High-level coexpression of the canine cardiac calcium pump and phospholamban in Sf21 insect cells

Phospholamban is a pentameric transmembrane phosphoprotein that regulates the activity of the Ca²⁺-transporting ATPase (SERCA2a) in cardiac sarcoplasmic reticulum. To better understand the structure and function of phospholamban and its mode of regulation of the ATPase, phospholamban and SERCA2a were coexpressed at high levels in Sf21 insect cells using the baculovirus expression system, SERCA2a was expressed as a functionally active Ca²⁺ pump, accounting for ≥ 20% of the total protein in Sf21 cell microsomes. Wild-type phospholamban, as well as phospholamban with different point mutations in the transmembrane region, inhibited both Ca²⁺ transport and ATP hydrolysis by the recombinant Ca²⁺ pump. The inhibition of SERCA2a activity,vas reversed by an anti-phospholamban monoclonal antibody. The phospholamban molecules studied decreased the apparent Ca²⁺ affinity of the Ca²⁺ pump, but had no effect on enzyme velocity measured at saturating Ca²⁺ concentration. Monomeric phospholamban produced by mutations in the leucine/isoleucine zipper domain decreased the apparent Ca²⁺ affinity the most, giving stronger inhibition of the Ca²⁺ pump than even wild-type phospholamban. Thus, the baculovirus cell expression system is ideally suited for examining functional interactions between phospholamban and SERCA2a. The results obtained suggest that the phospholamban monomer may be the active species inhibiting the Ca²⁺ pump in the cardiac sarcoplasmic reticulum membrane.