The Kv1-associated molecules TAG-1 and Caspr2 are selectively targeted to the axon initial segment in hippocampal neurons

Abstract

Caspr2 and TAG-1 (also known as CNTNAP2 and CNTN2, respectively) are cell adhesion molecules (CAMs) associated with the voltage-gated potassium channels Kv1.1 and Kv1.2 (also known as KCNA1 and KCNA2, respectively) at regions controlling axonal excitability, namely, the axon initial segment (AIS) and juxtaparanodes of myelinated axons. The distribution of Kv1 at juxtaparanodes requires axo-glial contacts mediated by Caspr2 and TAG-1. In the present study, we found that TAG-1 strongly colocalizes with Kv1.2 at the AIS of cultured hippocampal neurons, whereas Caspr2 is uniformly expressed along the axolemma. Live-cell imaging revealed that Caspr2 and TAG-1 are sorted together in axonal transport vesicles. Therefore, their differential distribution may result from diffusion and trapping mechanisms induced by selective partnerships. By using deletion constructs, we identified two molecular determinants of Caspr2 that regulate its axonal positioning. First, the LNG2-EGF1 modules in the ectodomain of Caspr2, which are involved in its axonal distribution. Deletion of these modules promotes AIS localization and association with TAG-1. Second, the cytoplasmic PDZ-binding site of Caspr2, which could elicit AIS enrichment and recruitment of the membrane-associated guanylate kinase (MAGuK) protein MPP2. Hence, the selective distribution of Caspr2 and TAG-1 may be regulated, allowing them to modulate the strategic function of the Kv1 complex along axons.

ADAM22 and ADAM23 modulate the targeting of the Kv1 channel-associated protein LGI1 to the axon initial segment

“…In particular, the AIS is a zone of restricted diffusion for the lateral mobility of transmembrane proteins, which are anchored to the ankyrin-spectrin-actin cytoskeleton. We previously reported that the Kv1-associated molecules TAG-1 and Caspr2 are co-transported in axonal vesicles whereas they are differently distributed along the axon (Bel et al, 2009;Pinatel et al, 2017). In the present study, we examined the role of ADAM proteins in the vesicular transport of distinct Kv1-associated CAMs, including TAG-1, Caspr2 and LGI1 and whether their interplay contributes to their targeting at the AIS.…”

“…In addition to juxtaparanodes, Caspr2 and TAG-1 are concentrated at the AIS of cortical and motor neurons, but they are not required for the recruitment of Kv1 channels at that site (Duflocq et al, 2011;Inda et al, 2006;Ogawa et al, 2008). In cultured hippocampal neurons, Kv1 channels are enriched at the AIS associated with TAG-1, whereas Caspr2 is targeted all along the axon (Pinatel et al, 2017). Other membrane proteins interacting with Kv1 channels may be localized at the AIS, including ADAM22, which is recruited at the AIS of cultured hippocampal neurons with PSD93 (also known as DLG2) (Ogawa et al, 2010).…”

In vivo CRISPRa decreases seizures and rescues cognitive deficits in a rodent model of epilepsy

“…We first asked if CRISPRa can be exploited to increase endogenous gene expression in glutamatergic neurons and decrease their excitability. As a proof of principle, we chose the Kcna1 gene encoding the Kv1.1 channel, which is important for the regulation of neuronal action potential firing and synaptic transmission (Pinatel et al, 2017;Vivekananda et al, 2017). Lentivirus-or adeno-associated virus-mediated overexpression of Kcna1 reduces neuronal excitability and, when targeted to principal cells, suppresses seizures in rodent models of epilepsy (Wykes et al, 2012;Snowball et al, 2019).…”

CNTNAP2 is targeted to endosomes by the polarity protein PAR3

“…It has been established that threonine 1,292 on CNTNAP2's intracellular domain regulates endocytosis through protein kinase C (Bel, Oguievetskaia, Pitaval, Goutebroze, & Faivre-Sarrailh, 2009) and that proper surface targeting of close family member neurexin-1 requires its PDZ-binding motif (Fairless et al, 2008). Furthermore, specific sites on CNTNAP2's extracellular region are also critical for proper secretory and axonal trafficking (Falivelli et al, 2012;Pinatel et al, 2017). Our immunocytochemistry experiments link PAR3-CNTNAP2 complexes with clathrin-coated endosomes, suggesting that PAR3 may, via PDZ binding, be another component critical for CNTNAP2 internalization from the surface membrane.…”