MIC channels are inhibited by internal divalent cations but not ATP

Abstract

TRPM7 channels are nonselective cation channels that possess a functional alpha-kinase domain. It has been proposed that heterologously expressed TRPM7 channels are activated (Runnels et al., 2001) or inhibited (Nadler et al., 2001) by dialyzing the cell with millimolar levels of ATP. The endogenous correlate of TRPM7 has been identified in T-lymphocytes and RBL (rat basophilic leukemia) cells and named MagNuM (for Mg(2+)-nucleotide-inhibited metal) or MIC (for Mg(2+)-inhibited cation). Here, we report that internal Mg(2+) rather than MgATP inhibits this current. Cytoplasmic MgATP, supplied by dialysis at millimolar concentrations, effectively inhibits only when a weak Mg(2+) chelator is present in the pipette solution. Thus, MgATP acts as a source of Mg(2+) rather than a source of ATP. Using an externally accessible site within the pore of the MIC channel itself as a bioassay, we show that equimolar MgCl(2) and MgATP solutions contain similar amounts of free Mg(2+), explaining the fact that numeric values of Mg(2+) and MgATP concentrations necessary for complete inhibition are the same. Furthermore, we demonstrate that Mg(2+) is not unique in its inhibitory action, as Ba(2+), Sr(2+), Zn(2+), and Mn(2+) can substitute for Mg(2+), causing complete inhibition. We conclude that MIC current inhibition occurs simply by divalent cations.

FIGURE 1

Inhibition of MIC current by internal MgATP is dependent on the type and amount of chelator. (A) The I/V relations of MIC current activated by dialysis with internal solutions containing 1 mM Mg²⁺ (10 mM EDTA), 0.5 mM Mg²⁺ (12 mM EGTA), and 5 mM MgATP (3 mM EGTA/2.5 mM HEDTA). The I/V shape did not vary with the composition of the internal solution. Traces are leak subtracted. (B) Summary of MIC current amplitudes. RBL cells were dialyzed with Mg²⁺- and MgATP-containing solutions; maximally activated MIC current amplitudes were measured at +80 mV. Maximal MIC current amplitudes were obtained by strongly chelating free Mg²⁺ with EDTA to an estimated 230 nM. An amount of 5 mM MgATP in the presence of 12 EGTA markedly inhibited the current, whereas the same amount of MgATP did not significantly inhibit when the internal chelator was 3 mM EGTA and 2.5 mM HEDTA. For comparison, ∼5.5 mM free [Mg²⁺] and no added MgATP are shown.

FIGURE 2

Monovalent MIC current as a bioassay for determining free Mg²⁺ concentrations. Solutions containing various amounts of MgCl₂ and MgATP were applied externally and the I/V relations compared. (A) MIC monovalent current with external Cs⁺-HEDTA solution was blocked to the same extent by 1 mM MgCl₂ (8 mM HEDTA) or by 1 mM MgATP (8 mM HEDTA). The calculated free [Mg²⁺] concentrations were 6.7 μM and 8.2 μM, respectively. (B) 2 mM MgCl₂ and MgATP (in the presence of 8 mM HEDTA) blocked the monovalent MIC current to the same extent. The calculated free [Mg²⁺] concentrations were 15.8 μM and 17.5 μM, respectively. (C) The internal solutions from the experiment described in Fig. 1 were applied externally to compare the degree of block: 0.5 mM MgCl₂ and 12 mM EGTA (calculated free [Mg²⁺] = 270 μM); compared with 5 mM MgATP and 3 mM EGTA/2.5 mM HEDTA (calculated free [Mg²⁺] = 270 μM). The current was allowed to run down completely to show the extent of block by 270 μM Mg²⁺.

FIGURE 3

Internal Sr²⁺, Ba²⁺, and Mn²⁺ can substitute for Mg²⁺ in inhibiting the MIC current in RBL cells. The maximal normalized current amplitudes (mean ± SE) during dialysis with ∼4 mM Sr²⁺ (n = 6 cells), Ba²⁺ (n = 5) and Mn²⁺ (n = 3) (1 mM EGTA), obtained as in Fig. 1 B, are compared to current inhibition by 5.5 mM Mg²⁺ (n = 3).

FIGURE 4

Millimolar concentrations of internal Ba²⁺ inhibit preactivated MIC current in rat PAS cells. (A) The MIC current I/V obtained 2 and 79 s after break-in with a pipette solution containing 5 mM BaCl₂ and 1 mM EGTA. The inhibition is complete at 79 s. (B) The MIC current I/V obtained 7 and 79 s after break-in with a pipette solution containing no internal divalents (12 mM EGTA). The current was substantially increased after 79 s.