Receptor-mediated regulation of the TRPM7 channel through its endogenous protein kinase domain

Abstract

TRPM7 is a ubiquitously expressed and constitutively active divalent cation-selective ion channel, whose basal activity is regulated by intracellular levels of Mg(2+) and Mg.ATP. We have investigated receptor-mediated mechanisms that may actively regulate TRPM7 activity. We here report that TRPM7 currents are suppressed by intracellular GTPgammaS, suggesting the involvement of heterotrimeric G proteins. TRPM7 currents are also inhibited by stimulating endogenous muscarinic receptors, which is mediated by G(i) because the inhibitory effect is blunted by pertussis toxin. Conversely, stimulation of endogenous G(s)-coupled beta-adrenergic receptors potentiates TRPM7 currents, whereas G(q)-coupled thrombin receptors have little effect. Consistent with the involvement of G(s)/G(i) in controlling adenylyl cyclase activity, elevations of intracellular cAMP levels enhance TRPM7 activity and prevent receptor-mediated modulation of TRPM7 activity by muscarinic and adrenergic agonists. This cAMP-dependent effect requires the functional integrity of both protein kinase A (PKA) and the endogenous kinase domain of TRPM7 because cAMP-mediated effects are abolished when treating cells with the PKA inhibitors H89 or KT5720 as well as in cells expressing phosphotransferase-deficient TRPM7 constructs. These mutant channels are also much less susceptible to GTPgammaS-mediated inhibition, suggesting that the main regulatory effect occurs through G(i)- and G(s)-mediated changes in cAMP. Taken together, our results demonstrate that TRPM7 activity is up- and down-regulated through its endogenous kinase in a cAMP- and PKA-dependent manner.

Fig. 1.

TRPM7 is modulated by G protein-coupled receptors. Whole-cell currents were recorded in HEK-293 cells induced to overexpress murine TRPM7 (A–E), except for F, which depicts uninduced HEK-293 cells. (A) Average inward and outward currents carried by TRPM7 at –80 and +80 mV, respectively (n = 5; note the different y axis scaling). Cells were perfused with standard internal solution (i.e., with no added Mg·ATP). (B) Representative current-voltage (I/V) relationship for fully activated TRPM7, derived from a high-resolution current record in response to a voltage ramp of 50 ms duration that ranged from –100 to +100 mV. (C) Cells were perfused with standard internal solution containing various concentrations of GTPγS as indicated in the legend (n = 5–11). (D) Cells were perfused with standard internal solution containing 0.3 mM GTP (n = 6). The bar indicates the time of application of the CCh-containing standard external solution (100 μM). (E) Cells were perfused with standard internal solution containing 0.5 mM GDPβS and stimulated by CCh as in D (n = 5). (F) Cells were perfused with standard internal solution as in D and stimulated by CCh (•, n = 6) or not (○, n = 5). MagNuM was inhibited by CCh in 6 of 13 cells (46% of cells).

Fig. 2.

Receptor-mediated calcium signals do not correlate with TRPM7 regulation. (A) Average calcium signals evoked by CCh (100 μM, n = 10) and thrombin (20 units/ml, n = 10) in intact, uninduced HEK-293 cells. Cells that did not respond with a change in [Ca²⁺]i were omitted. Preincubation of cells with the PLC inhibitor U73122 (10 μM, n = 10) abolished CCh responses. (B) Current amplitudes evoked by the standard ramp protocol recorded at +80 mV in HEK-293 cells overexpressing TRPM7. The bar indicates the time of application of either thrombin (20 units/ml) or CCh (100 μM). Thrombin has only mild inhibitory effects on TRPM7 currents (•, n = 9) as compared with CCh (○, same data as in Fig. 1C). (C) Current amplitudes evoked by the standard ramp protocol recorded at +80 mV in uninduced HEK-293 cells as in Fig. 1F. The bar indicates the time of application of thrombin (20 units/ml), with no significant effect on MagNuM (n = 10). (D) Current amplitudes evoked by the standard ramp protocol recorded at +80 mV in HEK-293 cells overexpressing TRPM7 and preincubated with U73122 (10 μM, n = 11). The bar indicates the time of application of CCh (100 μM). Note that the PLC inhibitor did not suppress the CCh-mediated inhibition of TRPM7. (E) Average calcium signals evoked by CCh (100 μM, n = 10) and thrombin (20 units/ml, n = 10) in intact, tetracycline-induced HEK-293 cells overexpressing TRPM7. Note the complete absence of calcium release with either agonist. (F) Average calcium signals evoked by intracellular InsP₃ (20 μM, n = 3) in intact, tetracycline-induced HEK-293 cells overexpressing TRPM7. Note that InsP₃-induced calcium release and subsequent store-operated Ca²⁺ influx is not compromised.

