A dynamic positive feedback mechanism, known as 'facilitation', augments L-type calcium-ion currents (ICa) in response to increased intracellular Ca2+ concentrations. The Ca2+-binding protein calmodulin (CaM) has been implicated in facilitation, but the single-channel signature and the signalling events underlying Ca2+/CaM-dependent facilitation are unknown. Here we show that the Ca2+/CaM-dependent protein kinase II (CaMK) is necessary and possibly sufficient for ICa facilitation. CaMK induces a channel-gating mode that is characterized by frequent, long openings of L-type Ca2+ channels. We conclude that CaMK-mediated phosphorylation is an essential signalling event in triggering Ca2+/CaM-dependent ICa facilitation.

Background-Calmodulin kinase (CaMK) II is linked to arrhythmia mechanisms in cellular models where repolarization is prolonged. CaMKII upregulation and prolonged repolarization are general features of cardiomyopathy, but the role of CaMKII in arrhythmias in cardiomyopathy is unknown. Methods and Results-We studied a mouse model of cardiac hypertrophy attributable to transgenic (TG) overexpression of a constitutively active form of CaMKIV that also has increased endogenous CaMKII activity. ECG-telemetered TG mice had significantly more arrhythmias than wild-type (WT) littermate controls at baseline, and arrhythmias were additionally increased by isoproterenol. Arrhythmias were significantly suppressed by an inhibitory agent targeting endogenous CaMKII. TG mice had longer QT intervals and action potential durations than WT mice, and TG cardiomyocytes had frequent early afterdepolarizations (EADs), a hypothesized mechanism for triggering arrhythmias. EADs were absent in WT cells before and after isoproterenol, whereas EAD frequency was unaffected by isoproterenol in TG mice. L-type Ca 2ϩ channels (LTTCs) can activate EADs, and LTCC opening probability (Po) was significantly higher in TG than WT cardiomyocytes before and after isoproterenol. A CaMKII inhibitory peptide equalized TG and WT LTCC Po and eliminated EADs, whereas a peptide antagonist of the Na ϩ /Ca 2ϩ exchanger current, also hypothesized to support EADs, was ineffective. Conclusions-These findings support the hypothesis that CaMKII is a proarrhythmic signaling molecule in cardiac hypertrophy in vivo. Cellular studies point to EADs as a triggering mechanism for arrhythmias but suggest that the increase in arrhythmias after ␤-adrenergic stimulation is independent of enhanced EAD frequency.

L-type Ca(2+) channels (LTCCs) are major entry points for Ca(2+) in many cells. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is associated with cardiac LTCC complexes and increases channel open probability (P(O)) to dynamically increase Ca(2+) current (I(Ca)) and augment cellular Ca(2+) signaling by a process called facilitation. However, the critical molecular mechanisms for CaMKII localization to LTCCs and I(Ca) facilitation in cardiomyocytes have not been defined. We show CaMKII binds to the LTCC beta(2a) subunit and preferentially phosphorylates Thr498 in beta(2a). Mutation of Thr498 to Ala (T498A) in beta(2a) prevents CaMKII-mediated increases in the P(O) of recombinant LTCCs. Moreover, expression of beta(2a)(T498A) in adult cardiomyocytes ablates CaMKII-mediated I(Ca) facilitation, demonstrating that phosphorylation of beta(2a) at Thr498 modulates native calcium channels. These findings reveal a molecular mechanism for targeting CaMKII to LTCCs and facilitating I(Ca) that may modulate Ca(2+) entry in diverse cell types coexpressing CaMKII and the beta(2a) subunit.

Insulin secretion from pancreatic β cells is stimulated by glucagon-like peptide-1 (GLP-1), a blood glucose-lowering hormone that is released from enteroendocrine L cells of the distal intestine after the ingestion of a meal. GLP-1 mimetics (e.g., Byetta) and GLP-1 analogs (e.g., Victoza) activate the β cell GLP-1 receptor (GLP-1R), and these compounds stimulate insulin secretion while also lowering levels of blood glucose in patients diagnosed with type 2 diabetes mellitus (T2DM). An additional therapeutic option for the treatment of T2DM involves the administration of dipeptidyl peptidase-IV (DPP-IV) inhibitors (e.g., Januvia, Galvus). These compounds slow metabolic degradation of intestinally released GLP-1, thereby raising post-prandial levels of circulating GLP-1 substantially. Investigational compounds that stimulate GLP-1 secretion also exist, and in this regard a noteworthy advance is the demonstration that small molecule GPR119 agonists (e.g., AR231453) stimulate L cell GLP-1 secretion while also directly stimulating β cell insulin release. In this review, we summarize what is currently known concerning the signal transduction properties of the β cell GLP-1R as they relate to insulin secretion. Emphasized are the cyclic AMP, protein kinase A, and Epac2 mediated actions of GLP-1 to regulate ATP-sensitive K+ channels, voltage-dependent K+ channels, TRPM2 cation channels, intracellular Ca2+ release channels, and Ca2+-dependent exocytosis. We also discuss new evidence that provides a conceptual framework with which to understand why GLP-1R agonists are less likely to induce hypoglycemia when they are administered for the treatment of T2DM.

