Quantification of Calcium Pump Structure Changes in Live Celss

Abstract

The sarcoendoplasmic reticulum ATPase (SERCA) is a calcium pump that undergoes conformational changes during catalytic cycling. To investigate SERCA structure changes in living cells we fused Cerulean to the A-domain and YFP to the N-, P- or TM-domain of SERCA2a and expressed these “2-color” SERCA constructs in AAV-293 cells. 2-color SERCA constructs were catalytically active as shown by ATPase activity in vitro and Ca uptake in live cells. Specifically, the transport activity of 2-color SERCA blunted IP3-mediated Ca transients and increased the size of the Tg-releasable Ca stores. Both of these effects were reversed by coexpression of exogenous phospholamban (PLB), suggesting that 2-color SERCA function and regulation were intact. All of the constructs exhibited dynamic FRET changes in response to the pump ligands Ca and thapsigargin (Tg). The Tg-dependent conformational change was not decreased by coexpression of PLB, nor did PLB slow the kinetics of Tg-binding. Notably, FRET in ionophore-permeabilized cells was higher with saturating calcium than with EGTA in the extracellular solution. This suggests a decrease in domain separation distance with the structure transition from E2 (Ca-free) to E1 (Ca-bound). The data are consistent with closure of the cytoplasmic headpiece with Ca-binding. These 2-color SERCA constructs provide insight into the structural dynamics of Ca transport and may also be useful for evaluating candidate small molecule regulators of Ca uptake activity.