Abstract
The arachidonate-regulated, Ca(2+)-selective ARC channels represent a novel receptor-activated pathway for the entry of Ca(2+) in nonexcitable cells that is entirely separate from the widely studied store-operated, Ca(2+) release-activated Ca(2+) channels. Activation of ARC channels occurs specifically at the low agonist concentrations typically associated with oscillatory Ca(2+) signals and appears to provide the predominant mode of Ca(2+) entry under these conditions (Mignen, O., Thompson, J. L., and Shuttleworth, T. J. (2001) J. Biol. Chem. 276, 35676-35683). In this study we demonstrate that ARC channels are present in a variety of different cell types including both cell lines and primary cells. Examination of their pharmacology revealed that currents through these channels are significantly inhibited by low concentrations (< 5 microm) of Gd(3+), are unaffected by 100 microm 2-aminoethyoxydiphenyl borane, and are not activated by the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (100 microm). Their selectivity for Ca(2+) was assessed by determining the EC(50) for external Ca(2+) block of the monovalent currents observed in the absence of external divalent cations. The value obtained (150 nm) indicates that the Ca(2+) selectivity of ARC channels is extremely high. Examination of the ability of various fatty acids, including arachidonic acid, to activate the ARC channels demonstrated that activation does not reflect any nonspecific membrane fluidity or detergent effects, shows a high degree of specificity for arachidonic acid over other fatty acids (especially monounsaturated and saturated fatty acids), and is independent of any arachidonic acid metabolite. Moreover, studies using the charged analogue arachidonyl coenzyme A demonstrate that activation of the ARC channels reflects an action of the fatty acid specifically at the internal face of the plasma membrane. Whether this involves a direct action of arachidonic acid on the channel protein itself or an action on some intermediary molecule is, at present, unclear.
Highlights
The generation and shaping of [Ca2ϩ]i signals in nonexcitable cells are profoundly influenced by the receptor-activated entry of Ca2ϩ
Biophysical analysis reveals that arachidonate-regulated Ca2ϩ (ARC) and Ca2ϩ release-activated Ca2ϩ (CRAC) channels represent entirely distinct conductances with several unique characteristics, the most important of which is that activation of the ARC channels is entirely independent of store depletion [17,18,19]
Cell Distribution— an arachidonic acid-dependent noncapacitative entry of Ca2ϩ has been described in a wide variety of different cells, to date the detailed characterization of the ARC channels has only been reported for HEK293 cells stably transfected with the human m3 muscarinic receptor (m3-HEK cells) [17]
Summary
Cell Culture—Cells from the human embryonic kidney cell line HEK293 stably transfected with the human m3 muscarinic receptor (m3-HEK cells; generous gift from Dr Craig Logsdon, University of Michigan) were cultured in Dulbecco’s modified Eagle’s medium with 10% calf serum and antibiotics in a 5% CO2 incubator at 37 °C as reported previously [15]. Data were sampled at 20 kHz during the voltage steps and at 5.5 kHz during the voltage ramps and digitally filtered off-line at 1 kHz. Initial currentvoltage relationships obtained immediately upon going whole cell (i.e. before activation of IARC) were averaged and used for leak subtraction of subsequent current recordings. The standard pipette (internal) solution contained the following (in mM): cesium acetate, 140; NaCl, 10; MgCl2, 1.22; CaCl2, 1.89; EGTA, 5; HEPES, 10; pH 7.2, unless otherwise specified. For Ca2ϩ concentrations below 0.5 M the external solution was supplemented with 2 mM EGTA In these same experiments, the pipette solution used contained the following (in mM): cesium acetate, 140; MgCl2, 8; CaCl2, 1.6; EGTA, 5; HEPES, 10; pH 7.2) so as to eliminate any contribution from the MagNuM (or MIC) channels (see “Results” for details).
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