Adrenergic regulation of CaV1.2 L-type calcium channels dynamics in neurons

In neurons of the central nervous system L-type CaV1.2 voltage gated calcium channels play a pivotal role in learning, memory, synaptic plasticity, and in excitation transcription coupling. CaV1.2s and beta adrenergic receptors (beta-ARs) have been independently shown to contribute to fear acquisition and extinction, and anxiety. The beta2-AR modulates the phosphorylation of CaV1.2 channels in brain. Interestingly, CaV1.2 channels and beta2-AR directly interact, are both anchored to the same signaling integrator AKAP79/150, and share downstream signaling to the nucleus via MAPK and CREB pathways. These findings are all consistent with CaV1.2 and beta2- AR being part of the same signaling complex in neurons. Intriguingly, our preliminary results suggest that the activation of beta2-AR induces changes in the localization and dynamics of CaV1.2s at the membrane. Therefore, we hypothesize that the formation of the signaling complex with CaV1.2 and beta2-AR is a dynamic and transient process that is regulated by adrenergic stimulation. To test this hypothesis, we will analyze the distribution, turnover rate, and lateral diffusion of CaV1.2 channels and beta2-AR with or without adrenergic stimulation. Our methods of choice are surface immunolabeling, fluorescence recovery after photobleaching (FRAP), and single particle tracking (SPT) analysis. To observe only membrane inserted CaV1.2 channels and beta2-AR receptors the experiments will be conducted on cultured hippocampal neurons heterologously transfected with extracellularly tagged CaV1.2 and beta2-AR. First, we will analyze the subcellular localization of the CaV1.2 and beta2-AR signaling complexes by surface immunolabeling experiments. Moreover, we will describe the dynamics of the CaV1.2/beta2-AR signaling complex formation using FRAP and SPT analysis. Second, we will determine whether and how the adrenergic stimulation modifies the localization of the signaling complex and its dynamics using surface immunolabeling, FRAP, and SPT experiments. Finally, we will analyze the potential molecular mechanisms underlying the changes in surface traffic of CaV1.2 channels. To address this point, we will generate CaV1.2 mutants where the CaV1.2 sequences plausibly involved in anchoring the CaV1.2 at the signaling complex are modified. Subsequently, we will determine whether these channel mutants present surface traffic properties similar to those of the control CaV1.2 during adrenergic stimulation. Altogether these findings will describe whether the formation of the signaling complex with CaV1.2 and beta2-AR is transient, subject to regulation by adrenergic stimulation, and what molecular domains of CaV1.2 are involved in the modulation of CaV1.2 surface traffic. Our results will indicate whether the dynamics of CaV1.2 channels at the membrane adapts to the adrenergic stimulus, thus revealing a novel mechanism of regulation of the CaV1.2 dependent signaling. This will meaningfully contribute to the understanding of neurophysiology processes and neurodegenerative diseases where dysfunction of L-type VGCCs is involved.

Proper activity of the brain allows us learning, building memory, generating and handling emotions, controlling behavior, and processing the enormous amount of information from the world around us. The CaV1.2 class of voltage gated calcium channels is responsible of these functions by initiating a variety of molecular mechanisms essential to the brain function. The activity of CaV1.2 channels is strongly modulated by norepinephrine (or activation of the adrenergic system) which is in turn important for arousal, behavioral acuity and learning in novel and emotionally challenged situations. The most important findings of this project rely on the understanding of how the adrenergic stimulation influences the activity of CaV1.2 channels in neurons at molecular level. We found that the CaV1.2 channels and adrenergic receptors by which norepinephrine exerts its function- are intimately connected and that their association is not only functional but also physical. This direct association, however, is dynamic and regulated by activation of the adrenergic system. Normally, in physiological conditions the CaV1.2 and adrenergic receptors interact. When the adrenergic system is activated, at first there is an enhancement of the CaV1.2 activity but soon after the adrenergic receptors is displaced to terminate the CaV1.2 response to the adrenergic challenge. We also found that adrenaline is responsible of activating the molecular mechanisms that drive the positioning of the CaV1.2 right at the loci in which they are active. This finding is of fundamental importance, as the perfect localization of the channel in the membrane is necessary to the fulfillment of its function. Failure of proper targeting of CaV1.2 to signaling complexes also means a failure of the brain function in which CaV1.2 is involved. Dysregulation of CaV1.2 channels is associated with devastating psychiatric disorders including schizophrenia, bipolar disorders, post-traumatic stress syndrome, for which no effective pharmacological treatment is available. We are confident that our results may represent an important contribution to designing of highly selective drugs that would help people affected by neuropsychiatric diseases and conditions in which memory capacity is heavily affected.