Enhancement of calcium signalling dynamics and stability by delayed modulation of the plasma‐membrane calcium‐ATPase in human T cells

In addition to its homeostatic role of maintaining low resting levels of intracellular calcium ([Ca 2+ ] i ), the plasma‐membrane calcium‐ATPase (PMCA) may actively contribute to the generation of complex Ca 2+ signals. We have investigated the role of the PMCA in shaping Ca 2+ signals in Jurkat human leukaemic T cells using single‐cell voltage‐clamp and calcium‐imaging techniques. Crosslinking the T‐cell receptor with the monoclonal antibody OKT3 induces a biphasic elevation in [Ca 2+ ] i consisting of a rapid overshoot to a level > 1 μM, followed by a slow decay to a plateau of ≈0.5 μM. A similar overshoot was triggered by a constant level of Ca 2+ influx through calcium‐release‐activated Ca 2+ (CRAC) channels in thapsigargin‐treated cells, due to a delayed increase in the rate of Ca 2+ clearance by the PMCA. Following a rise in [Ca 2+ ] i , PMCA activity increased in two phases: a rapid increase followed by a further calcium‐dependent increase of up to approximately fivefold over 10‐60 s, termed modulation. After the return of [Ca 2+ ] i to baseline levels, the PMCA recovered slowly from modulation (τ≈4 min), effectively retaining a ‘memory’ of the previous [Ca 2+ ] i elevation. Using a Michaelis‐Menten model with appropriate corrections for cytoplasmic Ca 2+ buffering, we found that modulation extended the dynamic range of PMCA activity by increasing both the maximal pump rate and Ca 2+ sensitivity (reduction of K M ). A simple flux model shows how pump modulation and its reversal produce the initial overshoot of the biphasic [Ca 2+ ] i response. The modulation of PMCA activity enhanced the stability of Ca 2+ signalling by adjusting the efflux rate to match influx through CRAC channels, even at high [Ca 2+ ] i levels that saturate the transport sites and would otherwise render the cell defenceless against additional Ca 2+ influx. At the same time, the delay in modulation enables small Ca 2+ fluxes to transiently elevate [Ca 2+ ] i , thus enhancing Ca 2+ signalling dynamics.