Apolipoprotein CIII hyperactivates β cell CaV1 channels through SR-BI/β1 integrin-dependent coactivation of PKA and Src
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- Shi, Y., Yang, G., Yu, J. et al. Cell. Mol. Life Sci. (2014) 71: 1289. doi:10.1007/s00018-013-1442-x
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Apolipoprotein CIII (ApoCIII) not only serves as an inhibitor of triglyceride hydrolysis but also participates in diabetes-related pathological events such as hyperactivation of voltage-gated Ca2+ (CaV) channels in the pancreatic β cell. However, nothing is known about the molecular mechanisms whereby ApoCIII hyperactivates β cell CaV channels. We now demonstrate that ApoCIII increased CaV1 channel open probability and density. ApoCIII enhanced whole-cell Ca2+ currents and the CaV1 channel blocker nimodipine completely abrogated this enhancement. The effect of ApoCIII was not influenced by individual inhibition of PKA, PKC, or Src. However, combined inhibition of PKA, PKC, and Src counteracted the effect of ApoCIII, similar results obtained by coinhibition of PKA and Src. Moreover, knockdown of β1 integrin or scavenger receptor class B type I (SR-BI) prevented ApoCIII from hyperactivating β cell CaV channels. These data reveal that ApoCIII hyperactivates β cell CaV1 channels through SR-BI/β1 integrin-dependent coactivation of PKA and Src.
KeywordsCa2+ channelIntegrinPancreatic β cellProtein kinaseScavenger receptor
Scavenger receptor class B type I
Voltage-gated calcium (CaV) channels are critical in pancreatic β cell physiology and pathophysiology [1, 2]. They not only take center stage in the regulation of insulin secretion but are also involved in β cell development, survival, and growth through the regulation of protein phosphorylation, gene expression, and the cell cycle [1, 2]. The function and density of β cell CaV channels are regulated by a wide range of mechanisms either shared by other cell types or specific to β cells, e.g., channel phosphorylation, interaction with other molecules, and glucose metabolism-derived signaling [1–3]. Dysfunctional CaV channels cause β cell malfunction and even death as manifested in the most common metabolic disorder diabetes mellitus [1, 2]. Indeed, a T-lymphocyte-mediated autoimmune attack plays a crucial role in β cell death in type 1 diabetes. In addition, exposure to type 1 diabetic serum results in unphysiological amounts of Ca2+ in the pancreatic β cell and consequent Ca2+-dependent apoptosis. This is, at least in part, due to excessive Ca2+ influx through hyperactivated CaV channels [4, 5]. Undoubtedly, this process aggravates the disease development on top of the autoimmune attack [1, 2].
It has been demonstrated that elevated apolipoprotein CIII (ApoCIII) acts as a diabetogenic serum factor to drive β cell destruction via hyperactivation of β cell CaV channels [5, 6]. Moreover, we have recently shown that in vivo suppression of ApoCIII delays the onset of diabetes in the BioBreeding rat, a rat model for human type 1 diabetes . Normally, ApoCIII is a blood plasma component. It is synthesized predominantly in the liver and to a minor extent in the intestine. Liver and intestinal cells release this apolipoprotein into the blood where it is situated on the surface of chylomicrons, very-low-density lipoproteins (VLDLs) and high-density lipoproteins (HDLs) [8, 9]. ApoCIII is composed of 79 amino acid residues that form six amphiphilic α-helixes, each containing about ten residues. The three-dimensional NMR structure and dynamics of ApoCIII have been resolved when it complexes with sodium dodecyl sulfate micelles, mimicking its natural lipid-bound state. The six amphiphilic α-helixes assemble into a necklace-like chain wrapping around the sodium dodecyl sulfate micelle surface . Dogmatically, ApoCIII serves as an effective inhibitor of triglyceride hydrolysis by inhibiting lipoprotein lipase and through interference with triglyceride-rich lipoproteins binding to the negatively charged cell surface where lipoprotein lipases and lipoprotein receptors reside [8, 9]. It impedes the selective uptake of cholesteryl esters from LDL and HDL by binding to the scavenger receptor class B type I (SR-BI), and hampers the endocytosis of cholesterol-rich LDL by prevention of apolipoprotein B binding to LDL receptors [10–12]. Elevated plasma ApoCIII concentration is a feature of dyslipidemia in obesity and observed in both type 1 and 2 diabetes [5, 13, 14], whereas a group of Ashkenazi Jews with reduced plasma ApoCIII concentration maintains cardiovascular health and greater insulin sensitivity with age and reaches exceptional longevity .
