Biology subjects

Hockerman, G. H.

Publications and source records attributed to Hockerman, G. H..

3 recordsLinked to original sources

bioRxiv2023

ERG1a K+ Channel Increases Intracellular Calcium Concentration through Modulation of Calsequestrin 1 in C2C12 Myotubes

The ERG1A K+ channel modulates the protein degradation that contributes to skeletal muscle atrophy by increasing intracellular calcium concentration ([Ca2+]i) and enhancing calpain activity, but the mechanism by which the channel regulates the [Ca2+]i is not known. Here, we have investigated the effect of human ERG1A (HERG) on [Ca2+]i in C2C12 myotubes, using Fura-2 calcium assays, immunoblot, RT-qPCR, and electrophysiology. We hypothesized that HERG would modulate L-type calcium channel activity, specifically the Cav1.1 channel known to carry signal from the sarcoplasmic membrane of skeletal muscle to the sarcomeres of the myofibrils. However, we find that HERG has no effect on the amplitude of L-type channel current nor does it affect the mRNA levels nor protein abundance of the Cav1.1 channel. Instead we find that, although the rise in [Ca2+]i (induced by depolarization) is greater in myotubes over-expressing HERG relative to controls, the difference between the KCl-stimulated Ca2+ increase in control and HERG over-expressing cells cannot be accounted for by L-type channel mediated Ca2+ influx, which suggests that HERG could modulate excitation coupled calcium entry (ECCE). Indeed, the HERG-enhanced increase in [Ca2+]i induced by depolarization is blocked by 2-APB, an inhibitor of ECCE (and SOCE). Further, we show data suggesting that HERG also modulates the activity of ryanodine receptors, a component of ECCE, as well as store operated calcium entry (SOCE). Therefore, we investigated the effect of HERG on calsequestrin1, a calcium buffering/binding protein known to modulate ryanodine receptor 1 and store operated Ca2+ entry activities. Indeed, we find that calsequestrin1 mRNA levels are decreased 0.83-fold (p<0.05) and the total protein abundance is lowered 77% (p<0.05) in myotubes over-expressing HERG relative to controls. In summary, the data show that ERG1A overexpression modulates [Ca2+]i in skeletal muscle cells by lowering the abundance of the calcium buffering/binding protein calsequestrin1.

physiology↗

bioRxiv2022

RyR2 regulates store-operated Ca2+ entry, phospholipase C activity, and electrical excitability in the insulinoma cell line INS-1

The ER Ca2+ channel ryanodine receptor 2 (RyR2) is required for maintenance of insulin content and glucose-stimulated insulin secretion, in part, via regulation of the protein IRBIT in the insulinoma cell line INS-1. Here, we examined store-operated and depolarization-dependent Ca2+entry using INS-1 cells in which either RyR2 or IRBIT were deleted. Store-operated Ca2+ entry (SOCE) stimulated with thapsigargin was reduced in RyR2KO cells compared to controls, but was unchanged in IRBITKO cells. STIM1 protein levels were not different between the three cell lines. Basal and stimulated (500 M carbachol) phospholipase C (PLC) activity was also reduced specifically in RyR2KO cells. Insulin secretion stimulated by tolbutamide was reduced in RyR2KO and IRBITKO cells compared to controls, but was potentiated by an EPAC-selective cAMP analog in all three cell lines. Cellular PIP2 levels were increased and cortical f-actin levels were reduced in RyR2KO cells compared to controls. Whole-cell Cav channel current density was increased by 65% in RyR2KO cells compared to controls, and barium current was reduced by acute activation of the lipid phosphatase pseudojanin preferentially in RyR2KO cells over control INS-1 cells. Action potentials stimulated by 18 mM glucose were more frequent in RyR2KO cells compared to controls, and insensitive to the SK channel inhibitor apamin. Taken together, these results suggest that RyR2 plays a critical role in regulating PLC activity and PIP2 levels via regulation of SOCE. RyR2 also regulates {beta}-cell electrical activity by controlling Cav current density, via regulation of PIP2 levels, and SK channel activation.

cell biology↗

bioRxiv2021

RyR2/IRBIT regulates insulin gene transcription, insulin content, and secretion in the insulinoma cell line INS-1

The role of ER Ca2+ release via ryanodine receptors (RyR) in pancreatic {beta}-cell function is not well defined. Deletion of RyR2 from the rat insulinoma INS-1 (RyR2KO) enhanced the Ca2+ integral (AUC) stimulated by 7.5 mM glucose, and rendered it sensitive to block by the IP3 receptor inhibitor xestospongin C, coincident with reduced levels of the protein IP3 Receptor Binding protein released with Inositol 1,4,5 Trisphosphate (IRBIT; aka AHCYL1). Deletion of IRBIT from INS-1 cells (IRBITKO) increased the Ca2+ AUC in response to 7.5 mM glucose and induced xestospongin sensitivity. Insulin content and basal (2.5 mM glucose) and 7.5 mM glucose-stimulated insulin secretion were reduced in RyR2KO cells and more modestly reduced in IRBITKO cells compared to controls. INS2 mRNA levels were reduced in both RyR2KO and IRBITKO cells, but INS1 mRNA levels were specifically decreased in RyR2KO cells. Nuclear localization of S-adenosylhomocysteinase (AHCY) was increased in RyR2KO and IRBITKO cells. DNA methylation of the INS1 and INS2 gene promotor regions was very low, and not different among RyR2KO, IRBITKO, and controls. In contrast, exon 2 of the INS1 and INS2 genes was more extensively methylated in RyR2KO and IRBITKO cells than in controls. Proteomics analysis using LC-MS/MS revealed that deletion of RyR2 or IRBIT resulted in differential regulation of 314 and 137 proteins, respectively, with 41 in common. These results suggest that RyR2 regulates IRBIT levels and activity in INS-1 cells, and together maintain insulin content and secretion, and regulate the proteome, perhaps via DNA methylation. One sentence SummaryDeletion of RyR2 from INS-1 cells had the unanticipated effect of reducing IRBIT proteins levels, and both RyR2 and IRBIT contribute to maintenance of glucose- stimulated insulin secretion.

cell biology↗