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

Stimulation of the gastric parietal cell results in a massiveredistribution ofH⁺-K⁺-ATPasefrom cytoplasmic tubulovesicles to the apical plasma membrane. Previousstudies have implicated the small GTPase rab11 in this process. Usingmatrix-assisted laser desorption mass spectrometry, we confirmed thatrab11 is associated withH⁺-K⁺-ATPase-enrichedgastric microsomes. A stoichiometry of one rab11 per six copies ofH⁺-K⁺-ATPasewas estimated. Furthermore, rab11 exists in at least three forms onrabbit gastric microsomes: the two most prominent resemble rab11a,whereas the third resembles rab11b. Using an adenoviral expressionsystem, we expressed the dominant negative mutant rab11a N124I inprimary cultures of rabbit parietal cells under the control of thetetracycline transactivator protein (tTA). The mutant was wellexpressed with a distribution similar to that of theH⁺-K⁺-ATPase.Stimulation of these cultures with histamine and IBMX was assessed bymeasuring the aminopyrine (AP) uptake relative to resting cells (APindex). In experiments on six culture preparations, stimulateduninfected cells gave an AP index of 10.0 ± 2.9, whereas parallelcultures expressing rab11a N124I were poorly responsive to stimulation,with a mean AP index of 3.2 ± 0.9. Control cultures expressing tTAalone or tTA plus actin responded equally well to stimulation, givingAP index values of 9.0 ± 3.1 and 9.6 ± 0.9, respectively. Thusinhibition by rab11a N124I is not simply due to adenoviral infection.The AP uptake data were confirmed by immunocytochemistry. In uninfectedcells,H⁺-K⁺-ATPasedemonstrated a broad cytoplasmic distribution, but it was cleared fromthe cytoplasm and associated with apically derived membranes onstimulation. In cells expressing rab11a N124I,H⁺-K⁺-ATPasemaintained its resting localization on stimulation. Furthermore, thiseffect could be alleviated by culturing infected cells in the presenceof tetracycline, which prevents expression of the mutant rab11. Wetherefore conclude that rab11a is the prominent GTPase associated withgastric microsomes and that it plays a role in parietal cell activation.