Molecular mimicking of C-terminal phosphorylation tunes the surface dynamics of CaV1.2 calcium channels in hippocampal neurons

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.

Dynamic L-type CaV1.2 channel trafficking facilitates CaV1.2 clustering and cooperative gating

“…More recently, the adapter protein Stac was found to regulate trafficking of Ca V 1.1 channels as well as Ca V 1.2 activity, which may have important implications for excitation-contraction coupling in skeletal and cardiac muscle, respectively [ 42 , 79 ]. The phosphorylation state of key residues on the channel also seems to influence Ca V 1.2 trafficking, dynamics and clustering, at least in neurons [ 80 ]. Whether either or all of these proteins and processes are involved in homotypic fusion of Ca V 1.2 vesicles, channel clustering at specific sites at/near the plasma membrane as well as cooperative gating is currently unknown but are most certainly important questions for future studies.…”

β‐adrenergic‐mediated dynamic augmentation of sarcolemmal CaV1.2 clustering and co‐operativity in ventricular myocytes

“…Do neurons then have a similarly sized functional reserve? Phosphorylation J Physiol 597.8 of neuronal Ca V 1.2 channels is considered to play a role in Ca V 1.2 trafficking and stabilization at the membrane (Folci et al, 2018), although, to our knowledge, the effect on channel cluster size remains to be clarified.…”

A homozygous missense variant in CACNB4 encoding the auxiliary calcium channel beta4 subunit causes a severe neurodevelopmental disorder and impairs channel and non-channel functions

“…Low-density cultures of hippocampal neurons were obtained from 16.5-18 day old embryonic BALB/c mice of either sex as described previously [101][102][103][104]. Dissociated neurons were plated at a density of~3500 cells/cm 2 on 18 mm glass coverslips (No 1.5; GML, Innsbruck, Austria) coated with poly-L-lysine (Sigma-Aldrich) in 60 mm culture dishes.…”