Activity-Dependent Cav1.2 Cluster Surface Expression in Insulin-Secreting Cells

BackgroundL-type Calcium channels evoke insulin secretion, and are particularly important for an initial rapid burst of hormone secretion in both rodents and human. The magnitude of this secretory burst depends on the number of Cav1.2 in plasma membrane (PM). However, the regulation of Cav1.2 dynamics in the PM has previously not been elucidadted.Methods4D confocal imaging performed in Cav1.2-GFP transfected INS-1 cells. The distance (R) between geometrical cell center and each Cav1.2 cluster, was determined for assessment of Cav1.2 surface dynamics. Flourescence Recovery After Photobleaching (FRAP) and Fluctuation Correlation Spectroscopy (FCS) were used for quantifying Cav1.2 kinetics in PM.ResultsStimulation with 20mM glucose or 70mM KCl for >15min lead to Cav1.2 cluster internalization as revealed by 4D confocal imaging. After 30min, the average R value decreased from 7.093±0.176 μm and 6.214±0.1082 μm to 5.76±0.202 μm and 5.23±0.156 μm, respectively (P<0.0001 and 0.001). The internalized Cav1.2 clusters co-localized with EEA-1, an early endosome marker. Cav1.2 internalization was also detectable by FCS. Stimulation with glucose decreased the number of Cav1.2 clusters in the PM by ∼ 40%. In addition, after glucose stimulation Cav1.2 clusters were loss mobile and diffusion times increased from 0.4±0.2s to 0.6±0.2s.eIF3e (eukaryotic translation initiation factor 3, subunit E) has been implicated in the regulation of activity-dependent Cav1.2 surface expression in neurons. Silencing of eIF3e counteracted Cav1.2 cluster internalization. FRAP experiments demonstrated that ablation of eIF3e increased the time constant of Cav1.2 recovery from 0.79s to 1.50s under resting conditions and from 0.21s to 0.63s in stimulated cells. These effects were associated with reduced insulin secretion, slower cell proliferation and increased apoptosis rates.ConclusionCav1.2 cluster surface distribution in insulin-secreting cells is activity-dependent. Cav1.2 traffic is regulated by eIF3e and affects both cell function and survival.