Expression of rab11a N124I in gastric parietal cells inhibits stimulatory recruitment of the H+-K+-ATPase

Expression of rab11a N124I in gastric parietal cells inhibits stimulatory r ecruitment of the H+-K+-ATPase. Am. J. Physiol. 277 (Cell Physiol. 46): C36 1-C372, 1999.-Stimulation of the gastric parietal cell results in a massive redistribution of H+-K+-ATPase from cytoplasmic tubulovesicles to the apic al plasma membrane. Previous studies have implicated the small GTPase rab11 in this process. Using matrix-assisted laser desorption mass spectrometry, we confirmed that rab11 is associated with H+-K+-ATPase-enriched gastric m icrosomes. A stoichiometry of one rab11 per six copies of H+-K+-ATPase was estimated. Furthermore, rab11 exists in at least three forms on rabbit gast ric microsomes: the two most prominent resemble rab11a, whereas the third r esembles rab11b. Using an adenoviral expression system, we expressed the do minant negative mutant rab11a N124I in primary cultures of rabbit parietal cells under the control of the tetracycline transactivator protein (tTA). T he mutant was well expressed with a distribution similar to that of the H+- K+-ATPase. Stimulation of these cultures with histamine and IBMX was assess ed by measuring the aminopyrine (AP) uptake relative to resting cells (AP i ndex). In experiments on six culture preparations, stimulated uninfected ce lls gave an AP index of 10.0 +/- 2.9, whereas parallel cultures expressing rab11a N124I were poorly responsive to stimulation, with a mean AP index of 3.2 +/- 0.9. Control cultures expressing tTA alone or tTA plus actin respo nded equally well to stimulation, giving AP index values of 9.0 +/- 3.1 and 9.6 +/- 0.9, respectively. Thus inhibition by rab11a N124I is not simply d ue to adenoviral infection. The AP uptake data were confirmed by immunocyto chemistry. In uninfected cells, H+-K+-ATPase demonstrated a broad cytoplasm ic distribution, but it was cleared from the cytoplasm and associated with apically derived membranes on stimulation. In cells expressing rab11a N124I , H+-K+-ATPase maintained its resting localization on stimulation. Furtherm ore, this effect could be alleviated by culturing infected cells in the pre sence of tetracycline, which prevents expression of the mutant rab11. We th erefore conclude that rab11a is the prominent GTPase associated with gastri c microsomes and that it plays a role in parietal cell activation.