Molecular Mimicking of Phosphorylation at S1928 and S1700-T1704 Confers Modified Surface Traffic Properties to CaV1.2 Voltage Gated Calcium Channels in Cultured Hippocampal Neurons

Abstract

Calcium influx via CaV1.2 L-type voltage gated calcium channels plays a pivotal role in neuronal functions. CaV1.2s distribute in clusters along the dendritic shafts and on the spines of cultured hippocampal neurons. An equilibrium of clustered and dynamic CaV1.2s at the membrane maintains stable calcium channel complexes involved in activity-dependent cell signaling. The molecular mechanisms underlying the dynamic properties of CaV1.2 channels are elusive. Here, we address the hypothesis that surface traffic of CaV1.2s depends on channel phosphorylation state. We modeled the phosphorylation at S1700 and T1704 by neutralizing two key arginines (R1696-1697Q) at the proximal C-terminus. Furthermore, we generated a CaV1.2 mutant mimicking the phosphorylated state on S1928 (S1928E). All channel mutants distribute in clusters at the dendritic membrane of young and mature neurons similar to controls in live immunolabeling experiments. However, the R1696-1697Q substitution significantly increases the amount of channels at the clusters in young neurons. To determine whether the R1696-1697Q and S1928E mutations modify the channel turnover rate and diffusive properties we performed fluorescence recovery after photobleaching (FRAP) experiments on neurons expressing N-terminal eGFP tagged CaV1.2 mutants. We observed steeper rising phases of FRAP curves with the R1696-1697Q mutant in young and mature neurons and with the S1928E only in young ones. Consistently, using single particle tracking analysis we found higher diffusion coefficients for both CaV1.2 channel mutants in young neurons; in mature ones the values increased only for the R1696-1697Q. Altogether, our data suggest that phosphorylation at S1700-T1704 promotes diffusive behavior of membrane inserted CaV1.2 channels in mature neurons. Furthermore, the specificity of phosphorylation site confers diverse dynamic properties to CaV1.2s in a developmental dependent manner. Support: FWF, grant #P25085 to V.D.B.