L30A Mutation of Phospholemman Mimics Effects of Cardiac Glycosides in Isolated Cardiomyocytes

To determine if mutations made to PLM could increase PLM-NKA binding we performed scanning mutagenesis of the transmembrane domain of PLM and measured FRET between each mutant and NKA. We observed increased binding to NKA for several PLM mutants compared to WT, including L27A, L30A, and I32A. In isolated cardiomyocytes, overexpression of WT PLM increased the amplitude of the Ca2+ transient compared to GFP control. Ca2+ transient amplitude was further increased by L30A PLM overexpression. L30A mutation also delayed Ca2+ extrusion and increased the duration of cardiomyocyte contraction. This mimics aspects of the effect of cardiac glycosides, which are known to increase contractility through inhibition of NKA. No significant differences between WT and L30A PLM expressing myocytes were observed after treatment with isoproterenol, suggesting that the superinhibitory effects of L30A are reversible with β-adrenergic stimulation. We also observed a decrease in PLM tetramerization with L30A compared to WT using F...