Mitochondrial control of calcium-channel gating: a mechanism for sustained signaling and transcriptional activation in T lymphocytes

Abstract

In addition to their well-known functions in cellular energy transduction, mitochondria play an important role in modulating the amplitude and time course of intracellular Ca(2+) signals. In many cells, mitochondria act as Ca(2+) buffers by taking up and releasing Ca(2+), but this simple buffering action by itself often cannot explain the organelle's effects on Ca(2+) signaling dynamics. Here we describe the functional interaction of mitochondria with store-operated Ca(2+) channels in T lymphocytes as a mechanism of mitochondrial Ca(2+) signaling. In Jurkat T cells with functional mitochondria, prolonged depletion of Ca(2+) stores causes sustained activation of the store-operated Ca(2+) current, I(CRAC) (CRAC, Ca(2+) release-activated Ca(2+)). Inhibition of mitochondrial Ca(2+) uptake by compounds that dissipate the intramitochondrial potential unmasks Ca(2+)-dependent inactivation of I(CRAC). Thus, functional mitochondria are required to maintain CRAC-channel activity, most likely by preventing local Ca(2+) accumulation near sites that govern channel inactivation. In cells stimulated through the T-cell antigen receptor, acute blockade of mitochondrial Ca(2+) uptake inhibits the nuclear translocation of the transcription factor NFAT in parallel with CRAC channel activity and [Ca(2+)](i) elevation, indicating a functional link between mitochondrial regulation of I(CRAC) and T-cell activation. These results demonstrate a role for mitochondria in controlling Ca(2+) channel activity and signal transmission from the plasma membrane to the nucleus.

Figure 1

Inhibition of mitochondrial Ca²⁺ uptake evokes CRAC channel inactivation. I_CRAC and [Ca²⁺]_i were measured in parallel using the perforated patch-clamp technique in the absence (A, B) or presence (C, D) of 1 μM CCCP (present throughout the experiments). After irreversible store depletion by TG in 0-Ca²⁺ Ringer's solution (see Materials and Methods), 2 mM Ca²⁺ was added to measure I_CRAC. A voltage step to −120 mV followed by a voltage ramp from −120 mV to + 60 mV was applied every 2 s from the holding potential (−40 mV). Current density at −100 mV is plotted in A and C (Lower), and leak-corrected ramp currents collected at the times indicated by the triangles are shown in B and D. (E) I_CRAC inactivation is plotted as the current measured 100 s after the peak (open triangles in A and C) relative to the peak current amplitude (solid triangles in A and C). 1 μM CCCP or 2 μM antimycin A1 + 1 μM oligomycin increase I_CRAC inactivation significantly relative to control (unpaired Student's t test, P < 0.007). The number of cells for each condition is indicated.

Figure 2

Energized mitochondria prevent Ca²⁺-dependent inactivation of CRAC channels. I_CRAC was measured in whole-cell recordings with 1.2 mM EGTA in the recording pipette. Stimulus protocol was identical to that used in Fig. 1. (A) I_CRAC measured at −100 mV before and after addition of 22 mM Ca²⁺ using the standard whole-cell internal solution (see Materials and Methods). (B) Leak-corrected ramp currents collected at the times indicated by the triangles in A. (C, D) I_CRAC recorded as in A and B with the addition of 2.5 mM malic acid/2.5 mM Na pyruvate/1 mM NaH₂PO₄/5 mM MgATP/0.5 mM Tris⋅GTP to the recording pipette to support mitochondrial function (“energized” conditions). (E, F) I_CRAC recorded as in C and D with addition of 1 μM CCCP/2 μM antimycin A1/1 μM oligomycin to the bath. (G) CRAC channel inactivation under various conditions measured as described in Fig. 1. Inactivation under energized conditions or in the presence of 12 mM internal EGTA (last two bars) was significantly less than under the other conditions shown (unpaired Student's t test, P < 0.007). The number of cells for each condition is indicated.

Figure 3

Mitochondria are necessary for sustained Ca²⁺ signaling and NFAT translocation. [Ca²⁺]_i and EGFP fluorescence were measured in parallel in fura-2-loaded Jurkat cells expressing NFATc1-EGFP. (A) Images of NFATc1-EGFP fluorescence are shown for cells before (Left) or 10 min after stimulation with OKT3 (Right; 1:75 ascites). In unstimulated cells, NFATc1-EGFP is mostly cytosolic, and OKT3 elicits extensive translocation of NFATc1-EGFP to the nucleus. (B, C) [Ca²⁺]_i and EGFP fluorescence measured in the absence (B) or presence (C) of 1 μM CCCP in single cells at room temperature. After depletion of Ca²⁺ stores for 10 min in 0-Ca²⁺ + 1 μM TG, 2 mM Ca²⁺ was readded as indicated. (D) [Ca²⁺]_i and nuclear NFATc1-EGFP intensity in OKT3-stimulated cells at 37°C. A maximally effective concentration of OKT3 (ascites diluted 1:75) was added to the cells as indicated under control conditions (solid trace) or in the presence of 1 μM CCCP (dotted trace). Each trace is an average of three experiments performed on a single day comprising a total of 151 (control) and 146 (+CCCP) cells.