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+](i)), 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+](i) 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+](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 [Ca2+](i) to baseline levels, the PMCA recovered slowly from modulation (tau approximately 4 min), effectively retaining a 'memory' of the previous [Ca2+](i) 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+](i) 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+](i) 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+](i), thus enhancing Ca2+ signalling dynamics.
Bautista
Diana M
DM
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, CA 94305, USA.
Hoth
Markus
M
Lewis
Richard S
RS
D000255:Adenosine Triphosphate / Q000378:metabolism; D000465:Algorithms; D015220:Calcium Channels / Q000502:physiology; D020013:Calcium Signaling / Q000502:physiology*; D000252:Calcium-Transporting ATPases / Q000502:physiology*; D002462:Cell Membrane / Q000201:enzymology / Q000502:physiology; D004558:Electric Stimulation; D004594:Electrophysiology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D018715:Microscopy, Video; D008928:Mitochondria / Q000378:metabolism; D018408:Patch-Clamp Techniques; D019831:Sodium-Calcium Exchanger / Q000378:metabolism; D013601:T-Lymphocytes / Q000201:enzymology* / Q000648:ultrastructure
D015220:Calcium Channels; D019831:Sodium-Calcium Exchanger; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases