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 (Ca2+), the plasma-membrane calcium-ATPase (PMCA) may actively contribute to the generation of complex Ca2+ signals. We have investigated the role of the PMCA in shaping Ca2+ 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 Ca2+ consisting of a rapid overshoot to a level > 1 microM, followed by a slow decay to a plateau of approximately 0.5 microM. A similar overshoot was triggered by a constant level of Ca2+ influx through calcium-release-activated Ca2+ (CRAC) channels in thapsigargin-treated cells, due to a delayed increase in the rate of Ca2+ clearance by the PMCA. Following a rise in Ca2+, 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 Ca2+ to baseline levels, the PMCA recovered slowly from modulation (tau approximately 4 min), effectively retaining a 'memory' of the previous Ca2+ elevation. Using a Michaelis-Menten model with appropriate corrections for cytoplasmic Ca2+ buffering, we found that modulation extended the dynamic range of PMCA activity by increasing both the maximal pump rate and Ca2+ sensitivity (reduction of K(M)). A simple flux model shows how pump modulation and its reversal produce the initial overshoot of the biphasic Ca2+ response. The modulation of PMCA activity enhanced the stability of Ca2+ signalling by adjusting the efflux rate to match influx through CRAC channels, even at high Ca2+ levels that saturate the transport sites and would otherwise render the cell defenceless against additional Ca2+ influx. At the same time, the delay in modulation enables small Ca2+ fluxes to transiently elevate Ca2+, thus enhancing Ca2+ signalling dynamics.