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 Ca2+ release‐activated Ca2+ current (ICRAC) 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 ICRAC. Differential suppression of ICRAC was achieved by buffering free [Ca2+]i to 90 nm and selective inhibition of MagNuM was accomplished by intracellular solutions containing 6 mm Mg.ATP, 1.2 mm free [Mg2+]i or 100 μm GTP‐γ‐S, allowing investigations on these currents in relative isolation. Removal of extracellular Ca2+ and Mg2+ caused both currents to be carried significantly by monovalent ions. In the absence or presence of free [Mg2+]i, ICRAC 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 ICRAC, these experimental conditions would have favoured the contribution of MagNuM to monovalent conductance and call for caution in interpreting results where both ICRAC and MagNuM are activated.

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

“…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.…”

“…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).…”

“…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). Monovalent currents that are unmasked by removal of divalent cations from the extracellular solution and are possibly due to TRPM7 have also been observed previously in myocytes (Mubagwa et al 1997), aortic smooth muscle cells (Zakharov et al 1999), and hippocampal neurons (Xiong et al 1997).…”

Amplification of CRAC current by STIM1 and CRACM1 (Orai1)

“…Whether or not the current exhibited the pharmacological profile of I CRAC , which is known to be enhanced by low concentrations of 2-aminoethoxydiphenylborate (2-APB) and inhibited at higher doses 13,14 , was also assessed. Treatment with 50 µM 2-APB, after an initial increase as 2-APB concentration built up, completely blocked the current.…”