Distinct Properties of CRAC and MIC Channels in RBL Cells

Abstract

In rat basophilic leukemia (RBL) cells and Jurkat T cells, Ca2+ release–activated Ca2+ (CRAC) channels open in response to passive Ca2+ store depletion. Inwardly rectifying CRAC channels admit monovalent cations when external divalent ions are removed. Removal of internal Mg2+ exposes an outwardly rectifying current (Mg2+-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 Mg2+, 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 Mg2+. Removal of internal Mg2+ induced MIC current despite widely varying Ca2+ and EGTA levels, suggesting that Ca2+-store depletion is not involved in activation of MIC channels. Increasing internal Mg2+ from submicromolar to millimolar levels decreased MIC currents without affecting rectification but did not alter CRAC current rectification or amplitudes. External Mg2+ 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 Mg2+ 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.

The Mg2+ and Mg2+-Nucleotide-Regulated Channel-Kinase TRPM7

“…The authors found that 1 mM of added free Mg 2+ reduced the currents of the T1428I mutant by approximately 50% compared to Mg 2+ -free solutions. This value on its own would not suggest an overly sensitive channel, since it is in fact very similar to the degree of Mg 2+ -induced inhibition observed in numerous studies on heterologous or native TRPM7 (Nadler et al 2001;Kozak et al 2002;Prakriya and Lewis 2002;Schmitz et al 2003;Demeuse et al 2006). However, in the context of that study, the same concentration of Mg 2+ produced a surprisingly small inhibition of wildtype TRPM7 (less than 20%), which is at variance with values obtained by others.…”

“…Under similar experimental conditions, Nadler et al (2001) arrived at a single-channel conductance of approximately 40 pS at +60 mV, and this is in good agreement with the single-channel conductance of approximately 40 pS obtained by Kerschbaum et al (1999) under symmetric divalent-free conditions at negative membrane voltages (Kerschbaum and Cahalan 1999). The latter study attributed these 40-pS single channels to store-operated calcium releaseactivated calcium (CRAC) channels; however, in retrospect, these channels have been identified as TRPM7 (Nadler et al 2001;Hermosura et al 2002;Kozak et al 2002;Prakriya and Lewis 2002). The asymmetrical permeation properties of TRPM7, with exclusive divalent ion influx at negative voltages and exclusive efflux of monovalent cations at high positive voltages, as well as the pronounced block of monovalent cation permeation by divalent cations, renders classical analyses of selectivity based on constant field equations inaccurate, since these rely on the assumption that the permeating ion species move through the channel independently of each other.…”

“…Both solutions reportedly caused a very similar inhibition of TRPM7 by approximate 75%. The strong suppression of TRPM7 by the reference solution containing 270 μM certainly is surprising, as it would suggest an IC 50 of free Mg 2+ that is well below the typical IC 50 values of approximately 700 μM observed by several laboratories (Nadler et al 2001;Kozak et al 2002;Prakriya and Lewis 2002;Schmitz et al 2003;Demeuse et al 2006). A recent study by Demeuse et al (2006) has reevaluated the experimental conditions employed by Kozak and Cahalan and confirmed that the MgATP-containing solution indeed causes approximately 80% inhibition of TRPM7 currents (Demeuse et al 2006).…”

“…Endogenous TRPM7 currents in various cell types are small and rarely exceed 20 pA/pF ( Fig. 1c, d; Nadler et al 2001;Hermosura et al 2002;Kozak et al 2002;Prakriya and Lewis 2002).…”

“…Nadler et al (2001) and Hermosura et al (2002) characterized native TRPM7-like currents in Jurkat T, RBL-2H3, and HEK-293 cells and named these endogenous currents MagNuM (for magnesium-nucleotide-regulated metal ion currents), because they were inhibited by high concentrations of MgATP and transport divalent metal ions. Unfortunately, two later publications (Kozak et al 2002;Prakriya and Lewis 2002) did not follow the original nomenclature and introduced the new term MIC (for Mg 2+ -inhibited cation current), which is based on the observation that Mg 2+ also inhibits TRPM7 as described by the same report that originally designated the current as MagNuM (Nadler et al 2001). Because MagNuM activates under the same experimental conditions typically used to study the store-operated Ca 2+ current I CRAC (Hermosura et al 2002;Kozak et al 2002;Prakriya and Lewis 2002), and both I CRAC and MagNuM will carry large monovalent cation currents when using divalent-free extracellular solutions (Hermosura et al 2002), endogenous TRPM7 channels had in fact been recorded before Cahalan 1998, 1999;Fomina et al 2000), but were erroneously interpreted to represent CRAC channels (Kerschbaum and Cahalan 1999).…”