CaV1.2 calcium channel dynamics in neuronal modulation

In neurons calcium currents through L-type CaV1.2 voltage-gated calcium channels regulate gene transcription and function in synaptic plasticity. However, it is still controversial whether neuronal activity or stimulation of G- protein coupled receptors affect the membrane expression of CaV1.2 channels in postsynaptic compartments of CNS neurons. The structural and functional interactions of CaV1.2 with beta2 adrenergic receptors (b2ARs) are well described. b2ARs are endocytosed upon receptor desensitation and recent experiments also demonstrated the activity-induced endocytosis of CaV1.2 channels, but the involved pathways are still elusive. Thus, we hypothesize that b2AR and CaV1.2 are constituents of a common signaling complex in dendritic spines and that sustained activation of b2AR causes the concomitant down-regulation of the receptor and the channel by clathrin-mediated endocytosis. To address this question we plan to use two experimental approaches in cultured hippocampal neurons transfected with extracellularly tagged CaV1.2 channels: First, high-resolution immunofluorescence analysis of membrane expressed CaV1.2-HA; a method that was previously established by the applicant to analyze the size and density of CaV1.2 clusters in dendrites of hippocampal neurons. Secondly, fluorescence recovery after photobleaching (FRAP) will reveal channel dynamics in the plasma membrane. In order to analyze exclusively membrane incorporated CaV1.2 channels, and also to distinguish recovery by lateral diffusion as opposed to de novo insertion, a CaV1.2 construct extracellularly tagged with a pH-sensitive GFP will be generated. Using these methods, the distribution and size of CaV1.2 clusters, and the lateral diffusion, turn-over, and endocytosis of CaV1.2 channels will be determined in untreated neurons, as well as in neurons activated with different physiological and pharmacological protocols. Most importantly, we will examine how these parameters are affected in response to b2AR activation by isoproterenol. In the postsynaptic compartment of CNS neurons L-type calcium currents regulate the expression of genes and thus play a vital function in processes leading to long-term memory. Determining whether membrane expression of these CaV1.2 calcium channels is itself controlled by neuronal activity and modulation, and if so, which mechanisms regulate it, is important for understanding the molecular machinery involved in learning and memory. The innovative fluorescence microscopy approach proposed here is expected to reveal unprecedented information about calcium channel dynamics in resting and activated neurons.