substrate for single-molecule studies. This technique was applied to determine the stoichiometry of α3β4 nicotinic receptors. The method provides the capability to extend single-molecule studies to previously inaccessible classes of receptors.

Dysregulated transcription, translation, and protein degradation are common features of cancer cells, regardless of specific genetic profiles. Several clinical anticancer agents take advantage of this characteristic vulnerability, and interfere with the processes of transcription and translation, or inhibit protein degradation. However, traditional assays that follow the process of protein production and removal require multi-step processing, and are not easily amenable to high-throughput screening (HTS). The use of recombinant fluorescent proteins provides a convenient solution to this problem, and moreover, photoconvertable fluorescent proteins allow for ratiometric detection of both new protein production and removal of existing proteins. Here, the photoconvertable protein Dendra2 is used in the development of in-cell assays of protein production and degradation that are optimized and validated for high-throughput screening. Conversion from the green to red emissive form can be achieved using a high intensity light emitting diode (LED) array, producing a stable pool of the red fluorescent form of Dendra2. This allows for rates of protein production or removal to be quantified in a plate reader or by fluorescence microscopy, providing a means to measure the potencies of inhibitors that affect these key processes.

A new approach is presented for the application of single‐molecule imaging to membrane receptors through the use of vesicles derived from cells expressing fluorescently labeled receptors. During the isolation of vesicles, receptors remain embedded in the membrane of the resultant vesicles, thus allowing these vesicles to serve as nanocontainers for single‐molecule measurements. Cell‐derived vesicles maintain the structural integrity of transmembrane receptors by keeping them in their physiological membrane. It was demonstrated that receptors isolated in these vesicles can be studied with solution‐based fluorescence correlation spectroscopy (FCS) and can be isolated on a solid substrate for single‐molecule studies. This technique was applied to determine the stoichiometry of α3β4 nicotinic receptors. The method provides the capability to extend single‐molecule studies to previously inaccessible classes of receptors.