TRPV5/V6 Channels Mediate Ca(2+) Influx in Jurkat T Cells Under the Control of Extracellular pH.

Regulation of cytoplasmic free calcium concentration [Ca(2+)]i is a key factor for the maintenance of cellular homeostasis in different cell types, including lymphocytes. During T lymphocyte activation as well as production of cytokines, sustained Ca(2+) influx is essential, however, it remains unclear how this influx is regulated. Previously, we reported the expression and functional activity of calcium channels TRPV5 and TRPV6 (transient receptor potential vanilloid type 5 and 6) in human leukemia Jurkat T cells. In this study, using single channel recordings, we found that activity of calcium channels TRPV5/V6 in Jurkat T cells is subject to strong control of external stimuli such as a low- or high-pH stressor. We showed that extracellular acidic pH reduces the activity of TRPV5/V6 channels, whereas alkaline pH increases the activity of TRPV5/V6 channels in Jurkat T cells. Using calcium imaging, we found that Ca(2+) influx in Jurkat T cells displayed sensitivity to extracellular pH, similar to that shown for the calcium channels TRPV5/V6. Double immunostaining of Jurkat T cells revealed that TRPV5 and TRPV6 channels colocalize with clathrin and the early endocytosis marker, EEA1. Moreover, we demonstrated that a specific inhibitor of clathrin-dependent endocytosis, dynasore, blocked TRPV5/V6 activity, and Ca(2+) influx into Jurkat T cells. Overall, our findings indicate that strong environmental cues may affect the intracellular calcium level in Jurkat T cells by influencing the traffic of TRPV5/V6 channels in lymphocytes.

Expression of transient receptor potential vanilloid channels TRPV5 and TRPV6 in human blood lymphocytes and Jurkat leukemia T cells.

Regulation of Ca(2+) entry is a key process for lymphocyte activation, cytokine synthesis and proliferation. Several members of the transient receptor potential (TRP) channel family can contribute to changes in [Ca(2+)](in); however, the properties and expression levels of these channels in human lymphocytes continue to be elusive. Here, we established and compared the expression of the most Ca(2+)-selective members of the TRPs, Ca(2+) channels transient receptor potential vanilloid 5 and 6 (TRPV5 and TRPV6), in human blood lymphocytes (HBLs) and leukemia Jurkat T cells. We found that TRPV6 and TRPV5 mRNAs are expressed in both Jurkat cells and quiescent HBLs; however, the levels of mRNAs were significantly higher in malignant cells than in quiescent lymphocytes. Western blot analysis showed TRPV5/V6 proteins in Jurkat T cells and TRPV5 protein in quiescent HBLs. However, the expression of TRPV6 protein was switched off in quiescent HBLs and turned on after mitogen stimulation of the cells with phytohemagglutinin. Inwardly directed monovalent currents that displayed characteristics of TRPV5/V6 currents were recorded in both Jurkat cells and normal HBLs. In outside-out patch-clamp studies, currents were reduced by ruthenium red, a nonspecific inhibitor of TRPV5/V6 channels. In addition, ruthenium red downregulated cell-cycle progression in both activated HBLs and Jurkat cells. Thus, we identified TRPV5 and TRPV6 calcium channels, which can be considered new candidates for Ca(2+) entry into human lymphocytes. The correlation between expression of TRPV6 channels and the proliferative status of lymphocytes suggests that TRPV6 may be involved in the physiological and/or pathological proliferation of lymphocytes.