CaV2.3 calcium channels control second-phase insulin release

Abstract

Concerted activation of different voltage-gated Ca 2+ channel isoforms may determine the kinetics of insulin release from pancreatic islets. Here we have elucidated the role of R-type Ca V 2.3 channels in that process. A 20% reduction in glucose-evoked insulin secretion was observed in Ca V 2.3-knockout (Ca V 2.3 -/-) islets, close to the 17% inhibition by the R-type blocker SNX482 but much less than the 77% inhibition produced by the L-type Ca 2+ channel antagonist isradipine. Dynamic insulin-release measurements revealed that genetic or pharmacological Ca V 2.3 ablation strongly suppressed second-phase secretion, whereas first-phase secretion was unaffected, a result also observed in vivo. Suppression of the second phase coincided with an 18% reduction in oscillatory Ca 2+ signaling and a 25% reduction in granule recruitment after completion of the initial exocytotic burst in single Ca V 2.3 -/-β cells. Ca V 2.3 ablation also impaired glucose-mediated suppression of glucagon secretion in isolated islets (27% versus 58% in WT), an effect associated with coexpression of insulin and glucagon in a fraction of the islet cells in the Ca V 2.3 -/-mouse. We propose a specific role for Ca V 2.3 Ca 2+ channels in second-phase insulin release, that of mediating the Ca 2+ entry needed for replenishment of the releasable pool of granules as well as islet cell differentiation. IntroductionSystemic glucose tolerance is orchestrated by the regulated release of insulin and glucagon from the β and α cells of the pancreatic islets of Langerhans. The α and β cells are electrically excitable and use electrical signals to couple changes in blood glucose concentration to stimulation or inhibition of hormone release. In both cell types, influx of extracellular Ca 2+ through voltage-gated Ca 2+ channels with resultant elevation of intracellular Ca 2+ concentration ([Ca 2+ ] i ) triggers exocytosis of the hormone-containing secretory granules. Like other electrically excitable cells, both α and β cells contain several types of voltage-gated Ca 2+ channel (1, 2). Assigning physiological functions to the respective Ca 2+ channels is central to the understanding of electrical and secretory activities in these cells.Voltage-gated Ca 2+ channels are divided into 3 subfamilies: (a) L-type high voltage-activated (HVA) Ca 2+ channel family that comprises the Ca V 1.1, 1.2, 1.3, and 1.4 channels and is inhibited by dihydropyridines (DHPs) (1, 3, 4); (b) non-L-type HVA channels Ca V 2.1 (P/Q-type), 2.2 (N-type), and 2.3 (R-type) that are sensitive to ω-agatoxin IVA and ω-conotoxin GVIA and SNX482, respectively (1, 4, 5); and (c) the low voltage-activated (LVA) T-type Ca 2+ channel family (Ca V 3.1, 3.2, and 3.3). The latter subtype differs electrophysiologically from the HVA Ca 2+ channels in opening transiently already upon modest depolarization (6, 7) and fulfilling important roles in pacemaker cells (8).

Newcomer insulin secretory granules as a highly calcium-sensitive pool

“…In addition, first-phase secretion from wild-type cells has been found to occur mainly from previously docked granules at Synt1A-rich locations, whereas second-phase secretion is mainly due to newcomer granules fusing away from Synt1A clusters (14). Because syntaxin-1 and L-type Ca 2ϩ channels colocalize (39), the results of Ohara-Imaizumi et al (14) show that first-phase secretion takes place mostly at L-type Ca 2ϩ channels, whereas second-phase fusion events occur away from L-type channels, in accordance with Ca 2ϩ -channel knockout experiments (10,11).…”

“…Resupply in this view is an element in the amplifying pathway because it increases the effectiveness of calcium (8,9). Knockout studies have shown that L-type Ca 2ϩ channels control first-phase secretion (10), whereas other types, such as R-type channels, are important for the second phase (11). Classically it has been thought that newly arrived vesicles must go through a sequence of steps, docking and priming, before fusing (12), but more recent data suggest that newcomers fuse with only a short delay during the second phase (13)(14)(15).…”

Glucose-dependent docking and SNARE protein-mediated exocytosis in mouse pancreatic alpha-cell

“…Our measurements where performed in the presence of cAMP where exocytosis is mainly dependent on influx though L-type Ca 2+ -channels [6,14]. The beta-cell Ca 2+ -current reduced by the antibody was a non-L-type and most likely a R-type Ca 2+ -current [36] and alpha-cells do not contain R-type Ca 2+ -channels [19]. Thus, it is not surprising that the antibody did not interfere with the Ca 2+ -influx in the alpha-cells.…”

“…Exocytosis of primed granules in the RRP of the alpha-cell is controlled by influx of Ca 2+ through N-type Ca 2+ -channels in the absence of cAMP, and through L-type Ca 2+ -channels in the presence of high levels of cAMP [6,14]. In mouse beta-cells, rapid exocytosis is under the control of L-type Ca 2+ -channels whereas refilling of granules is controlled by influx through Rtype Ca 2+ -channels [19]. Other than directly affecting exocytosis, SNAP-25 and syntaxin 1A have also been suggested to modulate the voltage-dependent Ca 2+ channels [9,16], however with conflicting results.…”

Polymorphisms in the gene encoding the voltage-dependent Ca2+ channel CaV2.3 (CACNA1E) are associated with type 2 diabetes and impaired insulin secretion

“…Pancreatic beta cells express several types of VDCCs in humans and rodents, including the L-type Ca V 1.2 and R-type Ca V 2.3 [15,16]. Studies in rodent models have demonstrated that these channels play distinct roles for the characteristic biphasic insulin release pattern and while Ca V 1.2 evokes the first phase [17], the Ca V 2.3 is considered responsible for sustained second-phase insulin release [18]. Mice lacking the Ca V 2.3 channel suffer from fasting hyperglycaemia and reduced glucose clearance [19,20].…”