Contribution of the Hydroxyl Group and Phenol Ring of Tyrosine 780 of the Alpha-Subunit to NA+ Binding by the Na/K Pump

Abstract

The Na/K pump is a heterodimeric(αβ) P-type ATPase that exports three Na+ and imports two K+ against their electrochemical gradients using the energy of ATP hydrolysis. Structural studies confirm three ion-binding sites in the catalytic α-subunit. Site-I and Site-II bind either Na+ or K+, while Site-III, the focus here, exclusively binds Na+. To determine the relative energetic contribution to Na+ binding of the hydroxyl group or the π-electrons of Tyr780 in site-III, we introduced the mutation Y780tag into the α-subunit cDNA for the purpose of using nonsense suppression. Y780tag-α- and β3-cRNA were co-injected in Xenopus oocytes with synthetic tRNA (ligated to Tyr, Phe or Phe derivatives) to compare the functional consequences of the chemical modifications at this position using two-electrode voltage clamp. Square voltage pulses in the absence of external K+ (K+o) and presence of external Na+ (Na+o) produce transient currents. The voltage dependence of steady-state charge moved by these transients is described by a Boltzmann distribution with a center (V1/2) related to the relative apparent affinity for Na+o (ΔV1/2= ­25 mV per 2-fold reduction in affinity). The centers of the distribution where V1/2= ­46.9 ±2.5 mV (n=10) for Tyr, V1/2= ­123 ± 9 mV for Phe, and V1/2= −54.6 ±2.1 mV (n=5) for mono-fluorinated at the 3’ position (mono-F)-Tyr. These results indicate ∼8-fold reduction in apparent affinity for Na+o by removal of the hydroxyl group and a smaller reduction (≪ 2-fold) by partial disruption of the cation-π interaction capability by monofluorination. Surprisingly, the apparent affinity for K+o estimated from the [K+]o dependence of pump current activation in the absence of Na+o was reduced by both Phe (∼4-fold) and mono-F-Tyr (∼1.5-fold). Measurements with other derivatives are underway. NSF-MCB 1515434.