Protein histidine phosphatase 1 negatively regulates CD4 T cells by inhibiting the K ⁺ channel KCa3.1

Abstract

The calcium activated K ؉ channel KCa3.1 plays an important role in T lymphocyte Ca 2؉ signaling by helping to maintain a negative membrane potential, which provides an electrochemical gradient to drive Ca 2؉ influx. We previously showed that nucleoside diphosphate kinase beta (NDPK-B), a mammalian histidine kinase, is required for KCa3.1 channel activation in human CD4 T lymphocytes. We now show that the mammalian protein histidine phosphatase (PHPT-1) directly binds and inhibits KCa3.1 by dephosphorylating histidine 358 on KCa3.1. Overexpression of wild-type, but not a phosphatase dead, PHPT-1 inhibited KCa3.1 channel activity. Decreased expression of PHPT-1 by siRNA in human CD4 T cells resulted in an increase in KCa3.1 channel activity and increased Ca 2؉ influx and proliferation after T cell receptor (TCR) activation, indicating that endogenous PHPT-1 functions to negatively regulate CD4 T cells. Our findings provide a previously unrecognized example of a mammalian histidine phosphatase negatively regulating TCR signaling and are one of the few examples of histidine phosphorylation/dephosphorylation influencing a biological process in mammals.nucleoside diphosphate kinase ͉ NM23-h2 ͉ PHPT-1 ͉ CRAC channel ͉ histidine kinase

Ion Channels in Innate and Adaptive Immunity

“…In the case of K Ca 3.1, TCR activation recruits and activates PI3K-C2β to the immunological synapse, increasing PI3P concentrations in the PM, thereby enabling the histidine kinase nucleoside diphosphate kinase B (NDPK-B) to activate K Ca 3.1 by phosphorylating histidine H358 in the C terminus of the channel (16, 18, 19) (Figure 5). Consistent with these findings, the PI3P phosphatase myotubularin–related protein 6 (MTMR6) and the histidine phosphatase phosphohistidine phosphatase-1 (PHPT-1) inhibit K Ca 3.1 function, TCR-stimulated Ca 2+ influx, and T cell proliferation by dephosphorylating PI3P and K Ca 3.1, respectively (20, 21). Thus, regulation of K Ca 3.1 is currently the best example of reversible histidine phosphorylation in mammals.…”

AMPKα1-Sensitivity of Orai1 and Ca²⁺ Entry in T - Lymphocytes

“…[Ca 2+ ] i increase upon T-cell receptor activation was determined in activated T-cell blasts as described earlier [60,61,62]. T lymphocytes were incubated with anti-CD3 (1 μl/ml; 145-2C11, R&D Systems) for 30 min on ice and subsequently loaded with Fura-2AM (2 µM, Molecular Probes, Goettingen, Germany) for 15 min at 37°C.…”

“…24h after cells were placed in the 96-well plate, 20 µl of the MTS/PMS solution was added directly to each well and incubated for 4h. The absorbance was measured at 490 nm with a reference wavelength at 690 nm using an ELISA plate reader (PowerWave XS2, BioTek, VT, USA) [60,61,62]. …”

Role of the potassium channel KCa3.1 in diabetic nephropathy

“…At the protein level, K Ca 3.1 can be directly activated by the binding of intracellular Ca 2 + to calmodulin, a Ca 2 + -binding protein that is constitutively associated with the C-terminus of the channel [11][12][13][14]. In addition, K Ca 3.1 activity is increased by PKA (protein kinase A) [15] and NDPK-B (nucleoside diphosphate kinase-B) [16] and inhibited by PKC (protein kinase C) [17], arachidonic acid [18], MTMR6 (myotubularin-related protein 6) [16], the histidine phosphatase PHPT1 (phosphohistidine phosphatase 1) [19] and AMPK (AMP-activated protein kinase) [20]. K Ca 3.1 was first described by Gardos [21] in erythrocytes in 1958 and has been demonstrated to be a target for sickle cell anaemia by preventing erythrocyte dehydration and regulating red blood cell volume [22,23].…”