Dissociation of the store-operated calcium current I(CRAC) and the Mg-nucleotide-regulated metal ion current MagNuM

Abstract

Rat basophilic leukaemia cells (RBL-2H3-M1) were used to study the characteristics of the store-operated Ca(2+) release-activated Ca(2+) current (I(CRAC)) and the magnesium-nucleotide-regulated metal cation current (MagNuM) (which is conducted by the LTRPC7 channel). Pipette solutions containing 10 mM BAPTA and no added ATP induced both currents in the same cell, but the time to half-maximal activation for MagNuM was about two to three times slower than that of I(CRAC). Differential suppression of I(CRAC) was achieved by buffering free [Ca(2+)](i) to 90 nM and selective inhibition of MagNuM was accomplished by intracellular solutions containing 6 mM Mg.ATP, 1.2 mM free [Mg(2+)](i) or 100 microM GTP-gamma-S, allowing investigations on these currents in relative isolation. Removal of extracellular Ca(2+) and Mg(2+) caused both currents to be carried significantly by monovalent ions. In the absence or presence of free [Mg(2+)](i), I(CRAC) carried by monovalent ions inactivated more rapidly and more completely than MagNuM carried by monovalent ions. Since several studies have used divalent-free solutions on either side of the membrane to study selectivity and single-channel behaviour of I(CRAC), these experimental conditions would have favoured the contribution of MagNuM to monovalent conductance and call for caution in interpreting results where both I(CRAC) and MagNuM are activated.

Figure 1. Differential activation time course of I_CRAC and MagNuM

A, average inward (•) and outward (○) I_CRAC and MagNuM at −80 and +80 mV, respectively (n = 8). I_CRAC and MagNuM were activated passively by omission of ATP and inclusion of 10 mm BAPTA in the pipette solution ([MgCl₂]_i = 1 mm, free [Mg²⁺]_i = 780 μm, extracellular [Ca²⁺] = 1 mm). The dotted line represents the inverted and scaled outward current to illustrate the difference in activation time course. B, current-voltage relationship derived from a high-resolution current record in response to a voltage ramp of 50 ms duration that ranged from −100 to +100 mV, taken at 300 s from a representative cell under experimental conditions described in A. Arrows indicate −80 and +80 mV. Inward currents are predominantly I_CRAC and outward currents are predominantly MagNuM. C, average inward (•) and outward (○) I_CRAC and MagNuM at −80 and +80 mV, respectively (n = 3). Experimental conditions and solutions as in A, except that 20 μm InsP₃ was added to the pipette solution to accelerate activation of I_CRAC. The dotted lines represent the scaled inward and outward currents depicted in A to illustrate the selective acceleration of I_CRAC activation time course. D, average inward (•) and outward (○) I_CRAC and MagNuM at −80 and +80 mV, respectively (n = 5). Experimental conditions and solutions as in A, except that the pipette solution lacked MgCl₂. The dotted line represents the scaled outward current taken from A to illustrate the difference in MagNuM activation time course.

