SUR1 Regulates PKA-independent cAMP-induced Granule Priming in Mouse Pancreatic B-cells

Abstract

Measurements of membrane capacitance were applied to dissect the cellular mechanisms underlying PKA-dependent and -independent stimulation of insulin secretion by cyclic AMP. Whereas the PKA-independent (Rp-cAMPS–insensitive) component correlated with a rapid increase in membrane capacitance of ∼80 fF that plateaued within ∼200 ms, the PKA-dependent component became prominent during depolarizations >450 ms. The PKA-dependent and -independent components of cAMP-stimulated exocytosis differed with regard to cAMP concentration dependence; the K d values were 6 and 29 μM for the PKA-dependent and -independent mechanisms, respectively. The ability of cAMP to elicit exocytosis independently of PKA activation was mimicked by the selective cAMP-GEFII agonist 8CPT-2Me-cAMP. Moreover, treatment of B-cells with antisense oligodeoxynucleotides against cAMP-GEFII resulted in partial (50%) suppression of PKA-independent exocytosis. Surprisingly, B-cells in islets isolated from SUR1-deficient mice (SUR1−/− mice) lacked the PKA-independent component of exocytosis. Measurements of insulin release in response to GLP-1 stimulation in isolated islets from SUR1−/− mice confirmed the complete loss of the PKA-independent component. This was not attributable to a reduced capacity of GLP-1 to elevate intracellular cAMP but instead associated with the inability of cAMP to stimulate influx of Cl− into the granules, a step important for granule priming. We conclude that the role of SUR1 in the B cell extends beyond being a subunit of the plasma membrane KATP-channel and that it also plays an unexpected but important role in the cAMP-dependent regulation of Ca2+-induced exocytosis.

Mathematical modeling and statistical analysis of calcium-regulated insulin granule exocytosis in β-cells from mice and humans

“…In single mouse β-cells, the exocytotic rate is higher for shorter than for longer pulses (Ammälä et al, 1993b;Barg et al, 2001;Eliasson et al, 2003). The drop in the exocytotic rate at sustained depolarizations has been suggested to result either from Ca 2+ -channel inactivation (Ammälä et al, 1993b;Engisch and Nowycky, 1996) or depletion of a finite pool of immediately releasable granules (Barg et al, 2001;Eliasson et al, 2003).…”

“…Capacitance measurements and Ca 2+ -entry data from mouse β-cells were taken from Eliasson et al (2003), where details on the experiments are provided. Briefly, using the perforated-patch technique, pulses of increasing length (5-850 ms) were applied to isolated single mouse β-cells, and resulting Ca 2+ -currents and increases in membrane capacitance were recorded.…”

“…Here we have carefully reanalyzed previously published data (Eliasson et al, 2003) with attention to the positive effect of forskolin on exocytosis by using a statistical mixed-effects model (Pinheiro and Bates, 2000). We find that the data show a linear relation between depolarization-evoked membrane capacitance increase (∆C m ) and the Ca 2+ -charge entering during a depolarization (Q), and argue that this indicates that pool depletion is negligible in response to short depolarizations in mouse β-cells.…”

“…Potentiation of insulin exocytosis by cAMP involves both activation of protein kinase A (PKA) and Epac 2 (Exchange protein associated with cAMP 2) (Ozaki et al, 2000). Using capacitance measurements, we have suggested that PKA stimulates mobilization whereas Epac 2 is involved in rapid exocytosis of IRP/RRP granules (Eliasson et al, 2003). Conditional for rapid cAMPdependent exocytosis is also the presence of the SNARE-protein SNAP-25, which binds to Epac 2 (Vikman et al, 2009).…”

“…Conditional for rapid cAMPdependent exocytosis is also the presence of the SNARE-protein SNAP-25, which binds to Epac 2 (Vikman et al, 2009). Epac has further been suggested to bind to piccolo (Fujimoto et al, 2002) and to associate with the plasma membrane sulphonylurea receptor SUR1 (Eliasson et al, 2003).…”