Role of a conserved ion-binding site tyrosine in ion selectivity of the Na+/K+ pump

Abstract

The essential transmembrane Na+ and K+ gradients in animal cells are established by the Na+/K+ pump, a P-type ATPase that exports three Na+ and imports two K+ per ATP hydrolyzed. The mechanism by which the Na+/K+ pump distinguishes between Na+ and K+ at the two membrane sides is poorly understood. Crystal structures identify two sites (sites I and II) that bind Na+ or K+ and a third (site III) specific for Na+. The side chain of a conserved tyrosine at site III of the catalytic α-subunit (Xenopus-α1 Y780) has been proposed to contribute to Na+ binding by cation–π interaction. We substituted Y780 with natural and unnatural amino acids, expressed the mutants in Xenopus oocytes and COS-1 cells, and used electrophysiology and biochemistry to evaluate their function. Substitutions disrupting H-bonds impaired Na+ interaction, while Y780Q strengthened it, likely by H-bond formation. Utilizing the non-sense suppression method previously used to incorporate unnatural derivatives in ion channels, we were able to analyze Na+/K+ pumps with fluorinated tyrosine or phenylalanine derivatives inserted at position 780 to diminish cation–π interaction strength. In line with the results of the analysis of mutants with natural amino acid substitutions, the results with the fluorinated derivatives indicate that Na+–π interaction with the phenol ring at position 780 contributes minimally, if at all, to the binding of Na+. All Y780 substitutions decreased K+ apparent affinity, highlighting that a state-dependent H-bond network is essential for the selectivity switch at sites I and II when the pump changes conformational state.

Structure and function of H+/K+ pump mutants reveal Na+/K+ pump mechanisms

“…Before determination of the Na + -bound crystal structures, it was proposed that the sodium-exclusive site III was formed by the side chains of Glu954 and Tyr771 together with other main-chain carboxyl groups, based on alterations in Na + affinity observed in several NKA mutations, including Glu954Ala 38 , 39 . Our findings clarify Na + coordination revealing the indirect effect of Glu954Ala in NKA, as destruction of the water-mediated hydrogen-bond network with Trp924 eliminates the stabilizing effect of the tryptophan on Gln923 (which also hydrogen bonds to Tyr771 32 ) and Asp926, drastically impairing Na + binding at the real site III. Thus, formation of Na + -exclusive site III in the ngHKA background requires the allosteric influence from the side chain at 943, which is not directly involved in Na + -coordination.…”

“…5e , triangles, V 1/2 = –85.5 ± 7.3 mV, n = 14) with respect to that for NKA (Fig. 5e , black line, V 1/2 = −51 ± 1 mV ( n = 12)), indicating a 2.7-fold reduced apparent affinity for extracellular Na + compared to NKA (25 mV/twofold reduction 31 , 32 ). This reduced apparent affinity for extracellular Na + , which competes with K + for extracellular sites, may contribute to the larger K 0.5,K+ of this mutant.…”

The phenotypic spectrum of pathogenic ATP1A1 variants expands: the novel p.P600R substitution causes demyelinating Charcot–Marie–Tooth disease

“…To increase the rate-limiting intracellular Na + concentration oocytes were incubated in Na + -loading solution (in mM, 90 NaOH, 20 TEA-OH, 40 HEPES, and 0.2 EGTA) with 10 µM ouabain one hour before the two-electrode voltage clamp (TEVC) experiments. Such ouabain concentration inhibited endogenous pumps for the duration of the experiments leaving exogenous pumps unaffected [ 19 ].…”

How a tyrosine primes the pump

“…These gradients are established and maintained by the Na + /K + pump, a P-type ATPase that expels three Na + ions and imports two K + ions with every round of ATP hydrolysis. In this issue of JGP , Spontarelli et al ( 1 ) probe the function of a critical tyrosine residue in the Na + /K + pump’s ion selectivity.…”

“…Artigas and colleagues, led by first author Kerri Spontarelli, generated versions of the Xenopus laevis Na + /K + pump in which this critical tyrosine residue—Y780—was mutated to other amino acids ( 1 ). They expressed these proteins in Xenopus oocytes or COS-1 cells and determined their affinities for cations using electrophysiology and, with the help of Bente Vilsen’s group at Aarhus University, biochemical assays.…”