Journal article
Role of a conserved ion-binding site tyrosine in ion selectivity of the Na+/K+ pump
The journal of general physiology, Vol.154(7), e202113039
07/04/2022
DOI: 10.1085/jgp.202113039
PMCID: PMC9171065
PMID: 35657726
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.
Details
- Title: Subtitle
- Role of a conserved ion-binding site tyrosine in ion selectivity of the Na+/K+ pump
- Creators
- Kerri Spontarelli - Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TXDaniel T Infield - University of IowaHang N Nielsen - Aarhus UniversityRikke Holm - Aarhus UniversityVictoria C Young - Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TXJason D Galpin - University of IowaChristopher A Ahern - University of IowaBente Vilsen - Aarhus UniversityPablo Artigas - Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX
- Resource Type
- Journal article
- Publication Details
- The journal of general physiology, Vol.154(7), e202113039
- DOI
- 10.1085/jgp.202113039
- PMID
- 35657726
- PMCID
- PMC9171065
- NLM abbreviation
- J Gen Physiol
- ISSN
- 0022-1295
- eISSN
- 1540-7748
- Grant note
- MCB-1515434 / National Science Foundation 7016-00193B / Danish Council For Independent Research R223-2016-595 / Lundbeck Foundation NINDS 5R24 NS104617-04 / National Institute of Health
- Language
- English
- Date published
- 07/04/2022
- Academic Unit
- Molecular Physiology and Biophysics; Iowa Neuroscience Institute
- Record Identifier
- 9984267660102771
Metrics
6 Record Views