We also performed measurements of membrane conductance on Xenopus oocytes expressing Bufo Na,K ATPase or Bufo ngH,K ATPase. The results in Fig. 3 showed that PTX produced a large increase of membrane conductance in oocytes expressing Na,K ATPase but not in those expressing either ngH,K ATPase or H,K ATPase 2 alone. Patch clamp experiments in HeLa cells showed that PTX produced a very large increase in conductance in cells expressing Na,K ATPase but no significant increase in conductance in cells expressing ngH,K ATPase or rat Na,K ATPase 1 subunit alone. We conclude from these studies that Na,K ATPase is the target of PTX action but not ngH,K ATPase. Rat ngH,K ATPase and rat Na,K ATPase structural models Figure 5 shows a marked difference between the N termini of rat Na,K ATPase and rat nongastric H,K ATPase models. This difference is due to a 40 residue shorter N terminus in nongastric H,K ATPase than the one in Na,K ATPase. The Na,K ATPase N terminus is situated close to the actuator domain, and that is thought to tilt the M1 helix by rotation of the A domain .
This change is thought to play a key role in the E1 to E2 conformational change of the enzyme. The absence of 40 residues in the H,K ATPase N terminus appears to SRC Inhibitor reduce the interaction between the A domain and TM1. This is evident since the short alpha helix and strand that loop around the A domain in the Na,K ATPase model are absent from the H,K ATPase model. The Na,K ATPase N terminus has been shown to play a role in PTXinduced channel inactivation . The absence of these 40 residues in ngH,KATPase N terminus may, therefore, account for the absence of a PTX effect on ngH,K ATPase. Additionally, the TM1 2 extracellular loop, which is critical for the high affinity binding of ouabain protrudes towards the extracellular region in a different way in the non gastric H,K ATPase and Na,K ATPase models. We suggest that this structural difference may account for the difference in sensitivity of ngH,K ATPase and Na,K ATPase to ouabain and or to PTX.
Experiments that directly measure PTX binding to the two proteins are warranted to test if this is the explanation of the difference Vemurafenib selleckchem in their responsiveness. If differences in binding affinity are found, it should be possible to test which regions are involved in PTX and ouabain binding by constructing chimeras of the two ATPases. It its well established that PTX binds to Na,K ATPase and opens a conducting pathway through it Non gastric H,K ATPase has been extensively studied since the successful cloning of its ? subunit in 1992 . There is controversy regarding its pharmacological sensitivity to ouabain and this complicates the interpretation of experiments in which both PTX and ouabain are present.