Dendritic computation, activity-dependent gene expression, synaptic plasticity, learning and memory are neuronal processes that are based in part upon Ca2+ influx through CaV1.2 voltage-gated L-type Ca2+ channels. The activity of CaV1.2 channels is modulated by their phosphorylation state. The scaffolding protein AKAP79 anchors both a regulating kinase (protein kinase A, PKA) and a phosphatase (calcineurin, CaN) to the channel. In hippocampal pyramidal neurons, norepinephrine binding to beta adrenergic receptors initiates a cascade that leads to cAMP production and PKA activation. Phosphorylation of CaV1.2 enhances channel activity, resulting in increased Ca2+ influx. The elevated Ca2+ near the intracellular mouth of the channel activates the CaN phosphatase, which acts to reverse channel phosphorylation and consequently down-modulate channel activity and reduce Ca2+ influx. We plan to obtain from single living cells on a microscope stage dynamic measurements of fluorescence resonance energy transfer (FRET) between the various partners in this signaling complex, in an effort to uncover intra- and intermolecular movements that underlie channel modulation. We will correlate in time these movements with changes in CaV1.2 current that we will simultaneously measure using whole-cell patch-clamp recording. To obtain FRET measurements, we will fuse fluorescent proteins (cyan CFP, yellow YFP) to various pairs of signaling partners (CaV1.2, AKAP79, PKA regulatory and catalytic subunits, CaN and its Ca2+-dependent activator calmodulin). These fluorescent fusion constructs will be transfected into HEK293 cells or cultured rat hippocampal neurons. We will use a ratiometric fluorescence approach (CFP intensity/YFP intensity) to measure dynamic changes in FRET between signaling partners in single live cells. The goal of the research plan is to gain substantial insight into the mechanics, at the molecular level, of modulation of CaV1.2 channel function. In addition, the dynamics of signaling within molecular complexes, such as the one proposed for study here, helps support higher order function in neurons, such as computation, and thus the general significance of the work is broad. Relevance to Public Health. Calcium channels help control activity of nerve cells, beating of heart cells, contraction of smooth muscle cells that are wrapped around blood vessels, and secretion of insulin from the pancreas. Calcium channels are therefore targets of drugs used to protect against nerve cell death following stroke, to treat cardiac arrhythmias and angina, and to combat high blood pressure. A mutation in the calcium channel studied here has been identified as the cause of Timothy syndrome in humans, symptoms of which are cognitive impairment and potentially fatal disturbance of heart rhythm.
