<p>  </p> <p>The role of the endoplasmic reticulum (ER) Ca2+ release channels ryanodine receptor 2 (RyR2) and inositol 1,4,5-triphosphate receptor (IP3R) in pancreatic b-cell function are emerging, but are not well defined. It has been demonstrated that ER stress brought about by RyR2 dysfunction leads to impaired insulin secretion and contributes to the etiology of type 2 diabetes (T2D). Our work contributes to the understanding of the role of RyR2 in physiological pancreatic b-cell function and how loss of RyR2 contributes to the pathophysiology of T2D.</p> <p>To investigate the role of RyR2 in pancreatic b-cell function, we utilized CRISPR-Cas9 to delete RyR2 from the rat insulinoma INS-1 cell line (RyR2KO). We found that RyR2KO cells displayed an enhanced glucose-stimulated Ca2+ integral (area under the curve; AUC) and were sensitive to inhibition by the IP3R antagonist, xestospongin C. Loss of RyR2 also resulted in a reduction in IRBIT protein levels. Therefore, we deleted IRBIT from INS-1 cells (IRBITKO) and found that IRBITKO cells also displayed an increased Ca2+ AUC in response to glucose stimulation. We discovered that total cellular insulin content and secretion were reduced in RyR2KO cells, but more modestly reduced in IRBITKO cells. We found that <em>INS2</em> mRNA levels were reduced in both RyR2KO and IRBITKO cells, but <em>INS1</em> mRNA levels were specifically decreased in RyR2KO cells. Additionally, nuclear localization of S-adenosylhomocysteinase (AHCY) was increased in both RyR2KO and IRBITKO cells. DNA methylation of exon 2 of the <em>INS1</em> and <em>INS2</em> genes was more extensively methylated in RyR2KO and IRBITKO cells compared to controls. Proteomics analysis revealed that deletion of RyR2 or IRBIT resulted in differential regulation of 314 and 137 proteins, respectively, with 41 in common. Our results suggest that RyR2 regulates IRBIT levels and activity, and together maintain insulin content and secretion, and regulate the INS-1 cell proteome, perhaps via DNA methylation.</p> <p>The role of interplay between RyR2 and IP3R in Ca2+ signaling and homeostasis in pancreatic b-cell function remains understudied. Stimulation with the sulfonylurea tolbutamide resulted in markedly delayed Ca2+ transients in both RyR2KO and IRBITKO cells. Xestospongin C significantly reduced the AUC of Ca2+ in RyR2KO and IRBITKO cells. Muscarinic receptor stimulation revealed a markedly increased AUC of Ca2+ in IRBITKO cells compared to both RyR2KO and control INS-1 cells. Assessment of PLC activity revealed that basal and stimulated PLC activity were reduced in the absence of RyR2 or IRBIT. Store-operated Ca2+ entry (SOCE) following ER Ca2+ depletion revealed a decreased SOCE amplitude only in RyR2KO cells. Given evidence that phosphatidylinositol-4,5-bisphosphate (PIP2) depletion from the plasma membrane can regulate voltage-gated Ca2+ channel inhibition, we explored electrophysiological properties of all three cell lines. The frequency of glucose-stimulated action potentials was doubled in RyR2KO cells. Additionally, whole-cell voltage-gated Ca2+ current density was doubled in RyR2KO cells, and this current was more sensitive to hydrolysis of PIP2. These results evidence crosstalk between RyR2 and IP3R, and that RyR2 plays a critical role in maintaining proper Ca2+ homeostasis, PLC activity, and electrophysiological properties in pancreatic b-cells.</p>