Kinetics by X-Ray Crystallography: Sequential Substitution of K+ Bound To Na+, K+-ATPase

Abstract

Na+, K+-ATPase is one of the most important members of the P-type ATPase family. It transports 3 Na+ from the cytoplasm into the extracellular medium and countertransports 2 K+ per ATP hydrolyzed in each reaction cycle, thereby establishing gradients for Na+ and K+ across the membrane. These ion gradients are used in many fundamental processes, notably, excitation of nerve cells. Na+, K+-ATPase is also regarded as a membrane receptor for cardiotonic steroids, such as ouabain and digoxin, which have been prescribed for treatment of heart failure for ∼200 years. Crystallographic studies of Ca2+-ATPase of sarcoplasmic reticulum have provided detailed information on the gating mechanism on the cytoplasmic side, but little on the gating on the other side. This is partly because H+ countertransported by Ca2+-ATPase is invisible to X-rays. In this regard, Na+, K+-ATPase has a fundamental advantage over Ca2+-ATPase, as it countertransports K+ and even its congeners of larger atomic numbers. Here, we carried out X-ray crystallography and isotopic measurements of the ATPase in a state analogous to E2·Pi·2K+ to visualize the substitution process of 2 bound K+ with congeners in the extracellular medium. Crystals were soaked in a buffer containing K+ congeners and anomalous diffraction data were collected. The electron density maps at various incubation times clearly show that the substitution of the 2 K+ with congeners occurs substantially faster at site II. An analysis of B-factors of protein atoms in the crystal shows that the M3-M4E helix pair opens and closes the ion pathway leading to the extracellular medium. These results indicate that site I K+ is the first cation to bind to the empty cation binding sites after releasing 3 Na+.