Figure 2. Selective activation of I_CRAC and MagNuM

A, average inward (•) and outward (○) currents at −80 and +80 mV, respectively (n = 4), recorded under experimental conditions that suppress MagNuM and favour I_CRAC activation. I_CRAC was activated passively by inclusion of 10 mm BAPTA in the pipette solution and MagNuM was suppressed by 6 mm Mg.ATP ([MgCl₂]_i = 0, free [Mg²⁺]_i = 550 μm). B, I-V relationship of I_CRAC derived from high-resolution current records in response to voltage ramps of 50 ms duration that ranged from −100 mV to +100 mV. Data are taken from a representative cell under experimental conditions described in A. C, average inward (•) and outward (○) currents at −80 and +80 mV, respectively (n = 7), recorded under experimental conditions that suppress I_CRAC and favour MagNuM activation. MagNuM was activated passively by omission of ATP from the pipette solution and I_CRAC was suppressed by buffering [Ca²⁺]_i to 90 nm using 10 mm EGTA and 3.6 mm CaCl₂ ([MgCl₂]_i = 1 mm, free [Mg²⁺]_i = 760 μm). D, I-V relationship of MagNuM derived from high-resolution current records measured with the same pulse protocol as in B. Data are taken from a representative cell under experimental conditions described in C. E, average inward (•) and outward (○) currents at −80 and +80 mV, respectively (n = 5), recorded under experimental conditions that suppress I_CRAC and favour MagNuM activation. MagNuM was activated passively by omission of ATP from the pipette solution and I_CRAC was suppressed by buffering [Ca²⁺]_i to 90 nm using 10 mm EGTA and 3.6 mm CaCl₂ (free [Mg²⁺]_i = 0). I_CRAC was activated by brief application (2–3 s) of 20 μm ionomycin applied at the time indicated by the arrow. F, I-V relationships derived from high-resolution current records, measured as in B. Data are taken from a representative cell under experimental conditions described in E before ionomycin application (300 s, thin line) and after I_CRAC had fully developed (400 s, thick line).

Figure 3. Monovalent I_CRAC and MagNuM

A, average inward (•) and outward (○) currents at −80 and +80 mV, respectively (n = 3), recorded under experimental conditions that suppress MagNuM and favour I_CRAC activation. I_CRAC was activated passively by inclusion of 10 mm BAPTA in the pipette solution and MagNuM was suppressed by 6 mm Mg.ATP ([MgCl₂]_i = 1 mm, free [Mg²⁺]_i = 1.1 mm). Cells were perfused with divalent-free (DVF) extracellular solution for the time indicated by the bars. B, I-V relationship derived from a high-resolution current record in response to a voltage ramp of 50 ms duration that ranged from −100 to +100 mV, taken at the peak of monovalent inward current (206 s) from a representative cell under experimental conditions described in A. Note the strong inward rectification of monovalent I_CRAC. C, average inward (•) and outward (○) currents at −80 and +80 mV, respectively (n = 4), recorded under experimental conditions that suppress I_CRAC and favour MagNuM activation. MagNuM was activated passively by omission of ATP from the pipette solution and I_CRAC was suppressed by buffering [Ca²⁺]_i to 90 nm using 10 mm EGTA and 3.6 mm CaCl₂ ([MgCl₂]_i = 1 mm, free [Mg²⁺]_i = 760 μm). Cells were perfused with DVF extracellular solution for the time indicated by the bars. D, I-V relationship derived from a high-resolution current record in response to a voltage ramp as detailed in B, taken at the peak of monovalent inward current (390 s) from a representative cell under experimental conditions described in C. Note the linear I-V relationship of monovalent MagNuM currents.

Figure 4. Monovalent I_CRAC and MagNuM in reduced intracellular Mg²⁺

In all panels, average inward (•) and outward (○) currents at −80 and +80 mV were acquired under defined free [Mg²⁺]_i. Left panels represent experimental conditions that suppress MagNuM and favour I_CRAC activation by inclusion of 6 mm Mg.ATP, 20 μm InsP₃ and 10 mm BAPTA in the pipette solution, whereas right panels represent conditions in which I_CRAC was suppressed by buffering [Ca²⁺]_i to 90 nm using 10 mm EGTA and 3.6 mm CaCl₂. MagNuM developed by omission of ATP. In all experiments, appropriate amounts of MgCl₂ were added to arrive at defined [Mg²⁺]_i, as indicated above each pair of panels. Cells were perfused with divalent-free extracellular solution for the times indicated by the bars. A, left panel: average responses of five cells ([MgCl₂]_i = 1 mm, free [Mg²⁺]_i = 1.1 mm). Right panel: average responses of nine cells ([MgCl₂]_i = 1.4 mm, free [Mg²⁺]_i = 1.1 mm). B, left panel: average responses of four cells ([MgCl₂]_i = 0, free [Mg²⁺]_i = 550 μm). Right panel: average responses of eight cells ([MgCl₂]_i = 740 μm, free [Mg²⁺]_i = 550 μm). C, left panel: average responses of five cells in [Mg²⁺]_i-free conditions where ATP and MgCl₂ were omitted from the pipette solution and MagNuM was suppressed by 100 μm GTP-γ-S. I_CRAC was activated by 20 μm InsP₃. Right panel: average responses of nine cells in [Mg²⁺]_i-free conditions. D, average responses of four cells in [Mg²⁺]_i-free conditions where ATP and MgCl₂ were omitted from the pipette solution. Therefore, both InsP₃-mediated I_CRAC and ATP-dependent MagNuM were activated. The dotted trace represents data taken from the left panel of C to illustrate the contribution of I_CRAC. E, I-V relationships derived from high-resolution current records in response to a voltage ramp of 50 ms duration that ranged from −100 to +100 mV, taken at the peak of monovalent inward currents from representative cells under experimental conditions described in C. Note the linear I-V relationship of monovalent MagNuM currents and inward rectification of I_CRAC.

