Molecular Biology Core. The field of ion channels has reached the stage where multidisciplinary approaches are routinely combined to devise experiments that address complex mechanisms at the cellular and molecular level. It is extremely difficult for any single laboratory to maintain the specialized equipment resources and level of technical expertise that is needed to fully exploit the wide range of existing technologies. Thus, the purpose of this core facility is to avoid unnecessary duplication of effort by providing a source of common technical and material support for the four participating laboratories in this Program Project. The core will establish shared resources in the areas of peptide/protein chemistry and purification, cell culture, molecular biology, and immunochemistry. This support function encompasses the following needs that are critical to the success of this project: 1. Production of peptide toxin derivatives suitable for specific biochemical detection and imaging of Slo K(Ca) channels. 2. Production and maintenance of stable mammalian cell lines expressing Slo K(Ca) and Slack channel constructs. 3. Harvesting of Xenopus oocytes and mRNA injection for Slo K(Ca) and Slack expression. 4. Construction of plasmid expression vectors for native and mutant Slo K(Ca) and Slack K channels. 5. Production and purification of anti-peptide antibodies specifically targeted to particular Slo K(Ca) and Slack domains. The molecular biology core will serve as a focal point for methods development and interaction among the four laboratories. Primary research personnel from each of the four laboratories will contact the core technical staff to obtain necessary cells, design molecular probes, and obtain unique molecular reagents for particular experiments. This will facilitate numerous interactions among the laboratories and provide common services for successful execution of the individual research objectives of the four projects.
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