title: 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 Ca2+ (CaV) 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 GH3 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 CaV 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 Cav3.1, the T-type channel isoform preferentially expressed in the GH3 cells. Consistent with this, transcriptional studies using a luciferase reporter assay suggested that insulin treatment does not affect the Cav3.1 promoter activity. In contrast, patch-clamp recordings on HEK-293 cells stably expressing Cav3.1 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 Cav3.1 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 CaV channels.
authors: Department of Cell Biology, Center for Research and Advanced Studies of the National Polytechnic Institute (Cinvestav-IPN), Mexico City, Mexico.
authors: Alicia
authors: A
authors: Toledo
authors: Alejandro
authors: A
authors: Sandoval
authors: Ricardo
authors: R
authors: González-Ramírez
authors: Traudy
authors: T
authors: Ávila
authors: Angélica
authors: A
authors: Almanza
authors: Eduardo
authors: E
authors: Monjaraz
authors: Juan Carlos
authors: JC
authors: Gomora
authors: Erika S
authors: ES
authors: Piedras-Rentería
authors: Ricardo
authors: R
authors: Felix
mesh_terms: D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D020747:Calcium Channels, T-Type / Q000235:genetics / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics; D009154:Mutation / Q000235:genetics; D018408:Patch-Clamp Techniques; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D015533:Transcriptional Activation / Q000187:drug effects / Q000235:genetics; D020691:rab GTP-Binding Proteins / Q000235:genetics; D000099193:rab11 GTP-Binding Proteins
chemical_list: D020126:Brefeldin A; D020747:Calcium Channels, T-Type; D007328:Insulin; D020559:Monomeric GTP-Binding Proteins; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C500994:Cacna1g protein, rat