Novel constitutively active non-store-operated Ca2+ current in T lymphocytes

Abstract

The Ca2+-release-activated Ca2+ (CRAC) channels is the only known mechanism mediating Ca2+ entry in T cells. However, using Mn2+ quench of Fura-2 fluorescence we observed a constitutive divalent cation influx in the absence of stimulated store-operated Ca2+ entry in Jurkat T lymphocytes. Suppression of CRAC channels activity either with blocking concentration of La3+ or by expression of dominant-negative Orai1 mutant did not affect the rate of constitutive Mn2+ quench. These data suggest the existence of an additional non-store-operated mechanism mediating Ca2+ entry in T lymphocytes. Consistently, a constitutively active current was recorded in metabolically intact T cells using perforated-patch technique. Whole cell and perforated patch experiments revealed that in the presence of extracellular Ca2+ both constitutively active and CRAC currents displayed inwardly rectifying current-voltage relationship, positive (> 50 mV) reversal potential, and were enhanced by increased concentrations of extracellular Ca2+. However, when the divalent cations were removed from the extracellular solution, the monovalent CRAC current displayed fast time-dependent inactivation, whereas the monovalent constitutively active current exhibited time-dependent activation and lack of inactivation. Equimolar substitution of Na+ with Cs+ in Ca2+-free solution reduced the amplitudes of monovalent CRAC current and constitutively-active current by > 90 % and < 40 % respectively. Taken together, these data indicate that the CRAC and constitutively active currents are carried via different types of Ca2+-selective channels. We speculate that in T lymphocytes the constitutively active Ca2+ entry channels may supply Ca2+ for maintaining resting cytosolic Ca2+ levels and/or for store refilling at unstimulated conditions. Supported by AHA Grant-in-Aid 0755086Y to A.F.F