In addition to the dogmatic roles in lipid metabolism, ApoCIII is also a multifaceted player in cell signaling. It can bind to distinct cell surface receptors including SR-BI and uncharacterized binding sites relaying corresponding signals to their downstream effectors, e.g., β1 integrin, pertussis toxin-sensitive G proteins, NF-κB, and protein kinases [10, 16–18]. However, nothing is known about the molecular mechanisms whereby ApoCIII hyperactivates β cell CaV channels. In the present study, we demonstrate that ApoCIII upregulates β cell CaV1 channels through SR-BI/β1 integrin-dependent coactivation of PKA and Src kinase.
Materials and methods
Cell culture and treatments
Islets of Langerhans were isolated from adult male and female mice and dispersed into single islet cells . RINm5F cells at about 70 % confluency were trypsinized. The resultant suspension of cells was seeded into Petri dishes or 12-well plates. The cells were cultivated in RPMI 1640 medium supplemented with 10 % fetal bovine serum, 2 mM l-glutamine, and 100 U/100 μg/ml penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) and maintained at 37 °C in a humidified 5 % CO2 incubator . They were grown overnight and then subjected to siRNA transfection. For patch-clamp analysis, cells underwent overnight treatment with ApoCIII, the PKA inhibitors H-89 (Calbiochem, La Jolla, CA, USA) and myristoylated PKI (14–22) (PKI, Sigma–Aldrich, St. Louis, MO, USA), the PKC inhibitor calphostin C (Calbiochem), the Src kinase inhibitor PP2 (Calbiochem) and the CaV1 channel blocker nimodipine (Calbiochem) in RPMI medium at final concentrations of 20 μg/ml, 0.5, 1, 0.1, 0.1, and 5 μM, respectively. ApoCIII was dissolved in 0.1 % trifluoroacetic acid (TFA) to make a stock solution of 1 mg/ml, PKI was dissolved in water to prepare a stock solution of 0.5 mM, whereas H-89, calphostin C, PP2, and nimodipine were dissolved in dimethyl sulfoxide (DMSO) to form stock solutions of 5, 1, 1, and 10 mM, respectively. As vehicle controls, 0.002 % TFA and/or 0.03 % DMSO were used.
siRNA design and transfection
Two pairs of 21-mer siRNA duplexes targeting the rat β1 integrin (β1 integrin siRNA #1, ID127971 and β1 integrin siRNA #2, ID127972) and SR-BI (ID128929) were designed and chemically synthesized by Applied Biosystems/Ambion (Austin, TX, USA). Their sequences were subjected to BLAST search to ensure their specificity. Silencer Select Negative Control siRNA (4390843), not targeting any gene product, and Silencer Select GAPDH Positive Control siRNA (4390849), efficiently silencing GAPDH in human, mouse, and rat cells, were purchased from Applied Biosystems/Ambion (Austin, TX, USA). RINm5F cells were reversely transfected with Lipofectamine RNAiMAX. Briefly, negative control siRNA, β1 integrin siRNA #1, β1 integrin siRNA #2 or SR-BI siRNA was mixed with Lipofectamine RNAiMAX followed by 20-min incubation at room temperature. Subsequently, cells were added to the siRNA/Lipofectamine RNAiMAX mixtures followed by gentle agitation and kept at 37 °C in a humidified 5 % CO2 incubator. After 72 h, the transfected cells were grown to about 70 % confluency and subjected to different treatments followed by immunoblot assay or electrophysiological analysis.
Total RNA was isolated from RINm5F cells using the RNeasy Micro Kit as recommended by the manufacturer (Qiagen, Valencia, CA, USA). RT-PCR primer pairs were synthesized by Sigma–Aldrich. The SR-BI primer pair consisted of the forward primer 5′-CAAGAAGCCAAGCTGTAGGG-3′ and the reverse primer 5′-CCCAACAGGCTCTACTCAGC-3′. The GAPDH primer pair comprised the forward primer 5′-TAGACAAGATGGTGAAGG-3′ and the reverse primer 5′-TCCTTGGAGGCCATGTAG-3′; 500 ng of total RNA was reverse transcribed with SuperScript II Reverse Transcriptase (Invitrogen) and Oligo(dT)12–18 Primer (Invitrogen). Polymerase chain reaction was carried out using the Platinum Taq DNA Polymerase (Invitrogen). It underwent 90 s at 94 °C for completely denaturing templates and activating the Taq DNA Polymerase, followed by 29 cycles of denaturing at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 30 s, and ending with a final extension at 72 °C for 5 min. The amplified PCR products were detected by agarose gel electrophoresis and ethidium bromide staining.
