Role of the synprint site in presynaptic targeting of the calcium channel Ca_V2.2 in hippocampal neurons

Abstract

Sequences in the cytoplasmic II-III loop of CaV2 voltage-gated calcium channels, termed the synaptic protein interaction (synprint) site, are considered important for the functional incorporation of presynaptic calcium channels into the synaptic vesicle fusion apparatus. Two novel CaV2.2 splice variants lack large parts of the cytoplasmic II-III loop (Delta1 R756-L1139, Delta2 K737-A1001) including the synprint protein-protein interaction domain. Here we expressed green fluorescent protein (GFP)-alpha1B subunit fusion constructs of CaV2.2 splice variants in mouse hippocampal neurons to study their distribution in distinct neuronal compartments and to address the question of whether and how the synprint site functions in the presynaptic targeting of N-type calcium channels. Similar to full-length GFP-alpha1B but divergent from the somatodendritic alpha1C-HA (CaV1.2) channel type, the splice variants GFP-alpha1B-Delta1 and GFP-alpha1B-Delta2 were targeted into the axons. Nevertheless, their ability to form bona fide presynaptic clusters was almost abolished for GFP-alpha1B-Delta1 and significantly reduced for GFP-alpha1B-Delta2. Thus, the synprint site is important for normal synaptic targeting of CaV2.2 but not essential. Conversely, insertion of the synprint site into the II-III loop of alpha1C-HA did not restore axonal targeting or synaptic clustering. Together these results indicate that protein-protein interactions with the synprint site must cooperate with other targeting mechanisms in the incorporation of CaV2.2 into presynaptic specializations of hippocampal neurons but are neither necessary nor sufficient for axonal targeting. The unique targeting properties of the splice variants lacking the synprint site are suggestive of specific functions of these calcium channels apart from activating fast synaptic transmission.

Reciprocal Interactions Regulate Targeting of Calcium Channel β Subunits and Membrane Expression of α1 Subunits in Cultured Hippocampal Neurons

“…␤ 1 clusters were primarily located adjacent to synapsin clusters, whereas a subset of ␤ 2 and ␤ 4 clusters was colocalized with synapsin (yellow in The localization of ␤ subunits along the dendritic shaft and at synaptic sites suggests their association with postsynaptic and presynaptic Ca V ␣ 1 subunits, respectively. For example, the overall ␤ subunit distribution along the dendrites was strikingly similar to the localization of endogenous L-type Ca V 1.2 (15) and Ca V 1.2-HA expressed in the membrane (15,42,49). Thus, we next investigated whether ␤ 1 and ␤ 4 subunits specifically colocalize with postsynaptic (Ca V 1.2) or presynaptic (Ca V 2.1) ␣ 1 subunits.…”

“…Previous analysis of Ca V 2.2 splices lacking the SYNPRINT domain suggested the existence of separate checkpoints for axonal targeting and the incorporation of channels into the synapse (49). Also, axonal ␤ subunits might differ in their ability to be incorporated into the presynaptic compartment.…”

“…In contrast to other Ca V targeting studies, we routinely express the ␣ 1 subunit without auxiliary ␣ 2 ␦ and ␤ subunits in cultured hippocampal neurons (15,42,49). As a consequence, subcellular localization, targeting, and membrane expression of these expressed subunits entirely depend on the interaction of the heterologous channel with endogenous ␤ and ␣ 2 ␦ subunits.…”

Old proteins, developing roles: The regulation of calcium channels by synaptic proteins

“…74 A third regulatory modality linked to the synprint motif is subcellular targeting of the channel. Work from Mochida et al 50,75 shows that the presence of the synaptic protein interaction site is an important determinant of subcellular targeting of P/Q-type channels. Similarly, splice variants of human N-type channels lacking the synprint region are still targeted to axonal compartments, but are excluded from the active zone.…”

“…Similarly, splice variants of human N-type channels lacking the synprint region are still targeted to axonal compartments, but are excluded from the active zone. 75 Conversely, R-type channels which do not contain a synprint region can still target to the presynaptic nerve terminal and participate in neurotransmitter release. 32 Collectively, these findings indicate that while the synprint region may be an important determinant of subcellular distribution of Ca V 2 channels in neurons, it is unlikely to be the only determinant, and its significance may vary with channel subtype.…”