Internalization of Cav1.3 Channels Control Glutamate-Induced Rise in Intracellular Ca2+ in Mouse Retina

Abstract

Purpose: : Glutamate, the major excitatory neurotransmitter in the retina, induces intracellular Ca²⁺ elevation by activating postsynaptic receptors. These include Ca-permeable ionotropic AMPARs and NMDARs, and family of G protein-coupled mGluRs that trigger Ca release from IP₃-sensitive internal stores. Glutamate-evoked depolarization also activates voltage-gated Ca channels. Understanding the mechanisms whereby glutamate-induced [Ca^2+]_i accumulation is regulated is equally important for physiology and pathophysiology of retina.

Methods: : We used immunocytochemistry, Ca imaging and Live/Dead viability assay on dissociated neurons and slices of mouse retina to demonstrate internalization of L-type channels and its implication in glutamate-induced excitotoxicity. High K⁺-induced signals were recorded in the presence of ω-conotoxin MVIIC and thapsigargin to block N and P/Q type channels and Ca²⁺ release from internal stores, respectively.

Results: : We found activation of ionotropic GluRs triggeres rapid internalization of Ca_v1.3 channels. In RGCs from mouse retinal slices loaded with fura-2, responses of RGCs to short (30 s) application of high K⁺ (20 mM) were reduced (~50%) after 3-5 min incubation with glutamate. High K⁺-induced Ca signals were eliminated by 5 µM nimodipine. This was observed in the presence of ω-conotoxin MVIIC and thapsigargin that block N and P/Q type channels and Ca release from internal stores, respectively. Responses to short (30 -40 s) application of glutamate also attenuated following long exposure to glutamate. Preincubation of retinal slices with 50 µM dynamin-inhibitory peptide, an inhibitor of endocytosis, prevented effect of long glutamate exposure. Incubation of mouse retina for 1h with 100 µM kainate resulted excitotoxic cell death in INL. This is in contrast to salamander retina where kainate affected cells in the GCL. Susceptibility of cells to kainate-induced excitotoxicity was significantly increased in slices pretreated with DIP. Nimodipine (5µM) significantly reduced kainate-induced cell death. Effect of nimodipine was more pronounced in DIP-treated retinas.

Conclusions: : Our data indicate that in mouse retinal ganglion cells activation of GluRs induce rapid internalization of Ca_v1.3 channels. This may serve as a negative feedback mechanism to control excess Ca²⁺ entry during glutamate receptor overactivation.

Keywords: calcium • ion channels • cell survival