SDS-PAGE and immunoblot analysis
RINm5F cells following different treatments were lysed in a lysis buffer (pH 7.5) consisting of 50 mM HEPES, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 10 % glycerol, 1 % triton X-100, 1 mM PMSF and a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). The lysate was centrifuged at 800 × g for 10 min at 4 °C to remove cell debris and nuclei. The protein concentration of the resulting samples was determined with Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA, USA). The samples were denatured by heating at 96 °C for 3 min in SDS sample buffer and then underwent sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis. Briefly, 50, 90, or 180 μg of protein were separated in discontinuous gels consisting of a 4 % acrylamide stacking gel (pH 6.8) and an 8 % acrylamide separating gel (pH 8.8). The separated proteins were then electroblotted to hydrophobic polyvinylidene difluoride membrane (Hybond-P; GE Healthcare, Uppsala, Sweden). The blots were blocked by incubation for 1 h with 5 % non-fat milk powder in a washing buffer, containing 50 mM Tris(hydroxymethyl)aminomethane, 150 mM NaCl and 0.05 % Tween 20 (pH 7.5). They were then incubated overnight at 4 °C with affinity-purified rabbit polyclonal antibodies to β1 integrin (1:500; Millipore, Billerica, MA, USA), SR-BI (1:2,500; Novus, Cambridge, UK), CaV1.2 (1:200) and CaV1.3 (1:200), respectively, and for 1 h at room temperature with mouse monoclonal antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:4000; Applied Biosystems/Ambion, Austin, TX, USA), respectively. After rinsing with the washing buffer, the blots were incubated with the secondary antibodies (either horseradish peroxidase-conjugated goat anti-rabbit IgG or horseradish peroxidase-conjugated goat anti-mouse IgG; 1:50,000; Bio-Rad) at room temperature for 45 min. The immunoreactive bands were visualized with the ECL plus Western blotting detection system (GE Healthcare, Uppsala, Sweden).
Mouse islet cells and RINm5F cells following different treatments were subjected to single-channel and whole-cell patch-clamp measurements . Cell-attached and perforated whole-cell patch-clamp configurations were employed . Electrodes were made from borosilicate glass capillaries, fire-polished and coated with Sylgard close to their tips. Some of them were filled with a solution containing (in mM) 110 BaCl2, 10 TEA-Cl, and 5 HEPES [pH 7.4 with Ba(OH)2] for single-channel measurements. Others were filled with a solution composed of (in mM) 76 Cs2SO4, 1 MgCl2, 10 KCl, 10 NaCl, and 5 HEPES (pH 7.35 with CsOH), as well as amphotericin B (0.24 mg/ml) for whole-cell current recordings. Electrode resistance ranged between 4 and 6 MΩ when they were filled with electrode solutions and immersed in bath solutions. The electrode offset potential was corrected in bath solutions prior to gigaseal formation. Single-channel recordings were performed with cells bathed in a depolarizing external recording solution, containing (in mM) 125 KCl, 30 KOH, 10 EGTA, 2 CaCl2, 1 MgCl2, and 5 HEPES–KOH (pH 7.15). This solution was used to bring the intracellular potential to 0 mV. For perforated whole-cell current measurements, the cells were bathed in a solution containing (in mM) 138 NaCl, 5.6 KCl, 1.2 MgCl2, 10 CaCl2, 5 HEPES (pH 7.4). Single-channel and whole-cell currents were recorded with an Axopatch 200B amplifier (Molecular Devices, Foster City, CA, USA) and an EPC-9 patch clamp amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany), respectively, at room temperature (about 22 °C). Acquisition and analysis of single channel and whole-cell current data were done using the software program pCLAMP 10 (Axon Instruments) and the software program PatchMaster/FitMaster (HEKA), respectively. To guarantee elimination of rapid transient Na+ currents appearing at the initial period of depolarization during whole-cell Ca2+ current recordings , we measured peak whole-cell Ca2+ currents within a time window from 30 to 100 ms after the start point of depolarization. The amplitude of whole-cell currents was normalized by the cell capacitance.
