Abstract
L-type voltage-gated CaV1.2 calcium channels (CaV1.2) are key regulators of neuronal excitability, synaptic plasticity, and excitation-transcription coupling. Surface-exposed CaV1.2 distributes in clusters along the dendrites of hippocampal neurons. A permanent exchange between stably clustered and laterally diffusive extra-clustered channels maintains steady-state levels of CaV1.2 at dendritic signaling domains. A dynamic equilibrium between anchored and diffusive receptors is a common feature among ion channels and is crucial to modulate signaling transduction. Despite the importance of this fine regulatory system, the molecular mechanisms underlying the surface dynamics of CaV1.2 are completely unexplored. Here, we examined the dynamic states of CaV1.2 depending on phosphorylation on Ser-1700 and Ser-1928 at the channel C terminus. Phosphorylation at these sites is strongly involved in CaV1.2-mediated nuclear factor of activated T cells (NFAT) signaling, long-term potentiation, and responsiveness to adrenergic stimulation. We engineered CaV1.2 constructs mimicking phosphorylation at Ser-1700 and Ser-1928 and analyzed their behavior at the membrane by immunolabeling protocols, fluorescence recovery after photobleaching, and single particle tracking. We found that the phosphomimetic S1928E variant increases the mobility of CaV1.2 without altering the steady-state maintenance of cluster in young neurons and favors channel stabilization later in differentiation. Instead, mimicking phosphorylation at Ser-1700 promoted the diffusive state of CaV1.2 irrespective of the differentiation stage. Together, these results reveal that phosphorylation could contribute to the establishment of channel anchoring mechanisms depending on the neuronal differentiation state. Finally, our findings suggest a novel mechanism by which phosphorylation at the C terminus regulates calcium signaling by tuning the content of CaV1.2 at signaling complexes.
Highlights
L-type voltage-gated CaV1.2 calcium channels (CaV1.2) are key regulators of neuronal excitability, synaptic plasticity, and excitation-transcription coupling
To assess whether pSer-1928 and pSer-1700 regulate the distribution of CaV1.2 at the dendritic membrane we generated CaV1.2 mutants mimicking pSer-1700 or pSer-1928, or both, and analyzed their surface expression by immunolabeling experiments in young and mature cultured hippocampal neurons, at DIV 10 and 20, respectively [10, 11]
To equalize channel expression all mutants were inserted into the same backbone plasmid optimized for neuronal expression [4, 5, 12,13,14]
Summary
To determine basal phosphorylation levels of Ser-1700 and Ser-1928, CaV1.2 channels were extracted from young and mature hippocampal cultures at 10 and 20 DIV, respectively [10, 11], in the presence of potent phosphatase inhibitors to prevent dephosphorylation. In surface immunolabeling experiments on young neurons CaV1.2-HA-RRQQ exhibited larger cluster size and higher amounts of channels at the membrane (Fig. 2, D and E, green circles) These data suggest that R1696Q,R1697Q could stabilize channels at cluster sites and that a putative impairment of channel internalization could be responsible for the increased membrane expression. The diffusion coefficients of individual channels, obtained by linear fitting of the first 4 points of MSD curves, increased for all mutants, indicating higher mobility and a weaker retention at anchoring sites (Fig. 4C, Table 1) (19 –21) Such faster local dynamics was consistent with the steeper rising phase of eGFP-CaV1.2-SE and eGFP-CaV1.2RRQQ FRAP curves and supports a potentiation of channel lateral mobility induced by S1928E and R1696Q,R1697Q mutations.
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