The membrane conductance of HeLa cells expressing rat Na,K ATPase and exposed to PTX averaged 2.69 0.24 nS; ?30 fold greater than the membrane conductance measured in HeLa cells exposed to PTX and expressing rat ngH,K ATPase or those transfected with 1 subunit alone . These results are summarized in Fig. 4B. Rat ngH,K ATPase and rat Na,K ATPase models If the failure of PTX to increase the conductance of ngH,K ATPase is a result of failure to bind to the protein, we would expect to find significant structural differences in the extracellular domains of ngH,K ATPase and Na,K ATPase that could account for this difference. In order to determine if there are structural differences beween ngH,K ATPase and Na,K ATPase that could be related to PTX binding, we constructed structural models of rat Na,K and rat ngH,K ATPase by alignment with SERCA. The alignments obtained correspond well with other alignments of type IIC ATPases with SERCA . These models are based on the known crystal structure of SERCA and were constructed for E2 P conformation as described in Methods. The Ntermini in type IIC ATPases are, in general, regions of low sequence identity. Indeed, Fig.
5 shows a clear difference between the long N terminus of the rat Na,K ATPase model and the short one of rat non gastric H,K ATPase. The Na,K ATPase N terminus is in a close proximity to the actuator domain, whereas the non gastric H,K ATPase N terminus is hardly in contact with the actuator domain. Fig.5 also shows differences of the shape of the protruding order Purmorphamine M1 2 loop that may account for differences in ouabain and or PTX binding affinity. Discussion The present study was designed to determine if PTX has an effect on non gastric H,K ATPases in cells in which they can be functionally expressed under experimental conditions in which the participation of endogenous Na,K ATPase can be prevented by prior application of a low dose of ouabain. The data in Fig. 1 demonstrate that exogenous ngHK ATPase and ouabain resistant 
NaK ATPase expressed in HeLa cells and oocytes are functional as measured by 86Rb uptake in cells in which endogenous NaK ATPase activity is blocked by 10 M ouabain.
A clear and simple demonstration of the effect of PTX on the morphology of confluent HeLa cells over expressing ouabain resistant rat Na,K ATPase is shown in Fig. 2A and B. After exposure to PTX the cells can no longer maintain their normal gradients of electrolytes. The cells develop intracellular granulations, become more rounded as they swell, detach from the substrate tumor and adjacent cells and eventually shrink to small spheres floating freely in the medium presumably because they lose their cellular contents across leaky surface membranes. HeLa cells expressing ngH,KATPase or Na,K ATPase 1 subunit alone that have their endogenous Na,K ATPase blocked by 20 M ouabain were not similarly affected.
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