The cells were then incubated at 37 C in a 5% CO2 atmosphere until confluence. The day after achieving confluence, steady state patch clamp currents were measured at room temperature . The patch pipette was filled with an intracellular solution containing : 85 Na sulfamate, 20 TEA Cl, 3 MgCl2, 5.5 dextrose, 10 EGTA, 10 HEPES, 5 Na pyruvate, 10 MgATP, and 7.9 phosphocreatine disodium salt. The K free bathing solution contained : 145 NaCl, 23 MgCl2, 2 BaCl2, 5.5 dextrose, 10 HEPES Na, and 0.2 CdCl2. The solution containinig 20 mM K to activate the Na K pump was the same as the K free solution but with equimolar replacement of NaCl by KCl. Ouabain was added to bathing solutions to inhibit endogenous sodium pumps before starting electrophysiological recordings. An aliquot of 100 M PTX was thawed just prior to each experiment and diluted to a final concentration of 100 nM in the external K free solution containing 0.002% BSA. All internal and external solutions used for patch clamp measurements had a pH of 7.4 0.
05 and osmolality price Vicriviroc of 280 300 mOsm kg. Direct PTX application to confluent HeLa cells The effect of palytoxin on cells over expressing rat ngH,K ATPase and Na,K ATPase was studied in HeLa cells grown in 35 mm2 Petri dishes to 50 60% confluency and then transiently transfected with rat NK?1 NK 1 cDNAs encoding for Na,K ATPase, rat ngHK?2 NK 2 cDNAs encoding for ngH,K ATPase, or rat NK 1 cDNA encoding for Na,K ATPase 1 subunit. Thirty six hours later, we treated all Petri dishes with 20 M oubain in 1 ml culture media for 30 minutes to inhibit endogenous HeLa Na,K ATPase. We then added 1 M PTX to each Petri dish and the cells were incubated for 90 minutes at 37 C, 5% CO2 atmosphere. Photographs were taken with a digital camera under phase contrast illumination at magnifications of 100X, 250X, and 400X. Petri dishes were photographed with phase contrast illumination at 250X and 400X. Modeling of rat Na,K ATPase and rat colonic H,K ATPase Modeller version 8.
2 was used to create structural models of rat colonic ngH,K ATPase and rat Na,K ATPase based on supplier MDV3100 selleckchem a template of SERCA in the E2 P conformation . Modeller?s SALIGN command was used to construct a global, multiple alignment that included sheep Na,K and rat gastric H,K sequences to provide a consensus alignment in regions of lower identity. SERCA and rat ngH,K ATPase share about 31% identity but there is a much higher similarity in the intracellular domains as well as in the transmembrane sections where ion binding and permeation occur. All images were prepared with the PyMOL program which is a molecular graphics system with a Python interpreter designed for real time visualization and generation of high quality molecular graphics images.