Study of energy consumption by Na+/K+ ATPase using PercevalHR
(English)Manuscript (preprint) (Other academic)
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.
Medical and Health Sciences
Research subject Biological Physics
IdentifiersURN: urn:nbn:se:kth:diva-204416OAI: oai:DiVA.org:kth-204416DiVA: diva2:1084467
QC 201703282017-03-242017-03-242017-03-28Bibliographically approved