Detection and quantification of Na,K-ATPase dimers in the plasma membrane of living cells by FRET-FCS

• Published in: Biochimica et biophysica acta. General subjects Vol. 1868; no. 7; p. 130619
• Main Authors: Nordahl, Linnea, Akkuratov, Evgeny E., Heimgärtner, Johannes, Schach, Katja, Meineke, Birthe, Elsässer, Simon, Wennmalm, Stefan, Brismar, Hjalmar
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.07.2024
• Subjects: Amino Acids; Cell Membrane - enzymology; fluorescence; fluorescence correlation spectroscopy; Fluorescence Resonance Energy Transfer; fluorescent dyes; FRET-FCS; HEK293 Cells; Humans; membrane potential; NaK-ATPase; Non-canonical amino acids; oligomerization; plasma membrane; Protein Multimerization; Single-Cell Analysis - methods; sodium; sodium-potassium-exchanging ATPase; Sodium-Potassium-Exchanging ATPase - metabolism; Spectrometry, Fluorescence; FRET-FCS; NaK-ATPase; Non-canonical amino acids
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2024.130619

The sodium potassium pump, Na,K-ATPase (NKA), is an integral plasma membrane protein, expressed in all eukaryotic cells. It is responsible for maintaining the transmembrane Na+ gradient and is the major determinant of the membrane potential. Self-interaction and oligomerization of NKA in cell membranes has been proposed and discussed but is still an open question. Here, we have used a combination of FRET and Fluorescence Correlation Spectroscopy, FRET-FCS, to analyze NKA in the plasma membrane of living cells. Click chemistry was used to conjugate the fluorescent labels Alexa 488 and Alexa 647 to non-canonical amino acids introduced in the NKA α1 and β1 subunits. We demonstrate that FRET-FCS can detect an order of magnitude lower concentration of green-red labeled protein pairs in a single-labeled red and green background than what is possible with cross-correlation (FCCS). We show that a significant fraction of NKA is expressed as a dimer in the plasma membrane. We also introduce a method to estimate not only the number of single and double labeled NKA, but the number of unlabeled, endogenous NKA and estimate the density of endogenous NKA at the plasma membrane to 1400 ± 800 enzymes/μm2. [Display omitted] •FRET-FCS is more sensitive than FCCS for detection of rare dual labeled molecules.•Na,K-ATPase can be found as both monomers and dimers in the plasma membrane.•The density of Na,K-ATPase in the HEK293T cell membrane is 1400 ± 800 enzymes/μm2.