Fig. 3.

TRPM7 is inhibited by G_i and facilitated by cAMP. Data are derived from whole-cell outward currents recorded at +80 mV in HEK-293 cells overexpressing TRPM7. (A) Cells were pretreated with 1 μg/ml PTX for 4–7h, perfused with standard internal solution containing 0.3 mM GTP, and stimulated by 100 μM CCh (•, n = 7). The currents measured in PTX-untreated cells are shown as control (○, same data as in Fig. 1D). (B) Average whole-cell currents in cells perfused with 3 mM Mg·ATP and stimulated with 100 μM CCh (line plot, n = 5) as well as 300 μM isoproterenol in the presence (○, n = 5) or absence (•, n = 5) of 10 μM H89 in the internal solution. Note that H89 completely abolishes isoproterenol-mediated facilitation of TRPM7. The H89 data have been shifted by –8pA/pF for display purposes. (C) Average whole-cell currents in cells perfused with intracellular solutions containing 3 mM Mg·ATP alone (○, n = 8) or additionally 100 μM cAMP (•, n = 8). Note the strong facilitation of TRPM7 currents by cAMP. (D) Data points correspond to averaged and normalized current amplitudes measured at +80 mV after 300 s of whole-cell recording, plotted as a function of intracellular Mg·ATP concentrations (n = 7–15). Current amplitudes evoked in the absence (○) and presence (•) of 100 μM intra-pipette cAMP. The data points at 3 and 4 mM Mg·ATP are statistically significant with P < 0.03 as determined by Student's paired t test. (E) Average whole-cell currents in cells perfused with intracellular solutions containing 3 mM Mg·ATP and 100 μM cAMP. Extracellular application of either 100 μM CCh (•, n = 11) or 300 μM isoproterenol (□, n = 8) now fail to modulate TRPM7 currents. (F) Typical examples of fluorescence quench of fura-2 fluorescence induced by Mn²⁺ in HEK-293 cells overexpressing TRPM7 in the absence (gray trace) or additional presence of 300 μM isoproterenol. The rate of Mn²⁺-induced quench of fura-2 during the time indicated by the first pair of arrows was 32 ± 8%/min (n = 5), and cells that were exposed to isoproterenol had average rates of 76 ± 6%/min (n = 5) during the time indicated by the second set of arrows.

Fig. 4.

Receptor-mediated regulation of TRPM7 is mediated by the kinase domain. Data are from HEK-293 cells overexpressing various constructs of human TRPM7. (A) Normalized average whole-cell currents at +80 mV (current amplitudes at 200 s were set to 1) in cells overexpressing WT hTRPM7 perfused with intracellular solutions containing 3 mM Mg·ATP (plus 0.3 mM GTP in the case of receptor stimulation) and stimulated by 300 μM isoproterenol (○, n = 6) or exposed to extracellular solution containing 5 mM dibutyryl-cAMP (•, n = 5). Note that both stimuli induce facilitation in hTRPM7. (B) Normalized average whole-cell currents at +80 mV (current amplitudes at 200 s were set to 1) in HEK-293 cells overexpressing phosphotransferase-deficient constructs caused by either a single point mutation of hTRPM7 (K1648R) or C-terminal truncation of the kinase domain of hTRPM7 (Δ-kinase) and stimulated by 300 μM isoproterenol. K1648 cells were perfused with intracellular solutions containing 3 mM Mg·ATP and 0.3 mM GTP (○, n = 5). Δ-Kinase cells were perfused with intracellular solutions with reduced free MgCl₂ (calculated free Mg²⁺ of 216 μM) containing 3 mM Na·ATP, 0.3 mM GTP, and free [Ca²⁺]i buffered to 100 nM (•, n = 5). Note that isoproterenol-induced facilitation is abolished in both phosphotransferase-deficient constructs. (C) Currents recorded from K1648R cells with the same experimental protocol as in A and stimulated with 100 μM CCh (•; n = 7) or not (○; n = 4). Note that CCh-induced inhibition normally observed in TRPM7 WT (see Fig. 1D) is almost completely abolished in the K1648R mutant. (D) Same experimental protocol as in Fig. 1 A. K1648R-overexpressing HEK-293 cells were perfused with the standard internal solution containing 300 μM GTPγS(•, n = 5). For comparison, the graph also plots WT human TRPM7 perfused with the same solution (○, n = 6). Note that inhibitory effects of GTPγS are greatly reduced in the K1648R mutant.