Date of Award

Fall 2011

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Supervisor

A.K.Grover

Language

English

Abstract

This work used biotechnology to invent new caloxins - allosteric peptide inhibitors of plasma membrane Ca2+ pumps (PMCA) needed to understand the Ca2+ signalling in coronary artery.

PMCA are encoded by genes PMCA1-4. Defects in PMCA expression have been associated with several pathologies. The major objectives of my thesis were to determine the expression of PMCA isoforms in the smooth muscle and the endothelium of coronary artery and to invent high affinity and specificity caloxins for the isoforms present in these tissues.

In Aim 1 it was determined that the total PMCA protein and activity was much greater in smooth muscle than in endothelium. Both tissues expressed only PMCA1 and PMCA4, with PMCA4 > PMCA1 in smooth muscle and PMCA1 > PMCA4 in endothelium. Therefore, the search for PMCA1 and 4 selective caloxins using phage display technique was conducted.

Aim 2 was to invent PMCA1 selective inhibitors. Caloxin 1b3 was invented as the first known PMCA1 selective inhibitor. It inhibited PMCA1 Ca2+-Mg2+-ATPase with higher affinity than PMCA2, 3 or 4. Aims 1 and 2 were consistent with the greater potency of caloxin 1b3 than a known PMCA4 selective caloxin 1b1 in increasing cytosolic Ca2+ concentration in endothelial cells.

Aim 3 was to obtain ultrahigh selectivity and affinity PMCA4 bidentate inhibitor using the previously invented PMCA4 selective caloxins 1c2 and 1b2. In the first step the affinity of caloxin 1b2 was improved by limited mutagenesis to obtain caloxin 1c4. Caloxin 1c4 had 5-6 times higher affinity than caloxin 1b2 for inhibiting PMCA4 activity. Optimization of the bidentate caloxins from caloxin 1c2 and 1c4 was also attempted.

The novel caloxins may aid in elucidating the role of PMCA1 and PMCA4 in the physiology and pathophysiology of coronary artery and other tissues.

McMaster University Library


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