Roles of voltage-gated Ca2+ channel subunits in pancreatic β cells

Abstract: Hallmarks of type 2 diabetes (T2D) include elevated blood glucose and free fatty acids (FFAs) as a result of impaired β cell insulin secretion and decreased β cell mass. The glucose-stimulated insulin secretion (GSIS) in β cells is triggered by depolarization-evoked Ca2+ entry through voltage-gated Ca2+ (CaV) channels. The majority of CaV channels are believed to reside in cholesterol-rich membrane microdomains called membrane rafts. CaV channels consist of the main pore-forming α1 subunit and three auxiliary subunits, β, α2δ, and γ. The roles of the CaV auxiliary subunits and the membrane rafts in pancreatic β cells are not fully understood, but we have recently shown that the TCF7L2 gene, associated with the strongest genetic risk factor of T2D, regulates Cacna2d1 (α2δ1). This thesis aims to elucidate the roles of β1, β2a, and α2δ1 subunits, as well as membrane rafts, in regulating the α1 subunit and, in turn, insulin secretion and β cell survival. Human islets from donors with T2D contained decreased membrane rafts. A similar phenotype was also observed in the diabetic rat model Goto Kakizaki (GK) rat islets. Cholesterol depletion in healthy human islets by cholesterol oxidase (CO) reduced membrane rafts, resembling islets from donors with T2D. Cholesterol depletion resulted in elevated basal insulin release in both human and rat islets. The reason for this appeared to be the declustering of CaV1.2, elevation in basal Ca2+ oscillations, and an increase in single-CaV channel activity as observed in patch-clamp experiments. When suppressing the Tcf7l2 gene, α2δ1 (mRNA and protein) was downregulated and intracellular Ca2+ was reduced as measured by confocal microfluorimetry. The decrease in Cacna2d1 expression resulted in CaV channel internalization in the recycling endosomes. This lowered the whole-cell Ca2+ current and decreased insulin secretion.Human gene expression analysis showed that both Cacnb1 (β1) and Cacnb2a (β2a) genes are abundant in pancreatic islets. When examining the GK rat islets, the expression of both genes was downregulated. Immunoblot experiments showed that high glucose treatment also reduced protein levels of β1 and β2a in INS-1 832/13 cells. Silencing the β1 subunit reduced insulin secretion, which may be due to the observed decrease in whole-cell Ca2+ currents. By contrast, β2a suppression did not affect insulin release. When comparing the palmitoylation state of β2a, cells overexpressing the non-palmitoylated β2a had a decreased membrane expression of both β2a and α1C. However, overexpression of palmitoylated β2a increased intracellular Ca2+, although without affecting secretion. FFA (palmitate) treatment reduced intracellular Ca2+ under stimulatory conditions thus decreasing GSIS. Cells that either lack β1 or express excess palmitoylated β2a have increased risk of apoptosis. These data reveal novel roles of membrane rafts and β1, β2a, and α2δ1 subunits in regulating CaV channel trafficking and activity, thus influencing β cell function and survival.