Distinct properties of CRAC and MIC channels in RBL cells

Abstract

In rat basophilic leukemia (RBL) cells and Jurkat T cells, Ca(2+) release-activated Ca(2+) (CRAC) channels open in response to passive Ca(2+) store depletion. Inwardly rectifying CRAC channels admit monovalent cations when external divalent ions are removed. Removal of internal Mg(2+) exposes an outwardly rectifying current (Mg(2+)-inhibited cation [MIC]) that also admits monovalent cations when external divalent ions are removed. Here we demonstrate that CRAC and MIC currents are separable by ion selectivity and rectification properties: by kinetics of activation and susceptibility to run-down and by pharmacological sensitivity to external Mg(2+), spermine, and SKF-96365. Importantly, selective run-down of MIC current allowed CRAC and MIC current to be characterized under identical ionic conditions with low internal Mg(2+). Removal of internal Mg(2+) induced MIC current despite widely varying Ca(2+) and EGTA levels, suggesting that Ca(2+)-store depletion is not involved in activation of MIC channels. Increasing internal Mg(2+) from submicromolar to millimolar levels decreased MIC currents without affecting rectification but did not alter CRAC current rectification or amplitudes. External Mg(2+) and Cs(+) carried current through MIC but not CRAC channels. SKF-96365 blocked CRAC current reversibly but inhibited MIC current irreversibly. At micromolar concentrations, both spermine and extracellular Mg(2+) blocked monovalent MIC current reversibly but not monovalent CRAC current. The biophysical characteristics of MIC current match well with cloned and expressed TRPM7 channels. Previous results are reevaluated in terms of separate CRAC and MIC channels.

Figure 1.

Development and run-down of CRAC and MIC currents in the presence and absence of external divalent ions. Internal solution contained 12 mM EGTA and no added Mg²⁺. (A) Outward (top) and inward (bottom) current development in 2 mM external Ca²⁺, followed at ∼400 s by exposure to Cs⁺– then Na⁺–HEDTA. MIC ran down gradually over 2,000 s. (B) Current development time course for inward and outward currents compared. The scaled and inverted inward current measured at −110 mV is shown superimposed with the outward current (80 mV). The inward current clearly precedes the development of the outward current, despite its smaller size. (C) I-V plots of MIC current in 2 mM Ca²⁺ plotted with two different current amplitude scales. Traces 1 and 2 correspond to times indicated in A. Trace 7 was collected 51 min after break-in after reintroduction of 2 mM Ca²⁺. (D) I-V plots of monovalent MIC current in Na⁺– and Cs⁺–HEDTA. Both Cs⁺ and Na⁺ are permeant through the channel. The indistinguishable traces 5 and 6 (taken 54 min after break-in) were obtained in Cs⁺ and Na⁺ solution, respectively, after complete run-down of MIC current.

Figure 2.

MIC current is permeable to external Mg²⁺. Internal solution contained 12 mM EGTA and no added Mg²⁺. External solution contained 2 mM Mg²⁺ and Na⁺ aspartate. (A) MIC-current development and run-down in 2 mM external Mg²⁺ with zero Ca²⁺. (B) I-V relations of MIC current in 2 mM Mg²⁺ obtained at various times after break-in (same cell as in A).

Figure 3.

MIC I-V shape does not depend on internal [Mg²⁺] or dialysis time after break-in. (A) Scaled and superimposed MIC I-V relations from three different cells with 0, 0.5, and 1 mM Mg²⁺ in pipette. Free [Mg²⁺] concentrations were estimated by calculation with Maxchelator: nominally zero, ∼280 μM, and ∼563 μM. External solution contained 2 mM Ca²⁺. (B) MIC current I-V from two different cells with 0 and 2 mM Mg²⁺ in the pipette with 2 mM external Ca²⁺. Note the different current axis scales, with the smaller, noisy current trace (2 mM internal Mg²⁺) corresponding to the pA scale. The internal solutions contained (mM): 12 EDTA + 0 Mg²⁺ (nominally Mg²⁺-free); and 12 EGTA + 2 total (∼1.15 free) Mg²⁺. (C) MIC I-V in the absence of external divalent ions, with 0 and 2 mM Mg²⁺ in the pipette as in B. External solution: Cs⁺–HEDTA to minimize CRAC current contamination (see Fig. 9). (D) Superimposed scaled traces from Fig. 2 B in 2 mM external Mg²⁺ at varying times following break-in, showing that the I-V characteristic of MIC current is time-invariant. Mg²⁺ was used as the permeant ion to minimize possible contamination by CRAC current. Internal solution: 12 mM EGTA, 0 Mg²⁺. (E and F) MIC current recorded with internal NMDG⁺ as the predominant cation. I-V curves in 2 mM Ca²⁺ and Na⁺–HEDTA, respectively. The low-EGTA internal solution contained NMDG⁺ as an impermeant cation substitute. External solutions were 2 mM Ca²⁺ (E) and Na⁺–HEDTA (F).

