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Study of energy consumption by Na+/K+ ATPase using PercevalHR
KTH, School of Engineering Sciences (SCI), Applied Physics. KTH.ORCID iD: 0000-0003-0374-4411
Karolinska Institutet.
KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
KTH, School of Engineering Sciences (SCI), Applied Physics.ORCID iD: 0000-0003-0578-4003
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Adenosine triphosphate (ATP) is an important macro molecule that is the prime supplier of energy to run cell metabolism and to regulate several physiological processes in a cell. Despite its central role, the number of non-invasive methods to study ATP on a single cell level in real time are limited. ATPases use energy derived from ATP hydrolysis to maintain cell membrane potential by regulating ion gradients across the plasma membrane. It is generally believed that the Na+/K+-ATPase (NKA) which belongs to the P-type ATPase superfamily represent the main energy consumer among the ATPases. In this study, we set out to quantify ATP consumption by NKA on a single cell level in human embryonic kidney cells (HEK293a) using PercevalHR, a genetically encoded fluorescent biosensor that reports changes in the ATP:ADP during live cell imaging. We demonstrate that ATP hydrolysis by NKA is faster at physiological temperatures (35 -37°C) compared to room temperature. K+ free KREBS pre-treatment increased the ATP consumption by NKA. The inhibition of NKA and SERCA reduced the overall ATP hydrolysis in HEK293a cells demonstrating that these ATPases consume a substantial amount of all ATP produced in the cell. We found that the inhibition of mitochondrial respiration reduced basal ATP:ADP in rat primary proximal tubule cells (PTC) but not in HEK293a cells. In contrast, an inhibition of glycolysis gave a more rapid reduction in basal ATP:ADP in HEK293a compared to PTC, illustrating the Warburg effect in cell lines, where the metabolism has been adapted to a high glucose and low oxygen environment. We demonstrate the use of PercevalHR as a robust tool for studies of dynamic energy consumption by NKA in both cell lines and primary cells. 

Keyword [en]
ATP, PercevalHR
National Category
Medical and Health Sciences
Research subject
Biological Physics
Identifiers
URN: urn:nbn:se:kth:diva-204416OAI: oai:DiVA.org:kth-204416DiVA: diva2:1084467
Note

QC 20170328

Available from: 2017-03-24 Created: 2017-03-24 Last updated: 2017-03-28Bibliographically approved
In thesis
1. Studies on molecular mechanisms in calcium signaling and cellular energy consumption
Open this publication in new window or tab >>Studies on molecular mechanisms in calcium signaling and cellular energy consumption
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Ion signaling plays fundamental role in cell survival. Na+ and Ca2+ are critical players in ion signaling. Cells spend the major amount of energy to maintain and regulate Na+ and Ca2+ gradients across the cell membrane. Any disruption in cellular energy consumption by plasma membrane ATPases affects ion signaling and vice versa. This thesis is a combination of four separate research studies. In the first study, We measured ATP consumption dynamics of Na+/K+-ATPase using a genetically encoded fluorescent indicator called Perceval HR. we demonstrate that PercevalHR is an excellent tool to visualize ATP:ADP in mammalian cells.

In the second study, We studied the role of calcium signaling and TRP channels in angiotensin II type 1 receptor  signaling cascade. We prove that low inhibition of CaV1.2 with physiological and therapeutically relevant concentration of Angiotensin II up regulate AT1R signaling.

In the third study, We studied the role of the TRPM5 channel in regulating insulin secretion, and cytoplasmic free calcium concentration in the rat β-cells by usingtriphenyl phosphine oxide, a selective inhibitor of the channel.

In the fourth study, We tested whether, the genetically engineered human β-cell line (EndoC-BH1) could be used as models for studying Ca2+signaling in the context of Type II Diabetes. We found that the EndoC-BH1 cells could be a relevant model to study stimulus-secretion coupling and Ca2+ signaling in the human β-cells.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2017. 76 p.
Keyword
ca2+ signaling, ATP, PercevalHR, NKA, diabetes, Angiotensin
National Category
Medical and Health Sciences
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-204418 (URN)978-91-7729-337-8 (ISBN)
Public defence
2017-04-19, Fire, Scilifelab, Tomtebodavägen 23a, Solna, 09:00 (English)
Opponent
Supervisors
Note

QC 20170328

Available from: 2017-03-28 Created: 2017-03-27 Last updated: 2017-03-28Bibliographically approved

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