Separation and characterization of currents through store-operated CRAC channels and Mg²⁺-inhibited cation (MIC) channels

Although store-operated calcium release-activated Ca²⁺ (CRAC) channels are highly Ca²⁺-selective under physiological ionic conditions, removal of extracellular divalent cations makes them freely permeable to monovalent cations. Several past studies have concluded that under these conditions CRAC channels conduct Na⁺ and Cs⁺ with a unitary conductance of ∼40 pS, and that intracellular Mg²⁺ modulates their activity and selectivity. These results have important implications for understanding ion permeation through CRAC channels and for screening potential CRAC channel genes. We find that the observed 40-pS channels are not CRAC channels, but are instead Mg²⁺-inhibited cation (MIC) channels that open as Mg²⁺ is washed out of the cytosol. MIC channels differ from CRAC channels in several critical respects. Store depletion does not activate MIC channels, nor does store refilling deactivate them. Unlike CRAC channels, MIC channels are not blocked by SKF 96365, are not potentiated by low doses of 2-APB, and are less sensitive to block by high doses of the drug. By applying 8-10 mM intracellular Mg²⁺ to inhibit MIC channels, we examined monovalent permeation through CRAC channels in isolation. A rapid switch from 20 mM Ca²⁺ to divalent-free extracellular solution evokes Na⁺ current through open CRAC channels (Na⁺-I_CRAC) that is initially eightfold larger than the preceding Ca²⁺ current and declines by ∼80% over 20 s. Unlike MIC channels, CRAC channels are largely impermeable to Cs⁺ (P_Cs/P_Na = 0.13 vs. 1.2 for MIC). Neither the decline in Na⁺-I_CRAC nor its low Cs⁺ permeability are affected by intracellular Mg²⁺ (90 μM to 10 mM). Single openings of monovalent CRAC channels were not detectable in whole-cell recordings, but a unitary conductance of 0.2 pS was estimated from noise analysis. This new information about the selectivity, conductance, and regulation of CRAC channels forces a revision of the biophysical fingerprint of CRAC channels, and reveals intriguing similarities and differences in permeation mechanisms of voltage-gated and store-operated Ca²⁺ channels.