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.
Skip Nav Destination
Article navigation
Article|
June 03 2022
Role of a conserved ion-binding site tyrosine in ion selectivity of the Na+/K+ pump
Kerri Spontarelli
,
Kerri Spontarelli
1
Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX
Search for other works by this author on:
Daniel T. Infield,
Daniel T. Infield
2
Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA
Search for other works by this author on:
Hang N. Nielsen,
Hang N. Nielsen
3
Department of Biomedicine, Aarhus University, Aarhus C, Denmark
Search for other works by this author on:
Rikke Holm,
Rikke Holm
3
Department of Biomedicine, Aarhus University, Aarhus C, Denmark
Search for other works by this author on:
Victoria C. Young
,
Victoria C. Young
1
Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX
Search for other works by this author on:
Jason D. Galpin
,
Jason D. Galpin
2
Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA
Search for other works by this author on:
Christopher A. Ahern
,
Christopher A. Ahern
2
Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA
Search for other works by this author on:
Bente Vilsen
,
3
Department of Biomedicine, Aarhus University, Aarhus C, Denmark
Bente Vilsen: bv@biomed.au.dk
Search for other works by this author on:
Pablo Artigas
1
Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX
Correspondence to Pablo Artigas: pablo.artigas@ttuhsc.edu
Search for other works by this author on:
Kerri Spontarelli
1
Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX
Daniel T. Infield
2
Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA
Hang N. Nielsen
3
Department of Biomedicine, Aarhus University, Aarhus C, Denmark
Rikke Holm
3
Department of Biomedicine, Aarhus University, Aarhus C, Denmark
Victoria C. Young
1
Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX
Jason D. Galpin
2
Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA
Christopher A. Ahern
2
Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA
Correspondence to Pablo Artigas: pablo.artigas@ttuhsc.edu
Bente Vilsen: bv@biomed.au.dk
Received:
October 19 2021
Revision Received:
April 19 2022
Accepted:
May 16 2022
Online Issn: 1540-7748
Print Issn: 0022-1295
Funding
Funder(s):
National Science Foundation
- Award Id(s): MCB-1515434,MCB-2003251
Funder(s):
National Institute of Health
- Award Id(s): NINDS 5R24 NS104617-04,NINDS 1R03 NS116433-01
Funder(s):
Lundbeck Foundation
- Award Id(s): R223-2016-595
Funder(s):
Danish Council For Independent Research
- Award Id(s): 7016-00193B
© 2022 Spontarelli et al.
2022
This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).
J Gen Physiol (2022) 154 (7): e202113039.
Article history
Received:
October 19 2021
Revision Received:
April 19 2022
Accepted:
May 16 2022
Connected Content
Commentary
How a tyrosine primes the pump
Citation
Kerri Spontarelli, Daniel T. Infield, Hang N. Nielsen, Rikke Holm, Victoria C. Young, Jason D. Galpin, Christopher A. Ahern, Bente Vilsen, Pablo Artigas; Role of a conserved ion-binding site tyrosine in ion selectivity of the Na+/K+ pump. J Gen Physiol 4 July 2022; 154 (7): e202113039. doi: https://doi.org/10.1085/jgp.202113039
Download citation file:
Sign in
Don't already have an account? Register
Client Account
You could not be signed in. Please check your email address / username and password and try again.
Sign in via your Institution
Sign in via your Institution
156
Views
0
Citations
Suggested Content
Route, mechanism, and implications of proton import during Na+/K+ exchange by native Na+/K+-ATPase pumps
J Gen Physiol (March,2014)
How a tyrosine primes the pump
J Gen Physiol (June,2022)
FXYD protein isoforms differentially modulate human Na/K pump function
J Gen Physiol (November,2020)
Advertisement