ATP from Subplasmalemmal Mitochondria Controls Ca2+-dependent Inactivation of CRAC Channels

Gema B Montalvo,Antonio R Artalejo,Juan A Gilabert

doi:10.1074/jbc.m603518200

Gema B Montalvo, Antonio R Artalejo + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m603518200

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Abstract

A sustained Ca2+ entry is the primary signal for T lymphocyte activation after antigen recognition. This Ca2+ entry mainly occurs through store-operated Ca2+ channels responsible for a highly selective Ca2+ current known as I(CRAC). Ca2+ ions act as negative feedback regulators of I(CRAC), promoting its inactivation. Mitochondria, which act as intracellular Ca2+ buffers, have been proposed to control all stages of CRAC current and, hence, intracellular Ca2+ signaling in several types of non-excitable cells. Using the whole-cell configuration of the patch clamp technique, which allows control of the intracellular environment, we report here that respiring mitochondria located close to CRAC channels can regulate slow Ca2+-dependent inactivation of I(CRAC) by increasing the Ca2+-buffering capacity beneath the plasma membrane, mainly through the release of ATP.

Highlights

Ca2ϩ ions themselves modulate the time course of ICRAC by several mechanisms: a fast inactivation process occurring in milliseconds [4] and a slower inactivation process operating in the range of seconds, which comprises both a store-dependent component due to the refilling of stores and a Ca2ϩ-dependent but store-independent component [5]
When store refilling is prevented by using inhibitors of the sarco/endoplasmic reticulum Ca2ϩ-ATPase (SERCA) pump [12] or mitochondria are metabolically potentiated with a mixture or cocktail containing respiratory substrates, ICRAC can be measured in cells dialyzed with low concentration of Ca2ϩ buffers [13]
When using the whole-cell configuration of the patch clamp technique and high concentrations of an exogenous Ca2ϩ buffer, InsP3, and thapsigargin in the intracellular solution, it is expected that after depletion of InsP3-sensitive stores the only regions experiencing abrupt changes in [Ca2ϩ]c are those around the mouth of CRAC channels

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—Human leukemic Jurkat T cells were cultured in RPMI 1640 culture medium supplemented with 2 mM L-glutamine, 10% fetal bovine serum, 10 mM HEPES, and penicillinstreptomycin (100 units/ml and 0.1 mg/ml, respectively) in a humidified atmosphere of 5% CO2 at 37 °C. Averaged current responses to voltage ramps applied every 2 s and measured at Ϫ80 mV in cells dialyzed with EGTA (n ϭ 12), BAPTA (n ϭ 17), or EGTA ϩ mitochondrial mixture (EGTAϩCktl) (n ϭ 16). For JC-1 labeling, Jurkat T cells (5 ϫ 105 cells/ml) were washed in fresh culture medium before being resuspended and incubated in a solution containing mitochondrial mixture in the presence or absence of oligomycin (5 ␮g/ml) for 25 min at 37 °C. The statistical differences between means were assessed by unpaired or paired StudentЈs t tests using GraphPad Prism௡ v. 4.00 software. ns, not significant; *, p Յ0.05; **, p Յ0.01; ***, p Յ0.001

RESULTS

ATP Released from Mitochondria

RR is a widely used inhibitor of the uniporter although it

DISCUSSION