P6: Does cGKI – through BK channels in the nuclear envelope – counteract activity-dependent acquired neuroprotection? (until 31.12.2023)

• Use isolated nuclei for biochemical characterization of nuclear cGKI and BK channel isoforms as well as for determining the molecular interactions between the two proteins.
• Employ voltage and Ca2+-sensitive dyes in isolated nuclei and nuclei of cultured hippocampal neurons from wild-type as well as BK channel-/- and cGKI-/- mice to validate BK channel and cGKI functions.
• In parallel, we will develop electrophysiological tools to observe cGKI-mediated modulation of nuclear BK channel currents directly and to distinguish between BK channels in the inner or outer leaflet of the nuclear envelope (excised patch).
• Nuclear Ca2+ and voltage will be observed in the presence or absence of pharmacological modulators of cGKI and BK channels. BK channel and downstream transcription factors like CREB will be isolated from nuclear fractions for biochemical analysis of phosphorylation by cGKI. cGKI and BK channel constructs will be transfected into neuronal cultures from cGKI-/- and BK channel-/- mice, respectively, as part of a molecular replacement strategy.
• Further, we will test how the putative regulation of BK channels by cGKI influences activity-dependent acquired neuroprotection of cultured hippocampal neurons in development, long-term survival, and ischemic cell death.

During the visit of the Roberts lab in Boston, the students will profit from its immense experience in examining nuclear cGKI function. This includes using

1. wide-field imaging and statistical modeling to localize proteins within sub-cellular compartments, such as the nucleus and endomembrane system, and
2. nuclear cGKI-regulated control of signaling using a genetically-encoded nuclear phospho-CREB biosensor probe and molecular imaging techniques.

Assoc. Prof. Jesse D. Roberts, MD

Link to Boston researcher lab