IP(3) receptor subtype-dependent activation of store-operated calcium entry through I(CRAC).

The store-operated, calcium release-activated calcium current I(CRAC) is activated by the depletion of inositol 1,4,5-trisphosphate (IP(3))-sensitive stores. The significantly different dose-response relationships of IP(3)-mediated Ca(2+) release and CRAC channel activation indicate that I(CRAC) is activated by a functionally, and possibly physically, distinct sub-compartment of the endoplasmic reticulum (ER), the so-called CRAC store. Vertebrate genomes contain three IP(3) receptor (IP(3)R) genes and most cells express at least two subtypes, but the functional relevance of various IP(3)R subtypes with respect to store-operated Ca(2+) entry is completely unknown. We here demonstrate in avian B cells (chicken DT40) that IP(3)R type II and type III participate in IP(3)-induced activation of I(CRAC), but IP(3)R type I does not. This suggests that the expression pattern of IP(3)R contributes to the formation of specialized CRAC stores in B cells

TRPM7 Provides an Ion Channel Mechanism for Cellular Entry of Trace Metal Ions

Trace metal ions such as Zn2+, Fe2+, Cu2+, Mn2+, and Co2+ are required cofactors for many essential cellular enzymes, yet little is known about the mechanisms through which they enter into cells. We have shown previously that the widely expressed ion channel TRPM7 (LTRPC7, ChaK1, TRP-PLIK) functions as a Ca2+- and Mg2+-permeable cation channel, whose activity is regulated by intracellular Mg2+ and Mg2+·ATP and have designated native TRPM7-mediated currents as magnesium-nucleotide–regulated metal ion currents (MagNuM). Here we report that heterologously overexpressed TRPM7 in HEK-293 cells conducts a range of essential and toxic divalent metal ions with strong preference for Zn2+ and Ni2+, which both permeate TRPM7 up to four times better than Ca2+. Similarly, native MagNuM currents are also able to support Zn2+ entry. Furthermore, TRPM7 allows other essential metals such as Mn2+ and Co2+ to permeate, and permits significant entry of nonphysiologic or toxic metals such as Cd2+, Ba2+, and Sr2+. Equimolar replacement studies substituting 10 mM Ca2+ with the respective divalent ions reveal a unique permeation profile for TRPM7 with a permeability sequence of Zn2+ ≈ Ni2+ >> Ba2+ > Co2+ > Mg2+ ≥ Mn2+ ≥ Sr2+ ≥ Cd2+ ≥ Ca2+, while trivalent ions such as La3+ and Gd3+ are not measurably permeable. With the exception of Mg2+, which exerts strong negative feedback from the intracellular side of the pore, this sequence is faithfully maintained when isotonic solutions of these divalent cations are used. Fura-2 quenching experiments with Mn2+, Co2+, or Ni2+ suggest that these can be transported by TRPM7 in the presence of physiological levels of Ca2+ and Mg2+, suggesting that TRPM7 represents a novel ion-channel mechanism for cellular metal ion entry into vertebrate cells

Dissociation of the store-operated calcium current ICRAC and the Mg-nucleotide-regulated metal ion current MagNuM

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