Topology of the Na,K-ATPase: Evidence for externalization of a labile transmembrane structure during heating

The topological organization of the Na,K-ATPase α subunit is controversial. Detection of extracellular proteolytic cleavage sites would help define the topology, and so attempts were made to find conditions and proteases that would permit digestion of Na,K-ATPase in sealed right-side- out vesicles from renal medulla. The β subunit is predominantly extracellular and could mask the surface of the α subunit. Most of the tested proteases cleaved β, and some digested it extensively. However, without further disruption of structure, there was still no digestion of the α subunit. Reduction (at 50 °C) of disulfide bonds that might stabilize the β subunit fragments, or heating alone at 55 °C, permitted tryptic digestion of a at a site close to the C terminus, while simultaneously increasing digestion of β. A 90-kDa N-terminal fragment of α was recovered, but the C-terminal fragment was further digested. Heating and reduction resulted in the extracellular exposure of a protein kinase A phosphorylation site, Ser-938, and the C terminus, both of which have been proposed to be located on the intracellular surface. At the same time, access to a distant protein kinase C phosphorylation site was not increased. The data suggest that the harsh treatment simultaneously resulted in alteration of the β subunit and the extrusion of a segment of α that normally spans the membrane, without causing complete denaturation or opening the sealed vesicles. Preincubation with Rb⁺ was protective, consistent with prior evidence that it stabilizes the protein segments in the C-terminal third of α. We conclude that this portion of the α subunit contains a transmembrane structure with unique lability to heating.