Figure 3.

MIC I-V shape does not depend on internal [Mg²⁺] or dialysis time after break-in. (A) Scaled and superimposed MIC I-V relations from three different cells with 0, 0.5, and 1 mM Mg²⁺ in pipette. Free [Mg²⁺] concentrations were estimated by calculation with Maxchelator: nominally zero, ∼280 μM, and ∼563 μM. External solution contained 2 mM Ca²⁺. (B) MIC current I-V from two different cells with 0 and 2 mM Mg²⁺ in the pipette with 2 mM external Ca²⁺. Note the different current axis scales, with the smaller, noisy current trace (2 mM internal Mg²⁺) corresponding to the pA scale. The internal solutions contained (mM): 12 EDTA + 0 Mg²⁺ (nominally Mg²⁺-free); and 12 EGTA + 2 total (∼1.15 free) Mg²⁺. (C) MIC I-V in the absence of external divalent ions, with 0 and 2 mM Mg²⁺ in the pipette as in B. External solution: Cs⁺–HEDTA to minimize CRAC current contamination (see Fig. 9). (D) Superimposed scaled traces from Fig. 2 B in 2 mM external Mg²⁺ at varying times following break-in, showing that the I-V characteristic of MIC current is time-invariant. Mg²⁺ was used as the permeant ion to minimize possible contamination by CRAC current. Internal solution: 12 mM EGTA, 0 Mg²⁺. (E and F) MIC current recorded with internal NMDG⁺ as the predominant cation. I-V curves in 2 mM Ca²⁺ and Na⁺–HEDTA, respectively. The low-EGTA internal solution contained NMDG⁺ as an impermeant cation substitute. External solutions were 2 mM Ca²⁺ (E) and Na⁺–HEDTA (F).

Figure 4.

Effects of external and internal Mg²⁺ on MIC current. (A) Time course of internal Mg²⁺ inhibition of preactivated MIC current. The pipette solution contained: 12 mM EGTA, 5 mM total (∼3 mM free) Mg²⁺. Outward current amplitude was measured at 85 mV, and the half-time of inhibition was 85 s. (B) Time course of external Mg²⁺ inhibition of MIC current. The pipette solution contained: 12 mM EGTA, 0 Mg²⁺. MIC current developed first in 2 mM Ca²⁺ external. Monovalent MIC current in Na⁺–HEDTA was blocked reversibly by 28 μM external Mg²⁺ (8 mM HEDTA + 3 mM MgCl₂).

Figure 5.

MIC and the endogenous IRK currents activate and run down in parallel. (A) Time course of development of MIC current measured at 70 mV and IRK current measured at −110 mV. (B) I-V shapes at various times. IRK was preactivated at break-in, whereas MIC was absent. The internal solution contained (mM): K⁺ glutamate, 1 EGTA, 0.5 Ca²⁺, 0 Mg²⁺. The external solution contained (mM) 4.5 KCl, 2 Ca²⁺, 1 Mg²⁺.

Figure 6.

Extracellular spermine blocks the MIC current in divalent-free solution. (A) 20 μM spermine was applied externally after MIC current had developed fully in divalent-free solution (Na⁺–and Cs⁺–HEDTA). Block was strongly voltage-dependent, blocking preferentially the inward current, but showing some relief of block at very negative potentials. Internal solution: 12 mM EGTA, 0 Mg²⁺. Spermine block was completely reversible (not shown). (B) Dose-response relationship for spermine block of Cs⁺ current (to minimize possible CRAC current contamination) at −100 mV. Data from nine cells are fitted with the Hill equation using a K _d value of 2.3 μM and a Hill coefficient of 1.1.

Figure 7.

SKF-96365 irreversibly inhibits MIC current. (A) 20 μM SKF was applied after development of MIC current in 2 mM Ca²⁺. (B) I-V relations in a different cell after break-in (1), after development of MIC current (2), and after SKF inhibition (3). Internal solution contained 2 mM EGTA.