Potential insulin secretagogue properties of an acetoxymethyl ester of a cAMP analog (8-pCPT-2′- O-Me-cAMP-AM) that activates the guanine nucleotide exchange factors Epac1 and Epac2 were assessed using isolated human islets of Langerhans. RT-QPCR demonstrated that the predominant variant of Epac expressed in human islets was Epac2, although Epac1 was detectable. Under conditions of islet perifusion, 8-pCPT-2′- O-Me-cAMP-AM (10 μM) potentiated first- and second-phase 10 mM glucose-stimulated insulin secretion (GSIS) while failing to influence insulin secretion measured in the presence of 3 mM glucose. The insulin secretagogue action of 8-pCPT-2′- O-Me-cAMP-AM was associated with depolarization and an increase of [Ca2+]i that reflected both Ca2+ influx and intracellular Ca2+ mobilization in islet β-cells. As expected for an Epac-selective cAMP analog, 8-pCPT-2′- O-Me-cAMP-AM (10 μM) failed to stimulate phosphorylation of PKA substrates CREB and Kemptide in human islets. Furthermore, 8-pCPT-2′- O-Me-cAMP-AM (10 μM) had no significant ability to activate AKAR3, a PKA-regulated biosensor expressed in human islet cells by viral transduction. Unexpectedly, treatment of human islets with an inhibitor of PKA activity (H-89) or treatment with a cAMP antagonist that blocks PKA activation (Rp-8-CPT-cAMPS) nearly abolished the action of 8-pCPT-2′- O-Me-cAMP-AM to potentiate GSIS. It is concluded that there exists a permissive role for PKA activity in support of human islet insulin secretion that is both glucose dependent and Epac regulated. This permissive action of PKA may be operative at the insulin secretory granule recruitment, priming, and/or postpriming steps of Ca2+-dependent exocytosis.

Calcium can be mobilized in pancreatic β-cells via a mechanism of Ca2+ -induced Ca 2+release (CICR), and cAMP-elevating agents such as exendin-4 facilitate CICR in β-cells by activating both protein kinase A and Epac2. Here we provide the first report that a novel phosphoinositide-specific phospholipase C-ε (PLC-ε) is expressed in the islets of Langerhans, and that the knockout (KO) of PLC-ε gene expression in mice disrupts the action of exendin-4 to facilitate CICR in the β-cells of these mice. Thus, in the present study, in which wild-type (WT) C57BL/6 mouse β-cells were loaded with the photolabile Ca 2+ chelator NP-EGTA, the UV flash photolysis-catalysed uncaging of Ca 2+ generated CICR in only 9% of the β-cells tested, whereas CICR was generated in 82% of the β-cells pretreated with exendin-4. This action of exendin-4 to facilitate CICR was reproduced by cAMP analogues that activate protein kinase A (6-Bnz-cAMP-AM) or Epac2 (8-pCPT-2 -O-Me-cAMP-AM) selectively. However, in β-cells of PLC-ε KO mice, and also Epac2 KO mice, these test substances exhibited differential efficacies in the CICR assay such that exendin-4 was partly effective, 6-Bnz-cAMP-AM was fully effective, and 8-pCPT-2 -O-Me-cAMP-AM was without significant effect. Importantly, transduction of PLC-ε KO β-cells with recombinant PLC-ε rescued the action of 8-pCPT-2 -O-Me-cAMP-AM to facilitate CICR, whereas a K2150E PLC-ε with a mutated Ras association (RA) domain, or a H1640L PLC-ε that is catalytically dead, were both ineffective. Since 8-pCPT-2 -O-Me-cAMP-AM failed to facilitate CICR in WT β-cells transduced with a GTPase activating protein (RapGAP) that downregulates Rap activity, the available evidence indicates that a signal transduction 'module' comprised of Epac2, Rap and PLC-ε exists in β-cells, and that the activities of Epac2 and PLC-ε are key determinants of CICR in this cell type. 2+ /calmodulin-regulated protein kinase II; cAMP-GEF, cAMP-regulated guanine nucleotide exchange factor; CICR, Ca 2+ -induced Ca 2+ release; Epac, exchange protein directly activated by cAMP; ER, endoplasmic reticulum; ESCA-AM, Epac-selective cAMP analogue acetoxymethyl ester; EYFP, enhanced yellow fluorescent protein; GLP-1, glucagon-like peptide-1; GLP-1R, glucagon-like peptide-1 receptor; GPCR, G protein-coupled receptor; KO, knockout; NAADP, nicotinic acid adenine dinucleotide phosphate; NP-EGTA, o-nitrophenyl ethylene glycol tetraacetic acid; Phosphate AM3, phosphate tris (acetoxymethyl) ester; PI, phosphoinositide; PKA, protein kinase A; PKC, protein kinase C; PLC-ε, phospholipase C-ε; Sp-8-pCPT-2 -O-Me-cAMPS, 8-(4-chlorophenylthio)-2 -O-methyladenosine-3 ,5 -cyclic monophosphorothioate; SES, standard extracellular saline; VDCC, voltage-dependent Ca 2+ channels; WT, wild-type.

