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.

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.

Regulation of Ca v 3.1 channels by glucocorticoids.

The activity of low voltage-activated Ca(2+) (Ca(V)3) channels is tightly coupled to neurotransmitter and hormone secretion. Previous studies have shown that Ca(V)3 channels are regulated by glucocorticoids (GCs), though the mechanism underlying channel regulation remains unclear. Here, using the pituitary GH(3) cell line as a model, we investigated whether Ca(V)3 channel expression is under the control of GCs, and if their actions are mediated by transcriptional and/or post-transcriptional mechanisms. RT-PCR and western blot analyses showed that Ca(V)3.1 but not Ca(V)3.2 and Ca(V)3.3 channels is expressed in the GH(3) cells, and patch clamp recordings confirmed that Ca(2+) currents through low voltage-activated channels were decreased after chronic treatment with GCs. Consistent with this, total plasma membrane expression of Ca(V)3.1 protein as analyzed by cell-surface biotinylation assays and semi-quantitative western blotting was also down-regulated, while quantitative real-time RT-PCR analysis revealed a significant decrease of Ca(V)3.1 mRNA expression in the treated cells. In contrast, patch-clamp recordings on HEK-293 cells stably expressing recombinant Ca(V)3.1 channels showed that Ca(2+) currents were not affected by GC treatment. These results suggest that decreased transcription is a likely mechanism to explain the inhibitory actions of GCs on the functional expression of native Ca(V)3.1 channels.

Up-regulation of high voltage-activated Ca(2+) channels in GC somatotropes after long-term exposure to ghrelin and growth hormone releasing peptide-6.

Activation of the growth hormone (GH)-secretagogue receptor (GHS-R) by synthetic GH-releasing peptides (GHRP) or its endogenous ligand (ghrelin) stimulates GH release. Though much is known about the signal transduction underlying short-term regulation, there is far less information on mechanisms that produce long-term effects. In the current report, using whole-cell patch-clamp recordings, we assessed the long-term actions of such regulatory factors on voltage-activated Ca(2+) currents in GH-secreting cells derived from a rat pituitary tumour (GC cell line). After 96 h in culture, all recorded GC somatotropes exhibited two main Ca(2+) currents: a medium voltage-activated (MVA; T/R-type) and a high voltage-activated (HVA; mostly dihydropyridine-sensitive L-type) current. Interestingly, L- and non-L-type channels were differentially up-regulated by GHRP-6 and ghrelin. Chronic treatment with the GHS induced a significant selective increase on Ba(2+) current through HVA Ca(2+) channels, and caused only a modest increase of currents through MVA channels. Consistent with this, in presence of D-(Lys(3))-GHRP-6, a specific antagonist of the GHS-R, the increase in HVA Ca(2+) channel activity after chronic treatment with the GHS was abolished. The stimulatory effect on HVA current density evoked by the secretagogues was accompanied by an augment in maximal conductance with no apparent changes in the kinetics and the voltage dependence of the Ca(2+) currents, suggesting an increase in the number of functional channels in the cell membrane. Lastly, in consistency with the functional data, quantitative real-time RT-PCR revealed that the expression level of transcripts encoding for the Ca(V)1.3 pore-forming subunit of the L-type channels was significantly increased after chronic treatment of the GC cells with ghrelin.

Transforming growth factor-beta1 and bone morphogenetic protein-2 downregulate CaV3.1 channel expression in mouse C2C12 myoblasts.

In the developing skeletal muscle, fusion of myoblasts and myotube formation is a process that involves Ca2+ influx through T-type (CaV3) channels. Treatment of myoblasts with transforming growth factor-beta1 (TGF-beta1) and bone morphogenetic protein-2 (BMP-2) decreases the number of CaV3 channels in the plasma membrane and reduces myotube formation. In the current report, we examined whether the inhibitory actions of TGF-beta1 and BMP-2 involve alterations in CaV3 mRNA expression in the myoblast C2C12 cell line. Using RT-PCR, we found that CaV3.1 but not CaV3.2 and CaV3.3 transcripts are present in either undifferentiated or fusion competent C2C12 myoblasts. Semi-quantitative analysis revealed a significant decrease of CaV3.1 mRNA expression in cells treated with TGF-beta1 and BMP-2. In contrast, patch-clamp recordings on HEK-293 cells stably expressing recombinant CaV3.1 channels showed that T-type currents were not affected by chronic exposure to the growth factors. These results suggest that muscle T-channel downregulation by TGF-beta1 and BMP-2 may be mediated by reduced transcription rather than through post-transcriptional modifications of CaV3.1 channels.

Two PEST-like motifs regulate Ca2+/calpain-mediated cleavage of the CaVbeta3 subunit and provide important determinants for neuronal Ca2+ channel activity.

An increase in intracellular Ca2+ due to voltage-gated Ca2+ (CaV) channel opening represents an important trigger for a number of second-messenger-mediated effects ranging from neurotransmitter release to gene activation. Ca2+ entry occurs through the principal pore-forming protein but several ancillary subunits are known to more precisely tune ion influx. Among them, the CaVbeta subunits are perhaps the most important, given that they largely influence the biophysical and pharmacological properties of the channel. Notably, several functional features may be associated with specific structural regions of the CaVbeta subunits emphasizing the relevance of intramolecular domains in the physiology of these proteins. In the current report, we show that CaVbeta3 contains two PEST motifs and undergoes Ca2+ -dependent degradation which can be prevented by the specific calpain inhibitor calpeptin. Using mutant constructs lacking the PEST motifs, we present evidence that they are necessary for the cleavage of CaVbeta3 by calpain. Furthermore, the deletion of the PEST sequences did not affect the binding of CaVbeta3 to the ion-conducting CaV2.2 subunit and, when expressed in human embryonic kidney-293 cells, the PEST motif-deleted CaVbeta3 significantly increased whole-cell current density and retarded channel inactivation. Consistent with this observation, calpeptin treatment of human embryonic kidney-293 cells expressing wild-type CaVbeta3 resulted in an increase in current amplitude. Together, these findings suggest that calpain-mediated CaVbeta3 proteolysis may be an essential process for Ca2+ channel functional regulation.