Phospholamban inhibits the cardiac calcium pump by reversing allosteric effects of ATP on calcium affinity

Phospholamban (PLB) is a transmembrane micropeptide that regulates the sarcoplasmic reticulum Ca2+ ATPase (SERCA) in cardiac muscle. PLB reduces the apparent Ca2+ sensitivity of SERCA, increasing the Ca2+ concentration required for pump cycling. However, PLB does not decrease Ca2+ binding to SERCA when ATP is absent, and the pump is not cycling. One prevailing explanation for these seemingly conflicting results is PLB may slow the Ca2+ binding step in the SERCA enzymatic cycle, altering the Ca2+ dependence of cycling without affecting true Ca2+ affinity. Alternatively, we speculated that equilibrium measurements of Ca2+ binding to non-cycling SERCA may overlook important allosteric effects of bound nucleotide. Specifically, ATP is known to have an allosteric effect on SERCA that is distinct from its role as a catalytic substrate and source of energy. When ATP binds, it increases the affinity of SERCA for Ca2+ - exactly the opposite of the PLB effect. We speculated that PLB may inhibit SERCA by reversing pre-activation of SERCA Ca2+ affinity by ATP. To test this hypothesis, we used a 2-color SERCA biosensor expressed in microsomal membranes to measure Ca2+ binding to the pump reported by changes in intramolecular FRET. We quantified the Ca2+ affinity of non-cycling SERCA in the absence of ATP, then mimicked ATP binding while preventing enzymatic cycling using the non-hydrolyzable ATP-analog AMPPCP. Interestingly, nucleotide activation by AMPPCP increased the Ca2+ affinity of 2-color SERCA, but this effect was reversed by co-expression of PLB. PLB had no effect on Ca2+ affinity in the absence of nucleotide. These results may reconcile the seemingly paradoxical effects of PLB. We propose that PLB reduces the true Ca2+ affinity of SERCA by reversing allosteric activation of the pump by ATP.