Reactivity of cysteines in the transmembrane region of the Na, K-ATPase alpha subunit probed with Hg(2+).
To gain insight into the structure and conformational coupling in the Na,K-ATPase, this study characterized the reaction of the alpha1 subunit transmembrane cysteines with a small probe. Intact HeLa cells expressing heterologous Na,K-ATPase were treated with (microm) HgCl(2) after placing the enzyme predominantly in either of two conformations, phosphorylated E2P.Na/E2P or dephosphorylated ATP.E1. K/ATP.E1. Under both conditions the treatment led to enzyme inactivation following a double exponential kinetic as determined by ouabain-sensitive K(+) uptake measurements. However, the rate constant of the slow reacting component was ten times larger when the protein was probed in a medium that would favor enzyme phosphorylation. Enzymes carrying mutations of cysteines located in the alpha1 subunit transmembrane region were used to identify the reacting-SH groups. Replacement Cys104Ser reduced enzyme inactivation by removing the slow reacting component under both treatment conditions. Replacement of Cys964 reduced the inactivation rate constant of the fast reacting component (79%) and removed the slow reacting component when the dephosphorylated enzyme was treated with Hg(2+). Moreover, Cys964Ser substituted enzyme was insensitive to Hg(2+) when treated under phosphorylation conditions. These results indicate that Cys964 is involved in the fast inactivation by Hg(2+). Although the double mutant Cys964, 104Ser was still partially inactivated by treatment under nonphosphorylating conditions, an enzyme devoid of transmembrane cysteines was insensitive to Hg(2+) under all treatment conditions. Thus, this enzyme provides a background where accessibility of engineered transmembrane cysteines can be tested.