INVOLVEMENT OF G-ALPHA(I2) IN THE MAINTENANCE AND BIOGENESIS OF EPITHELIAL-CELL TIGHT JUNCTIONS

yPolarized epithelial cells have highly developed tight junctions (TJ) to maintain an impermeant barrier and segregate plasma membrane funct ions, but the mechanisms that promote TJ formation and maintain its in tegrity are only partially defined. Treatment of confluent monolayers of Madin-Darby canine kidney (MDCK) cells with AlF4- (activator of het erotrimeric G protein alpha subunits) results in a 3-4-fold increase i n transepithelial resistances (TER), a reliable indicator of TJ integr ity. MOCK cells transfected with activated G alpha(0) (Q205L) have acc lerated TJ formation (Denker, B. M., Saha, C., Khawaja, S., and Nigam, S. J. (1996) J. Biol. Chem. 271, 25750-25753). G alpha(i2) has been l ocalized within the tight junction, and a role for G alpha(i2) in the formation and/or maintenance of the tight junction was studied by tran sfection of MDCK cells with vector without insert (PC), wild type G al pha(i2), or a GTPase-deficient mutant (constitutively activated), Q205 L alpha(i2). Tryptic conformational analysis confirmed expression of a constitutively active G alpha(i2) in Q205L alpha(i2)-MDCK cells, and confocal microscopy showed a similar pattern of G alpha(i2) localizati on in the three cell lines. Q205L alpha(i2)-MDCK cells had significant ly higher base-line TER values than wild type G alpha(i2)- or PC-MDCK cells (1187 +/- 150 versus 576 +/- 89 (G alpha(i2)); 377 +/- 52 Omega . cm(2) (PC)), and both G alpha(i2)- and Q205L alpha(i2)-transfected c ell lines more rapidly develop TER in the Ca2+ switch, a model widely used to study the mechanisms of junctional assembly. Treatment of cell s with AlF4- during the Ca2+ switch had little effect on the kinetics of TER development in G alpha(i2)- or Q205L alpha(i2)-MDCK cells, but PC cells reached half-maximal TER significantly sooner in the presence of AlF4- (similar times to G alpha(i2)-transfected cells). Base-line TER values obtained after the switch were significantly higher for all three cell lines in the presence of AlF4-. These findings indicate th at G alpha(i2) is important for both the maintenance and development o f the TJ, although additional G alpha subunits are likely to play a ro le.