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. 2008 Jun;294(6):E1178-86.
doi: 10.1152/ajpendo.90237.2008. Epub 2008 Apr 22.

Differential Modulation of L-type Calcium Channel Subunits by Oleate

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Free PMC article

Differential Modulation of L-type Calcium Channel Subunits by Oleate

Yingrao Tian et al. Am J Physiol Endocrinol Metab. .
Free PMC article

Abstract

Nonesterified fatty acids such as oleate and palmitate acutely potentiate insulin secretion from pancreatic islets in a glucose-dependent manner. In addition, recent studies show that fatty acids elevate intracellular free Ca(2+) and increase voltage-gated Ca(2+) current in mouse beta-cells, although the mechanisms involved are poorly understood. Here we utilized a heterologous system to express subunit-defined voltage-dependent L-type Ca(2+) channels (LTCC) and demonstrate that beta-cell calcium may increase in part from an interaction between fatty acid and specific calcium channel subunits. Distinct functional LTCC were assembled in both COS-7 and HEK-293 cells by expressing either one of the EYFP-tagged L-type alpha(1)-subunits (beta-cell Cav1.3 or lung Cav1.2) and ERFP-tagged islet beta-subunits (ibeta(2a) or ibeta(3)). In COS-7 cells, elevations in intracellular Ca(2+) mediated by LTCC were enhanced by an oleate-BSA complex. To extend these findings, Ca(2+) current was measured in LTCC-expressing HEK-293 cells that revealed an increase in peak Ca(2+) current within 2 min after addition of the oleate complex, with maximal potentiation occurring at voltages <0 mV. Both Cav1.3 and Cav1.2 were modulated by oleate, and the presence of different auxiliary beta-subunits resulted in differential augmentation. The potentiating effect of oleate on Cav1.2 was abolished by the pretreatment of cells with triacsin C, suggesting that long-chain CoA synthesis is necessary for Ca(2+) channel modulation. These results show for the first time that two L-type Ca(2+) channels expressed in beta-cells (Cav1.3 and Cav1.2) appear to be targeted by nonesterified fatty acids. This effect may account in part for the acute potentiation of glucose-dependent insulin secretion by fatty acids.

Figures

Fig. 1
Fig. 1
Oleate increases intracellular Ca2+ in single COS-7 cells expressing L-type voltage-dependent calcium channels (LTCC). Representative traces of changes in intracellular free calcium concentration ([Ca2+]i) in fura 2-loaded COS-7 cells as specified in MATERIALS AND METHODS in the presence of 10 mM glucose, 2 mM CaCl2, and 0.05% BSA. Reagents were added sequentially at the times indicated as 500 µl bolus to the dish with a starting volume of 1 ml. A: COS-7 cells expressing Cav1.3-iβ2a for 48-h posttransfer of transgenes. B: COS-7 control cells in the absence of any heterologous expression of transgenes.
Fig. 2
Fig. 2
Oleate increases intracellular Ca2+ in single COS-7 cells expressing Cav1.3 plus mut iβ2a or iβ3. Representative traces, generated with 5-point moving average, of fura 2-loaded COS-7 cells expressing Cav1.3-mut iβ2a (A) and Cav1.3-iβ3 (B) imaged 48 h posttransgenes’ transfer in the presence of 10 mM glucose, 2 mM CaCl2, and 0.05% BSA and monitored for [Ca2+]i changes after addition of 500 µl bolus of each reagent at the ndicated times.
Fig. 3
Fig. 3
Mean oleate-induced increase in intracellular [Ca2+]i in LTCC-expressing COS-7 cells. Intracellular free [Ca2+]i increases for each condition were calculated from the fluorescent ratio for each parameter after subtraction of the equivalent value from control cells. A Kd of 224 nm/l for [Ca2+]i binding to free fura-2 was used in the calculations. Data are means ± SE from ≥8 single cells and P values calculated using Student’s paired t-test.
Fig. 4
Fig. 4
Oleate potentiates voltage-sensitive calcium current in HEK-293 cells expressing the calcium channel subunits Cav1.3 or Cav1.2 plus iβ2a and β2. A: representative whole cell calcium current traces recorded from HEK-293 cells expressing the calcium channel subunits Cav1.3-iβ2a2δ. Currents were elicited by a voltage step from −65 to 0 mV in the presence and absence of 100 µM oleate. B: peak calcium current measured at 0 mV in the presence of oleate was normalized to the peak current measured in the absence of oleate. Application of 100 µM oleate increased mean calcium current amplitude at 0 mV by 18.1 ± 8.3% compared with control (n = 17, P < 0.05). C: representative whole cell calcium current traces recorded from HEK-293 cells expressing the calcium channel subunits Cav1.2-iβ2a2δ in response to a voltage step from −65 to 0 mV. D: application of 100 µM oleate increased mean calcium current amplitude at 0 mV by 23.3 ± 10.0% compared with control (n = 24, P < 0.05).
Fig. 5
Fig. 5
The expression of different β-subunit isoforms modulates the extent of oleate current potentiation. A: peak calcium currents were measured at voltages ranging from −70 to −50 mV in HEK-293 cells expressing the calcium channel subunits Cav1.2-iβ2a2δ Current-voltage (I–V) curves are shown for peak currents measured in the presence (gray circles) and absence (■) of 20 µM oleate. B: mean normalized calcium current amplitude at 0 mV was plotted for currents measured in the presence and absence of oleate. Application of 20 µM oleate increased current amplitude by 106 ± 21% compared with control (n = 9, P < 0.002). C: I–V curves are shown for peak currents measured at voltages ranging from −70 to −50 mV in HEK-293 cells transfected with the calcium channel subunits Cav1.2-iβ32δ in the presence (gray circles) and absence (■) of 20 µM oleate. D: application of 20 µM oleate increased mean calcium current amplitude at 0 mV by 16 ± 2% compared with control (n = 8, P < 0.01).
Fig. 6
Fig. 6
Triacsin C abolishes calcium current potentiation by oleate. A: HEK-293 cells expressing the calcium channel subunits Cav1.2-iβ2a-α2δ were treated with 96 µM triacsin C for 20 min. Peak calcium currents were then measured at voltages ranging from −70 to −50 mV in the presence (gray circles) or absence (■) of 20 µM oleate. B: mean calcium current amplitude measured at 0 mV in the presence of oleate was normalized to control mean current amplitude without oleate. Application of 20 µM oleate did not significantly alter mean calcium current amplitude in triacsin C-treated cells (n = 9, P > 0.05).

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