All data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA, followed by least significant difference (LSD) test. When two groups were compared, unpaired Student’s t test or Mann–Whitney U test was employed. The significance level was set to 0.05 or 0.01.
Apolipoprotein CIII increases CaV1 channel density and conductivity in the β cell
Pharmacological ablation of CaV1 channels prevents apolipoprotein CIII-induced hyperactivation of β cell CaV channels
Apolipoprotein CIII hyperactivates β cell CaV channels via coactivation of PKA and Src kinase
The increase in open probability of β cell CaV1 channels by ApoCIII and the mediating role of protein kinases in ApoCIII signaling suggest that ApoCIII may signal upstream of some protein kinases to hyperactivate β cell CaV channels [16, 22–25]. Therefore, we explored the involvement of PKA, PKC, and Src kinase in ApoCIII-induced hyperactivation of β cell CaV channels.
Second, we tested the effect of the PKC inhibitor calphostin C (CalpC) on ApoCIII-induced hyperactivation of β cell CaV channels in RINm5F cells. We observed that cells incubated with ApoCIII and ApoCIII/CalpC-cotreated cells displayed similar whole-cell Ca2+ currents, which were larger than those acquired in vehicle-treated cells (Fig. 3c). Mean Ca2+ current densities in ApoCIII-treated cells (filled circles, n = 33) at the voltage range of 10–50 mV and cells exposed to ApoCIII/CalpC (filled triangles, n = 33) at a voltage range from 20 to 50 mV increased significantly in comparison with vehicle-treated control cells (open circles, n = 33) (Fig. 3d). There is no difference between ApoCIII-treated cells and ApoCIII/CalpC-cotreated cells with regard to the Ca2+ current density (Fig. 3d). Furthermore, cells exposed to control vehicle were similar to CalpC-treated cells in terms of Ca2+ current density (Supplementary Fig. S1C and D). These data demonstrate that PKC inhibition does not affect ApoCIII-induced hyperactivation of β cell CaV channels.
Third, we evaluated the effect of the Src kinase inhibitor PP2 on ApoCIII-induced hyperactivation of β cell CaV channels in RINm5F cells. We found smaller and larger whole-cell Ca2+ currents in cells following incubation with vehicle solution and ApoCIII-incubated cells, respectively (Fig. 3e). Cells exposed to ApoCIII and PP2 fell between vehicle control cells and cells treated with ApoCIII with regard to whole-cell Ca2+ currents (Fig. 3e). Whole-cell Ca2+ current densities quantified in cells treated with ApoCIII (filled circles, n = 40) at the voltage range 10–50 mV were significantly elevated as compared with those determined in vehicle control cells (open circles, n = 40); (Fig. 3f). Cells subjected to cotreatment of ApoCIII and PP2 (filled triangles, n = 40) showed significantly larger Ca2+ currents at the voltage range 20–40 mV compared to vehicle-treated control cells (open circles, n = 40). However, the difference in the Ca2+ current density between ApoCIII/PP2-cotreated cells and cells incubated with vehicle solution is less prominent than that between cells treated with ApoCIII and vehicle-treated control cells (Fig. 3f). Moreover, vehicle-treated cells (open circles, n = 20) and cells incubated with PP2 (filled circles, n = 19) exhibited similar Ca2+ current densities (Supplementary Fig. S1E and F). The results suggest that Src kinase inhibition has a tendency to decrease ApoCIII-induced hyperactivation of β cell CaV channels.
The marginal effect of PKA or Src kinase inhibitors alone on whole-cell Ca2+ currents inevitably raised the question if coinhibition of PKA and Src kinase is sufficient to prevent ApoCIII-induced hyperactivation of β cell CaV channels. We answered the question by analyzing whole-cell Ca2+ currents in RINm5F cells following cotreatment with H-89 and PP2. We observed that whole-cell Ca2+ currents in ApoCIII-treated cells were larger than those in control cells or cells subjected to treatment with ApoCIII in the presence of H-89 and PP2 (Fig. 5c). Significantly higher densities of whole-cell Ca2+ currents appeared in the ApoCIII group (filled circles, n = 26) in comparison with control group (open circles, n = 26) or the group subjected to incubation with ApoCIII in the presence of H-89 and PP2 (filled triangles, n = 27); (Fig. 5d). Moreover, whole-cell Ca2+ currents in control cells resembled those observed in cells treated with H-89 and PP2 (Supplementary Fig. S2C and D). To rule out the possibility of non-specific effects of H-89, we replaced H-89 with PKI. In fact, PKI produced similar results to those above-described in the presence of H-89 (Fig. 4; Supplementary Fig. S3). These data reveal that ApoCIII enhances whole-cell Ca2+ currents via coactivation of PKA and Src Kinase.
