Abstract
Calcium is an essential signal carrier that enables cellular processes such as muscle contraction, secretion, and metabolism. Consequently, calcium dysregulation is associated with many human diseases, including heart failure, cancer, diabetes, and many others.
Considering heart failure alone, 26 million people currently suffer worldwide. The principal site for cellular calcium homeostasis is the endo- and sarcoplasmic reticulum (ER & SR), where resident calcium pumps (SERCA) actively maintain cytosolic calcium. SERCA is so important for cellular function that it is controlled by a number of tissue-specific master regulators – phospholamban (PLN), sarcolipin (SLN) and newly identified Dworf. These SERCA regulators control cardiac contractility, thermogenesis, and skeletal muscle performance. We will present mutagenesis and structure-function studies of PLN, SLN, and Dworf, with an emphasis on clinically relevant mutations.
As genome sequencing projects rapidly expand the spectrum of human genetic variants in PLN and SLN, this information provides a basis for targeted structure-function analyses. Focusing on missense variants of these peptides, we use membrane reconstitution, functional assays and biophysical approaches – circular dichroism (CD) spectroscopy of PLN and SLN variants and electron cryo-microscopy (cryoEM) of two-dimensional crystals of the SERCA-PLN and SERCA-SLN complexes.
Some of the mutants investigated include Ala15-Thr, Arg9-Cys, and Arg14-deletion. The Ala15-Thr mutant was identified in a 4-year female with dilated cardiomyopathy. This mutant has comparable inhibitory activity to wild-type PLN, but the interaction with protein kinase A (PKA) is altered. The Arg9-Cys mutant was identified in an extended family with dilated cardiomyopathy and death at approximately 25 years of age. This mutant is a dominant-negative regulator of SERCA and PKA, with severe consequences for cellular calcium homeostasis and cardiac contractility. The Arg14-deletion mutation is highly prevalent in the Netherlands, with consequences on SERCA function and PKA phosphorylation. Our current progress on PLN, SLN, Dworf, and associated mutations will be presented.
About Dr Howard Young
Dr Howard Young is an Associate Professor at the Department of Biochemistry, University of Alberta, Canada. He has a PhD from the University of Connecticut in Molecular Biology and Biochemistry.