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The sarcoplasmic reticulum (SR) of striated muscle plays a central role in control of contractile activity. It acts as an intracellular sink for calcium during relaxation and releases cav2 + during contraction. This highly differentiated endoplasmic reticulum is a self-contained network with a continuous hollow interior which surrounds each muscle fibril (Fig. 1) (Porter, 1961). The SR is fragmented at longitudinal intervals of one sarcomere in length by the transverse tubular or T-tubular system (Porter, 1961; Franzini-Armstrong, 1980). On each side of the T-tubules there are enlarged areas known as cisternae, with branched areas between the cisternae known as the longitudinal elements. The juncture of the three membranes (two terminal cisternae and one Ttubule) is referred to as the triad. The junction between the SR and • 0 the T-tubules is known as a junctional gap and has.a width of 100-200 A. Periodic densities, referred to as 'feet', cross the junctional gap to join the SR and T-tubular membranes (Franzini-Armstrong, 1980). Direct communication between the SR, the T-tubules and the feet have been postulated (Schneider· and Chandler, 1973; Mathias et al., 1979). The view has developed, starting with the experiments of Huxley and Taylor (1958) and Huxley and Straub (1958), that, under physiological conditions, contraction in skeletal muscle is triggered by depolarisation of the membranes of the T-tubules. This results in the release of ca 2 + into the myoplasm from its intracellular storage location, the SR, and thus activating the contractile proteins (Schneider and Chandler, 1973). (See Fuchs (1974) and Ebashi (1980) for a review on the possible mechanism involved in excitation-contraction coupling.)

<p>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.</p> <p>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.</p> <p>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.</p> <p>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.</p> <p>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.</p> <p>The novel caloxins may aid in elucidating the role of PMCA1 and PMCA4 in the physiology and pathophysiology of coronary artery and other tissues.</p> / Doctor of Philosophy (PhD)