Biochemical modifications and regulation of the gastric H,K-ATPase

The alpha/beta heterodimeric gastric H,K-ATPase is the enzyme responsible for gastric HCl secretion by the parietal cell. It is an integral membrane protein of the extensive system of intracellular membranes known as tubulovesicles of resting parietal cells, and it is trafficked to the apical plasma membrane upon stimulation by a regulated fusion of tubulovesicles with the apical membrane. There are, however, further mechanisms that may regulate HCl secretion, and these will be explored in this doctoral dissertation. Very little is known about the direct regulation of the H,K-ATPase and the mechanisms that regulate the enzymatic activity of the enzyme. Furthermore, little has been reported about certain posttranslational modifications that may occur on the H,K-ATPase, specifically ubiquitination and glutathiolation, which may have implications in the regulation of the activity of the H,K-ATPase within the parietal cell. The regulation of membrane recycling, particularly the tight packing of tubulovesicles in a resting parietal cell is also poorly understood.;Here we present evidence for the direct regulation of H,K-ATPase enzymatic activity by phosphatidylinositol phosphates and their metabolites. Two posttranslational modifications, ubiquitination and glutathiolation, were also determined to play a potential role in H,K-ATPase regulation. Ubiquitin generally targets cellular proteins for degradation via the lysosome or proteasome. Glutathione potentially serves several functions when modified to the cysteine residues of proteins, including protection from irreversible oxidation. These biochemical modifications and potential regulatory mechanisms of H,K-ATPase activity are novel findings.;Finally, a potential regulatory mechanism of parietal cell tubulovesicular membrane structure and function in vitro was discovered to be dependent on pH. Tubulovesicles were found to reversibly cluster in vitro at low pH, and H,K-ATPase activity was inhibited. A majority of the original H,K-ATPase activity was able to be restored upon reversal of the clustering, suggesting that the clustering may be physiologically significant.;By gaining an understanding of regulatory mechanisms and biochemical modifications of the gastric H,K-ATPase in vitro, we can better understand how it may function in vivo. This research has the potential to lead to novel drug targets for the H,K-ATPase that may aid in the control of acid secretion -- a problem from which millions worldwide suffer.