Elucidating mechanisms regulating neuronal survival and plasticity is relevant for understanding normal learning and memory as well as cognitive impairments in mental retardation, aging and Alzheimer?s. Influx of calcium ions (Ca2+) through L-type voltage-gated calcium channels (LTCCs) can influence long-term changes in synaptic plasticity and neuronal survival by turning on and off gene transcription in the nucleus. While it is known that signaling very near the site of Ca2+ influx is required for regulation of both LTCC activity and gene expression, molecular mechanisms that organize channel proximal signals and transduce them to the nucleus are largely unknown. One important pathway by which LTCC activity in neurons is regulated involves b-adrenergic receptor-mediated stimulation of cAMP production by adenylyl cyclase and activation of the kinase PKA. Previous studies in the heart suggest that efficient regulation of LTCC activity by PKA requires phosphorylation of the channel protein and localization of PKA near the channel through binding to A-kinase-anchoring proteins (AKAP). However, little is known about the roles of AKAPs or the opposing actions of protein phosphatases in neuronal LTCC regulation. In postsynaptic neurons one AKAP that may play a key role in regulating LTCC phosphorylation and signaling to transcription factors in the nucleus is AKAP79/150. Our overall hypothesis is that AKAP79/150 targets PKA and CaN to LTCCs to bi-directionally regulate channel activity and signaling to the nucleus. We will test this hypothesis in the context of a model in which anchored CaN strongly opposes cAMP-PKA regulation of the channel currents to function as a Ca2+ negative feedback mechanism. In addition, we will explore a novel role for dynamic anchoring of PKA and CaN to AKAP79/150 in these plasma membrane localized Ca2+ signaling events that also control downstream activation of NFAT and CREB transcription factors. Thus, our studies will characterize a novel molecular assembly that coordinates plasma membrane LTCC Ca2+ signaling to regulate both local and distal responses that are important in neuronal plasticity. We will use biochemical, cell biological and electrophysiological approaches in HEK-293 cells and hippocampal neurons to study AKAP79/150-LTCC regulation:
(Aim 1) Molecular and functional characterization of a direct interaction between AKAP79/150 and the LTCC CaV1.2 in neuronal channel regulation;
(Aim 2) Role of dynamic PKA and CaN anchoring to AKAP79/150 in neuronal LTCC regulation;
(Aim 3) Role of the AKAP79/150 channel-associated signaling complex in regulating neuronal LTCC excitation-transcription coupling.
Excitation induced entry of calcium ions (Ca2+) into neurons through L-type voltagegated Ca2+ channels (LTCC) influences synaptic plasticity and neuronal survival by regulating gene expression. Elucidating mechanisms regulating neuronal cell survival and plasticity is relevant for understanding normal learning and memory as well as learning deficits in mental retardation, aging and neurodegenerative conditions such as Alzheimer?s. Changes in LTCC regulated synaptic plasticity and channel modulation by protein kinase A (PKA) phosphorylation have been implicated in cognitive impairments associated with aging, and deficits in LTCC Ca2+ regulation of transcription factors involved in gene expression have been identified in Down Syndrome. Previous studies have demonstrated that highly localized signaling processes occur near the channel that are important for both channel modulation and downstream signaling to transcription factors in the nucleus. However, the molecular mechanisms that coordinate phosphorylation regulation of neuronal LTCCs and coupling to transcription factors are not well understood. The scaffold protein AKAP79/150 could help provide these functions by promoting channel phosphorylation by PKA, by providing a novel Ca2+-activated calcineurin (CaN) phosphatase negative feedback loop to oppose this regulation, and by positioning both CaN and PKA near the channel to regulate downstream transcriptional pathways. Understanding how AKAP79/150 coordinates LTCC signaling may lead to development of novel therapeutics to treat cognitive impairments.