Apolipoprotein CIII does not influence β cell CaV1 channel expression
Apolipoprotein CIII upregulates β cell CaV channels via β1 integrin
Apolipoprotein CIII hyperactivates β cell CaV channels via SR-BI
The gross conductivity of CaV channels depends on the density and activity of functional channels in the plasma membrane of the cell. Enhancement of whole-cell Ca2+ currents by type 1 diabetic serum and its factor ApoCIII can result from enriched density and/or increased conductivity of functional CaV channels in the β cell plasma membrane [4, 5]. However, all studies [1, 2, 5, 27] except one  have so far examined the effect of type 1 diabetic serum on CaV channels only at the whole cell level. In the study by Juntti-Berggren et al.  the increase in β cell CaV channel activity by type 1 diabetic serum was characterized at both the single channel and the whole-cell level. However, this work did not analyze whether type 1 diabetic serum could alter the density of functional CaV channels in the β cell plasma membrane . Although we have previously revealed that ApoCIII serves as a type 1 diabetic serum factor, hyperactivating β cell CaV channels, only whole-cell patch-clamp analysis was performed . Undoubtedly, detailed examination of biophysical properties of single CaV channels in ApoCIII-treated cells should be implemented to mechanistically dissect hyperactivation of β cell CaV channels by this apolipoprotein. Interestingly, cell-attached single channel recordings in the present work reveal that incubation with ApoCIII not only augments the activity of individual β cell CaV1 channels but also enriches the number of functional CaV1 channels in the recorded area of the β cell plasma membrane. The augmentation of single CaV1 channel activity is visualized as an increased open probability attributed to the prolonged mean open time and shortened mean closed time. Enrichment of number of functional CaV1 channels is verified by appearance of more levels of single CaV1 channel conductance.
The insulin-secreting RINm5F cell is equipped with CaV1, CaV2 and CaV3 channels [1, 2]. We investigated if ApoCIII selectively hyperactivates CaV1 channels or indiscriminately impacts all these three types of CaV channels in this insulin-secreting cell. It turned out that ApoCIII-induced hyperactivation of β cell CaV channels could no longer take place following pharmacological ablation of CaV1 channels. This means that ApoCIII selectively hyperactivates CaV1 channels, which are the major CaV channel type playing a predominant role over other types of CaV channels in β cell physiology and pathophysiology. Give that ApoCIII acts as the actual factor in type 1 diabetic serum to drive Ca2+-dependent β cell death that is prevented by the CaV1 channel blocker verapamil, the selective hyperactivation of β cell CaV1 channels observed in the present work most likely accounts for the pathophysiological role of this apolipoprotein in Ca2+-dependent β cell [1, 2, 4, 5].
A series of protein kinases, such as PKA and PKC, can effectively phosphorylate CaV channels resulting in increases in the open channel density and activity due to phosphorylation-induced conformational changes in these channels [3, 28, 29]. Increases in the number and open probability of functional CaV channels by ApoCIII might be mediated by protein kinases. ApoCIII has been demonstrated to activate PKC through β1 integrin in monocytic cells . Furthermore, β1 integrin activation can also upregulate CaV1 channels in neurons, ventricular myocytes and vascular smooth muscle cells through stimulation of PKA, PKC and Src kinase [22–25]. All these components are present in β cells [2, 30–33] and may suggest that ApoCIII employs the β1 integrin-PKA/PKC/Src kinase cascade to hyperactivate β cell CaV channels. Indeed, the present work shows that complex inhibition of PKA, PKC and Src kinase effectively abrogates ApoCIII-induced hyperactivation of β cell CaV channels and that coinhibition of PKA and Src kinase is enough for this effect. However, individual inhibition of PKA, PKC or Src kinase only produced, if anything, a marginal effect on ApoCIII-induced hyperactivation of β cell CaV channels. Hence, we conclude that ApoCIII relies on parallel PKA and Src pathways to upregulate β cell CaV channels.
Occurrence of ApoCIII-induced hyperactivation of β cell CaV channels requires overnight incubation. Hence, the effect might be accounted for by an increase in CaV channel expression. Therefore, we quantified immunoreactivities of CaV1.2 and CaV1.3 subunits in RINm5F cells following overnight incubation with ApoCIII. However, the incubation had no influence on β cell CaV1 channel expression. We therefore excluded the possibility that ApoCIII elevates β cell CaV1 channel expression.
