ABSTRACT
Na(+)-K(+)-ATPase (NKA) establishes the transmembrane [Na(+)] gradient in cells. In heart, phospholemman (PLM) inhibits NKA activity by reducing its apparent Na(+) affinity, an effect that is relieved by PLM phosphorylation. The NKA crystal structure suggests regions of PLM-NKA interaction, but the sites important for functional effects in live cells are not known. We tested wild type (WT) and CFP-NKA-α1 point mutants (alanine substitution at F956, E960, L964, and F967) for fluorescence resonance energy transfer (FRET) with WT-PLM-YFP in HEK293 cells. NKA-PLM FRET was unaltered with F956A or F967A, reduced with L964A, and nearly abolished with E960A. Mutating the PLM site (F28A) identified by structural analysis to interact with E960-NKA also nearly abolished NKA-PLM FRET. In contrast, NKA-PLM coimmunoprecipitation was only slightly reduced by E960A-NKA or F28A-PLM mutants, consistent with an additional interaction site. FRET titrations indicate that the additional site has higher affinity than that between E960-NKA and F28-PLM. To test whether the FRET-preventing mutations also prevent PLM functional effects, we measured NKA-mediated Na(+)-transport in intact cells. For WT-NKA, PLM reduced apparent Na(+)-affinity of NKA and PLM phosphorylation reversed the effect. In contrast, for E960A-NKA the apparent Na(+)-affinity was unaltered by either PLM or forskolin-induced PLM phosphorylation. We conclude that E960 on NKA and F28 on PLM are critical for PLM effects on both NKA function and NKA-PLM FRET, but also there is at least one additional site that is critical for tethering PLM to NKA.
Subject(s)
Membrane Proteins/chemistry , Membrane Proteins/metabolism , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Sodium-Potassium-Exchanging ATPase/chemistry , Sodium-Potassium-Exchanging ATPase/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Crystallography, X-Ray , Dogs , Fluorescence Resonance Energy Transfer , HEK293 Cells , Humans , Ion Transport , Kinetics , Membrane Proteins/genetics , Mice , Models, Biological , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphoproteins/genetics , Protein Conformation , Protein Interaction Domains and Motifs , Rats , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Sharks , Sodium/metabolism , Sodium-Potassium-Exchanging ATPase/geneticsABSTRACT
Intussusception is relatively uncommon, occurring in 0.5 to 2.3 cases per 1,000 live births in the USA. Radiology residents, therefore, have few opportunities to participate in intussusception reduction during training, and practicing radiologists encounter it infrequently. Training is essential, as successful reduction avoids surgery. The judgment involved in reducing an intussusception is best gained with experience. We developed a training device that simulates fluoroscopic intussusception reduction with air. The device consists of a doll that contains a cylinder with similar stress and strain characteristics to the human colon. The trainee pumps air into the cylinder through a rectal tube using a standard hand-held air reduction pump. A sensor measures the pressure within the chamber and transmits readings to a computer, which displays images from actual intussusception reductions based on the pressure maintained within the device. A random component in the software gives the user a new experience each time and models uncertainties in the actual reduction process, including perforation. This intussusception reduction simulator can enhance resident education, giving residents the opportunity to practice this technique before employing it on a real patient. The simulator can also help practicing radiologists become more comfortable with intussusception air reduction.
