Insulin-mediated upregulation of T-type Ca2+ currents in GH3 cells is mediated by increased endosomal recycling and incorporation of surface membrane Cav3.1 channels.

Abstract Growth factors and hormones have both short- and long-term regulatory effects on the functional expression of voltage gated Ca 2+ (Ca V ) channels. In particular, it has been reported that chronic treatment with insulin upregulates T-type channel membrane expression, leading to an increase in current density in clonal pituitary GH 3 cells. Though this regulatory action may result from alterations in gene expression, recent studies have demonstrated also that endosomal trafficking provides a mechanism for dynamic changes in Ca V channel membrane density. Therefore, in the present work we sought to determine whether the actions of insulin on T-type channel functional expression are mediated by transcriptional and/or post-transcriptional mechanisms. Using real-time RT-PCR and semi-quantitative western blot we found no changes after treatment in the transcript and protein levels of Ca v 3.1, the T-type channel isoform preferentially expressed in the GH 3 cells. Consistent with this, transcriptional studies using a luciferase reporter assay suggested that insulin treatment does not affect the Ca v 3.1 promoter activity. In contrast, patch-clamp recordings on HEK-293 cells stably expressing Ca v 3.1 channels showed a significant increase in current density after treatment, suggesting that the effects of insulin may require post-transcriptional regulation. In line with this, disruption of the endosomal recycling pathway using Brefeldin A and a dominant negative mutant of the small GTPase Rab11a prevented the stimulatory effects of insulin on Ca v 3.1 channels in HEK-293 cells. These results may help explain the effects of insulin on T-type channels and contribute to our understanding of how endosomal recycling impacts the functional expression of Ca V channels.