The TCF7L2-dependent high-voltage activated calcium channel subunit α2δ-1 controls calcium signaling in rodent pancreatic beta-cells

Highlights

• Tcf7l2 controls expression of Cacna2d1/α2δ-1.

• Silencing of Cacna2d1 prevents trafficking of Cav1.2 to the plasma membrane.

• Silencing of Cacna2d1 retains Cav1.2 in recycling endosomes.

• Silencing of Cacna2d1 affects Ca²⁺ signaling and exocytosis.

• Overexpression of α2δ-1 partially counteracts the effect of Tcf7l2 silencing.

Abstract

The transcription factor TCF7L2 remains the most important diabetes gene identified to date and genetic risk carriers exhibit lower insulin secretion. We show that Tcf7l2 regulates the auxiliary subunit of voltage-gated Ca²⁺ channels, Cacna2d1 gene/α2δ-1 protein levels. Furthermore, suppression of α2δ-1 decreased voltage-gated Ca²⁺ currents and high glucose/depolarization-evoked Ca²⁺ signaling which mimicked the effect of silencing of Tcf7l2. This appears to be the result of impaired voltage-gated Ca²⁺ channel trafficking to the plasma membrane, as Cav1.2 channels accumulated in the recycling endosomes after α2δ-1 suppression, in clonal as well as primary rodent beta-cells. This impaired the capacity for glucose-induced insulin secretion in Cacna2d1-silenced cells. Overexpression of α2δ-1 increased high-glucose/K⁺-stimulated insulin secretion. Furthermore, overexpression of α2δ-1 in Tcf7l2-silenced cells rescued the Tcf7l2-dependent impairment of Ca²⁺ signaling, but not the reduced insulin secretion. Taken together, these data clarify the connection between Tcf7l2, α2δ-1 in Ca²⁺-dependent insulin secretion.

Introduction

Type 2 diabetes develops when the demand for insulin exceeds what the pancreatic beta cells can deliver. Glucose-stimulated insulin secretion occurs by Ca²⁺-dependent exocytosis and in this respect, Ca²⁺ entering via voltage-gated calcium channels (VGCCs) plays a particularly important role (Rorsman et al., 2000). Pancreatic β-cells are equipped with at least six types of calcium channels, including Cav1.2, Cav1.3, Cav2.1, Cav2.2, Cav2.3 and Cav3.1 (Yang and Berggren, 2006), of which L-type calcium channels (Cav1.2 and Cav1.3) conduct ~50% of the whole Ca²⁺ currents (Schulla et al., 2003). Nitert et al. reported that the mRNA level of Cav1.2 exceeded that of Cav1.3 and Cav2.3 two-fold in INS-1 832/13 cells, and suggested that Cav1.2 rather than Cav1.3 is critical to glucose-stimulated insulin secretion in INS-1 832/13 cells (Nitert et al., 2008). Rorsman et al. concluded that Cav1.2 is the principal L-type Ca²⁺-channels subtype in mouse β-cells (Rorsman et al., 2012). VGCCs are multi-subunit proteins consisting of the pore-forming α1 subunit, which exists in ten different isoforms in mammalian genomes and is the major VGCC subunit determining the main physiological and pharmacological properties. In addition, a number of auxiliary subunits exist in the genome, each in different isoforms, which attach to the α1 pore subunit and modulate its functions. Of the auxiliary subunits, the function of the γ subunits remains largely unknown, whereas the α2δ and β subunits have been suggested to control VGCC trafficking to plasma membrane (PM), but also to influence certain of the channels’ biophysical properties (Dolphin, 2013). Accordingly, in heterologous expression systems the α2δ subunits increase the maximum current density of Cav1 and Cav2 VGCCs (Dolphin, 2003). The main mechanism has been suggested that increased expression of Cav1 and Cav2 complexes at the plasma membrane coupled to a decrease in their turnover (Canti et al., 2005; Bernstein and Jones, 2007). Interestingly, it was demonstrated in mouse that genetic ablation of α2δ-1 (the major variant in pancreatic islets) reduces Ca²⁺ influx via all types of functional VGCCs in the pancreatic beta cells, which resulted in reduced insulin secretion and impaired glucose tolerance, at least in male mice (Mastrolia et al., 2017). However, a detailed characterization of the cellular effects at the single beta cell level remains to be performed.

TCF7L2 (T-cell factor 7-like 2, also known as TCF4) harbors the single nucleotide polymorphism (SNP) rs7903146, which remains the most significantly common genetic variation associated with human type 2-diabetes (Grant et al., 2006). This genetic variant is linked to reduced gene expression, in particular of certain splice variants (Osmark et al., 2009), and reduced capacity for insulin secretion (Lyssenko et al., 2007), but pathophysiological role of TCF7L2 in development of type 2-diabetes is elusive. Recently, report showed that TCF7L2 is a master regulator of insulin production and processing (Zhou et al., 2014). By contrast TCF7L2 has no major influence over genes involved in the control of Ca²⁺ signaling and exocytosis, with one exception: Cacna2d1 gene expression is markedly reduced in Tcf7l2-silenced cells (Zhou et al., 2014). However, the functional consequences in terms of protein levels and beta cell function are still missing. Therefore, we decided to extend on these previous reports by verifying the regulatory role of the diabetes gene Tcf7l2 on α2δ-1 expression, as well as its consequences for Ca²⁺ signaling and insulin secretion in the beta cell.