Regulation of CRAC channels activity is mediated by functional and structural microdomains in Jurkat T cells

Gema B. Montalvo, Antonio R. Artalejo, Juan A. Gilabert

Department of Toxicology and Pharmacology, Facultad de Veterinaria, Universidad Complutense, Madrid, Spain

A sustained Ca2+ entry through CRAC channels, a class of store-operated Ca2+ channels, is the initial signal in T cells to become activated after antigen recognition. As occurs with voltage-dependent Ca2+ channels, local Ca2+ microdomains in the vicinity of the channel act as negative feedback regulators of Ca2+ entry by promoting the inactivation of Ca2+-release activated Ca2+ current (ICRAC).

The aim of this work has been to study the ability of different cellular components to exert a local modulation on CRAC channels. In particular, to study the role of subplasmalemal mitochondria to control the extent of the local Ca2+ concentration around the channel (functional microdomains) and the role of plasma membrane composition (structural microdomains) on CRAC channels activity.

ICRAC was measured in Jurkat T cells by using the whole cell configuration of the patch-clamp technique, which permits the control of the intracellular solutions. The current was activated by depletion of InsP3- sensitive intracellular Ca2+ stores using thapsigargin (2 μM), a blocker of the SERCA pump; a supramaximal concentration of InsP3 (30 μM) and high Ca2+ buffering capacity (10 mM) to study the influence of local Ca2+ microdomains. In some cells, a cocktail of respiratory substrates (2 mM piruvate, 2 mM malate and 1 mM NaH2PO4) was used to stimulate mitochondrial function.

Previous results showed that kinetic properties of two exogenous Ca2+ chelators (EGTA, a slow Ca2+ buffer or BAPTA, a fast Ca2+ buffer), with similar affinity for Ca2+, determined the extent of Ca2+ microdomains and hence, the rate of inactivation of CRAC channels without to affect their activation. Thus, internal dialysis of cells with either EGTA, BAPTA or EGTA plus “mitochondrial cocktail” produced changes in the number of cells displaying CRAC inactivation (92, 47 and 37% respectively) and the extent of this inactivation (62.45±6.75, 36.85±6.12, and 30.23±6.14%, respectively; P<0.05 using Student&apos;s t test) with no alteration in inactivation kinetics. Thus, in the presence of high buffer capacity, energized mitochondria are able to reduce the slow Ca2+-dependent inactivation of ICRAC by increasing subplasmalemmal Ca2+ buffering capacity mainly through a localized ATP production, a dominant mechanism over direct mitochondrial buffering of Ca2+ through the uniporter (for details, see Montalvo et al., 2006). On the other hand, the role of regulatory proteins as PKC on slow inactivation was confirmed using an activator (PMA) or inhibitors added to the extracellular solution once the current reached its maximum. Thus, PKC activation with 100 nM PMA increased inactivation (69.99±6.21% vs 44.19±9.34%; P<0.05). ICRAC was also studied in cells whose membrane cholesterol composition was altered using different β-cyclodextrins (βCD). Cholesterol enrichment using 100 μM of hydroxypropyl-βCD, increased CRAC inactivation compared to control cells (42.93±5.85% vs 16.93±6.56%; P<0.05) without affecting activation parameters. These results support the idea of a functional relationship between CRAC channels and a diffusible factor (ATP) from peripheral mitochondria. In addition, the alteration of structural cholesterol mediated-microdomains (or lipid rafts) on the plasma membrane could determine the localization of some other elements involved in ICRAC regulation, indicating their complexity to keep sustained high levels of Ca2+ which are crucial to start a T cell long-lasting activation.

Montalvo GB et al. (2006) ATP from subplasmalemmal mitochondria controls Ca2+-dependent inactivation of CRAC channels. J Biol Chem 281: 35616-35623.