Epac1 and Epac2 are guanine nucleotide exchange factors activated by adenosine-3Ј,5Ј-cyclic monophosphate (cAMP), and which are known to be expressed in numerous mammalian cell types (1, 2). An accumulating body of evidence indicates that the existence of Epac may explain novel protein kinase A (PKA) 2 independent actions of cAMP that underlie cellular responsiveness to hormones, neurotransmitters, and pharmacological agents of therapeutic importance (3). Selective activation of Epac may be achieved through the use of 8-(4-chlorophenylthio)-2Ј-O-methyladenosine-3Ј,5Ј-cyclic monophosphate, also known as 8-pCPT-2Ј-O-Me-cAMP (4, 5). This cAMP analog, which incorporates a 2Ј-O-methyl group on the ribose ring of the nucleotide, as well as a 4-chlorophenylthio group on position 8 of the adenine moiety, acts as a "superactivator" of Epac while having a greatly diminished ability to activate PKA (4). Thus, 8-pCPT-2Ј-O-Me-cAMP is an Epac-selective cAMP analog (ESCA) (6).8-pCPT-2Ј-O-Me-cAMP can cross the plasma membrane and is able to alter diverse cellular functions that include Rap1 GTPase activity, PKB, and ERK1/2 protein kinase activity, phospholipase C⑀ activity, Ca 2ϩ signaling, ion channel activity, exocytosis, cell adhesion, and gene expression (7-9). Although no selective antagonist of Epac activation exists, these effects of 8-pCPT-2Ј-O-Me-cAMP are believed to be Epac-mediated because they are observed under conditions in which PKA activity is blocked, whereas they are reduced or eliminated when Epac gene expression is down-regulated. Furthermore, such actions of 8-pCPT-2Ј-O-Me-cAMP are measurable in cells that do not express the cyclic nucleotide-regulated ion channels that constitute an alternative target of cAMP action.Interestingly, published findings exist in which cAMP-elevating agents were found to exert actions not attributable to * This work was supported, in whole or in part, by National Institutes of Health Grants DK045817 and DK069575 (to G. G. H.). This work was also supported by a American Diabetes Association Research Award (to C. A. L. -89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; Luc, luciferase; PDE, cyclic nucleotide phosphodiesterase; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein; SES, standard extracellular saline; RIP1, rat insulin 1 gene promoter; Epac, exchange protein directly activated by cAMP.

Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is potentiated by cAMP-elevating agents, such as the incretin hormone glucagon-like peptide-1 (GLP-1), and cAMP exerts its insulin secretagogue action by activating both protein kinase A (PKA) and the cAMP-regulated guanine nucleotide exchange factor designated as Epac2. Although prior studies of mouse islets demonstrated that Epac2 acts via Rap1 GTPase to potentiate GSIS, it is not understood which downstream targets of Rap1 promote the exocytosis of insulin. Here, we measured insulin secretion stimulated by a cAMP analog that is a selective activator of Epac proteins in order to demonstrate that a Rap1-regulated phospholipase C-epsilon (PLC-ε) links Epac2 activation to the potentiation of GSIS. Our analysis demonstrates that the Epac activator 8-pCPT-2'-O-Me-cAMP-AM potentiates GSIS from the islets of wild-type (WT) mice, whereas it has a greatly reduced insulin secretagogue action in the islets of Epac2 (-/-) and PLC-ε (-/-) knockout (KO) mice. Importantly, the insulin secretagogue action of 8-pCPT-2'-O-Me-cAMP-AM in WT mouse islets cannot be explained by an unexpected action of this cAMP analog to activate PKA, as verified through the use of a FRET-based A-kinase activity reporter (AKAR3) that reports PKA activation. Since the KO of PLC-ε disrupts the ability of 8-pCPT-2'-O-Me-cAMP-AM to potentiate GSIS, while also disrupting its ability to stimulate an increase of β-cell [Ca2+]i, the available evidence indicates that it is a Rap1-regulated PLC-ε that links Epac2 activation to Ca2+-dependent exocytosis of insulin.