Figure 8.

Divalent and monovalent selectivity of CRAC channels. Internal solution contained 12 mM EGTA, with 6 mM total (∼3.67 free) Mg²⁺ to block MIC current development. (A) Time course of inward and outward currents showing inward CRAC Ca²⁺ and monovalent currents. Recording was started in 2 mM Mg²⁺; the time-dependent activation of Mg²⁺ MIC current was absent. Adding 2 mM Ca²⁺ revealed CRAC current. The inward monovalent current showed partial inactivation in Na⁺ - HEDTA, and was greatly reduced in Cs⁺ - HEDTA. (B) I-V curves in 2 mM external Mg²⁺ and Ca²⁺. (C) I-V curves in Na⁺ - and Cs⁺ - HEDTA.

Figure 9.

CRAC current inward rectification is independent of internal Mg²⁺. All pipette solutions contained 12 mM EGTA. (A) Trace 1 shows current after break-in. Traces 2 and 3 show CRAC current in 2 and 5 mM external Ca²⁺. Internal solution: 5 mM total (∼3 mM free) Mg²⁺. The current is strongly inwardly rectifying and shows no outward current at 80 mV, indicating that 3 mM free Mg²⁺ is sufficient to inhibit MIC current completely. The reversal potential is above 40 mV. Note the difference in the I-V shape compared with Fig. 1 C. (B) CRAC current in 2 mM external Ca²⁺. Internal solution contained 2 mM total (∼1.15 free) Mg²⁺. (C) CRAC current in 5 mM external Ca²⁺ with 1 mM (∼563 μM free) Mg²⁺ in pipette. (D) CRAC current in 5 mM external Ca²⁺ with 150 μM (∼83 μM free) Mg²⁺ in pipette. Traces shown in B–D were obtained after run-down of MIC current.

Figure 10.

Pharmacological properties of monovalent CRAC current. CRAC current was allowed to develop in external Ca²⁺ and Na⁺ current through CRAC channels was recorded in Na⁺–HEDTA. The internal solution contained: 12 EGTA, and 6 (3 mM free) Mg²⁺. (A) Effect of 20 μM SKF-96365 on Na⁺ current through CRAC channels. SKF block was fully reversible. (B) I-V of Na⁺ current through CRAC channels in the presence and absence of 20 μM spermine. (C) I-V of Na⁺ current through CRAC channels in the presence and absence of 28 μM external Mg²⁺, a free Mg²⁺ concentration that effectively blocks MIC current (Fig. 4 B). At the end of the experiment (B and C) external Na⁺–HEDTA was substituted by Cs⁺–HEDTA; the loss of inward current confirms that the monovalent current is through CRAC channels.

Figure 11.

Separation of MIC and CRAC currents using SKF-induced run-down of MIC with low Mg²⁺ inside. (A) Development of MIC and CRAC currents in the same cell. External solution was 2 mM Ca²⁺ switched to 5 mM to increase inward current. SKF-96365 application caused slow reduction of both the outward and inward currents. The outward current ran down completely in presence of SKF. Removal of SKF did not increase outward current but reversed the inhibition of the inward current. The remaining inward current in 5 mM Ca²⁺ after washout of SKF is CRAC current. External solution is switched from Na⁺– to Cs⁺–HEDTA. Internal solution contained 12 EGTA, 0.5 mM Mg²⁺. (B) MIC current isolated by use of 2.5 mM external Mg²⁺. SKF caused irreversible run-down of both inward and outward currents, leaving no residual CRAC current in the absence of external Ca²⁺.

Figure 12.

Change in the monovalent current selectivity after MIC current run-down induced by SKF-96365. Internal solution was 12 mM EGTA, 0.5 mM (280 μM free) Mg²⁺. (A) Time course of CRAC and MIC currents. SKF-96365 (20 μM) is applied in Cs⁺ and the MIC current was allowed to run down completely. After washout of SKF, reintroduction of Na⁺ shows an increased inward current. (B) Same as A with expanded current scale. (C and D) I-V of the combined current before MIC current run-down in 2 mM Ca²⁺ (C, same trace at different scale in D) and Na⁺– and Cs⁺–HEDTA (D). (E) I-V of monovalent CRAC in Na⁺– and Cs⁺–HEDTA after MIC run-down.