Date of Graduation

2004

Document Type

Thesis

Abstract

The goal of this research project was to elucidate the physical mechanism of Ca2+-ATPase regulation by phospholamban in cardiac muscle. The Ca2+-ATPase transports calcium into the sarcoplasmic reticulum in order to promote muscle relaxation. Phospholamban regulates the Ca 2+-ATPase in the heart by decreasing the apparent calcium affinity of the Ca2+-ATPase, resulting in an inhibition of Ca 2+-ATPase activity. At present, the physical mechanism by which phospholamban regulates the Ca2+-ATPase is unknown. Our central hypothesis was that phospholamban disrupts the Ca2+-ATPase conformational transition from the low Ca2+ affinity state (the E2 intermediate) to the high Ca2+ affinity state (the E1 intermediate), with concomitant inhibition of enzyme activity. First, we developed the baculovirus-insect cell system to produce microsomes containing cardiac Ca2+-ATPase alone or Ca2+-ATPase co-expressed with cardiac phospholamban in sufficient quantities for kinetic and spectroscopic experiments. The enzymatic properties of the expressed Ca2+-ATPase were similar to those observed in native cardiac SR vesicles, and when co-expressed with phospholamban, the Ca2+-ATPase was functionally coupled to phospholamban similar to that observed in cardiac SR vesicles. Next, we measured the effect of phospholamban on Ca2+-ATPase E2 phosphorylation by inorganic phosphate (P i). The results showed that phospholamban decreased the amount of Ca 2+-ATPase phosphorylated by Pi without affecting the [P i]-dependence of phosphorylation. We then used fluorescence spectroscopy to determine the effect of phospholamban on the Ca2+-dependent Ca2+-ATPase E2 to E1 conformational equilibrium. We found that phospholamban decreased the amount of Ca2+-ATPase undergoing the E2 to E1(Ca2) transition in response to calcium, but phospholamban did not alter the [Ca2+]-dependence of the transition. Taken together, phospholamban decreased the number of enzymes undergoing a transition without affecting the affinity of the enzyme for the ligand that stimulated the transition. This suggested that phospholamban inhibits Ca2+-ATPase by sequestering the enzyme in a conformation which resists the transition to E2P in the presence of Pi or to E1(Ca2) in the presence of Ca2+. We propose that phospholamban stabilizes the Ca2+-ATPase into an E2-like conformation, E, which is physically distinct from E2.

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