The transmembrane receptor β1 integrin is noncovalently associated with other integrins to form a set of heterodimers. They recognize a large number of soluble and surface-bound proteins to mediate cell–cell, cell-extracellular matrix and cell-pathogen interactions . β1 Integrin is situated downstream of ApoCIII and upstream of PKA/PKC/Src kinase in some cell types [16, 22–25]. This made us investigate whether the ApoCIII-β1 integrin-PKA/PKC/Src kinase pathway operates in the β cell as the mechanism whereby this apolipoprotein hyperactivates CaV1 channels. Interestingly, knockdown of β1 integrin does not influence β cell CaV channel activity in the absence of ApoCIII, but significantly abrogates ApoCIII-induced hyperactivation of β cell CaV channels. The results clearly verify that β1 integrin plays a significant role in mediating the action of ApoCIII on β cell CaV1 channel activity.
Although β1 integrin can couple ApoCIII to the corresponding downstream effectors PKA, PKC and Src kinase, β1 integrin is unlikely to directly interact with this apolipoprotein [16, 22–25]. Previous work shows that SR-BI not only physically associates with ApoCIII but also interacts with β1 integrin [10, 26]. This pinpoints the possibility that SR-BI may serve as a molecular bridge between ApoCIII and β1 integrin with regard to β cell CaV channel hyperactivation. Indeed, in the present study we could demonstrate that SR-BI serves as a molecular bridge since SR-BI gene silencing efficiently nullifies ApoCIII-induced hyperactivation of β cell CaV channels. This generates a complete picture of the novel cascade of β cell CaV channel hyperactivation, namely ApoCIII-SR-BI-β1 integrin-PKA/Src.
It is worth noting that ApoCIII-induced hyperactivation of β cell CaV1 channels observed in the present work occurred when cells were depolarized to more positive potentials than +10 mV. It seems that β cell CaV1 channels in vivo never experience such strong depolarization and thus the effect of ApoCIII could hardly happen since the action potential of the β cell peaks at about −20 mV [35–37]. However, the effect of ApoCIII was detected by using the perforated whole-cell patch-clamp recording mode under experimental conditions where 10 mM Ca2+ was added in extracellular solution to obtain optimal Ca2+ currents. Such a high concentration of extracellular Ca2+ (10 mM) in comparison with physiological concentration of extracellular Ca2+ (2.5 mM) can significantly shift the I–V curve to more positive potentials [38–40]. The perforated whole-cell patch-clamp recording mode has a similar effect. Hence, under in vivo conditions ApoCIII is likely to affect β cell CaV1 currents within the physiological membrane potential range [38–40].
In conclusion, our findings demonstrate that ApoCIII selectively hyperactivates β cell CaV1 channels through parallel PKA and Src kinase pathways in a SR-BI/β1 integrin-dependent fashion. ApoCIII-induced hyperactivation of β cell CaV1 channels is characterized by the enriched density and increased activity of functional CaV1 channels in the β cell plasma membrane. Undoubtedly, this novel signal-transduction pathway has a potential to serve as an innovative drug discovery platform for the prevention of Ca2+-dependent β cell death in association with diabetes.
This work was supported by grants from Berth von Kantzow’s Foundation, Diabetes Research and Wellness Foundation, EuroDia (FP6-518153), the Family Erling-Persson Foundation, Fredrik and Ingrid Thuring’s Foundation, Funds of Karolinska Institutet, the Knut and Alice Wallenberg Foundation, Magn. Bergvall’s Foundation, Novo Nordisk Foundation, Skandia Insurance Company, Ltd., the Stichting af Jochnick Foundation, Strategic Research Program in Diabetes at Karolinska Institutet, the Swedish Alzheimer Association, the Swedish Diabetes Association, the Swedish Foundation for Strategic Research, the Swedish Research Council, the Swedish Society of Medicine, Torsten and Ragnar Söderberg Foundation, VIBRANT (FP7-228933-2) and Åke Wiberg’s Foundation. P.-O. Berggren is founder of the Biotech Company BioCrine AB and is also a member of the board of this company. S.-N.Yang is a consultant to BioCrine AB. BioCrine AB is developing ApoCIII as a novel druggable target for the treatment of diabetes.