Figure 5. Differential permeation of Ca²⁺ and Mg²⁺ through I_CRAC and MagNuM

A, average inward (•) and outward (○) currents at −80 and +80 mV, respectively (n = 3), recorded under experimental conditions that suppress MagNuM and favour I_CRAC activation. I_CRAC was activated passively by inclusion of 10 mm BAPTA in the pipette solution and MagNuM was suppressed by 6 mm Mg.ATP ([MgCl₂]_i = 1 mm, free [Mg²⁺]_i = 1.1 mm). Cells were perfused with nominally Ca²⁺-free, but Mg²⁺-containing extracellular solution for the time indicated by the bars. Note the complete block of I_CRAC when Ca²⁺ was removed. B, average inward (•) and outward (○) currents at −80 and +80 mV, respectively (n = 3), recorded under experimental conditions that suppress I_CRAC and favour MagNuM activation. MagNuM was activated passively by omission of ATP and MgCl₂ from the pipette solution and I_CRAC was suppressed by buffering [Ca²⁺]_i to 90 nm using 10 mm EGTA and 3.6 mm CaCl₂ (free [Mg²⁺]_i = 0). Cells were perfused with nominally Ca²⁺-free, but Mg²⁺-containing extracellular solution for the time indicated by the bars. Note that MagNuM inward current is not abolished, but carried by Mg²⁺ and that outward currents are enhanced.

Figure 6. Differential pharmacology of I_CRAC and MagNuM

A, average inward (•) and outward (○) I_CRAC and MagNuM at −80 and +80 mV, respectively (n = 4). I_CRAC and MagNuM were activated passively by omission of ATP and inclusion of 20 μm InsP₃ and 10 mm BAPTA in the pipette solution ([MgCl₂]_i = 1 mm, free [Mg²⁺]_i = 780 μm). After full activation of I_CRAC, 100 μm 2-APB was applied extracellularly for the time indicated by the bar and a second application was performed after MagNuM had developed. Note the differential reversibility of 2-APB-mediated inhibition of outward and inward currents. B, average inward (•) and outward (○) currents at −80 and +80 mV, respectively (n = 3), recorded under experimental conditions that suppress MagNuM and favour I_CRAC activation. I_CRAC was activated passively by inclusion of 10 mm BAPTA in the pipette solution and MagNuM was suppressed by 6 mm Mg.ATP ([MgCl₂]_i = 1 mm, free [Mg²⁺]_i = 1.1 mm). After full activation of I_CRAC, 10 μm Gd³⁺ was applied extracellularly for the time indicated by the bar. C, average inward (•) and outward (○) I_CRAC and MagNuM at −80 and +80 mV, respectively (n = 3). I_CRAC and MagNuM were activated passively by omission of ATP and MgCl₂ and inclusion of 10 mm BAPTA in the pipette solution. After full activation of both I_CRAC and MagNuM, 10 μm Gd³⁺ was applied extracellularly for the time indicated by the bar. Note the complete inhibition of inward current and the lack of effect on outward current.