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A High Affinity Extracellular ATP Sensor for Studying Purinergic Signaling
thesisposted on 13.08.2019, 16:59 by Daniel Cholger
Adenosine Triphosphate (ATP) can be released as a signal between cells in an autocrine and paracrine manner that binds purinergic receptors. Highly conserved, purinergic receptors expressed on the cell surface of neurons and astrocytes are capable of being activated across eight orders of magnitude from hundreds of nanomolar ATP to millimolar. Genetically encoded fluorescent protein biosensors have been used to detect ATP outside the cell, but a high affinity extracellular ATP sensor is required to study the ATP signaling dynamics from nanomolar to micromolar magnitudes. Previously, our lab developed a first generation sensor of extracellular ATP called ECATS1 (Conley et al.). To develop an improved sensor, we caried out site-directed mutagenesis of the sensor's ATP binding site and identified a mutant that exhibited a 4-fold increase in ATP binding affinity in solution. We then optimized the membrane-tethering of the sensor to achieve the 4-fold increase in extracellular ATP binding affinity when measured on live cell.s This second-generation sensor was dubbed ECATS2. As a proof-of-concept application, we sought to detect ATP release from cells using in vitro models of edema. We subjected HEK293A cells to hypo-osmotic shock (HOS), revealing ATP release at micromolar levels. Then we tested HOS in cultured cortical astrocytes, also revealing micromolar ATP release. However, when we tested neuron-astrocyte co-cultures, we no longer observed ATP release in response to HOS. Interestingly, this implies that co-culture either entirely prevented ATP release from astrocytes or dampened it into the nanomolar range below the limit of ECATS2 detection. Thus, we have validated the development of a higher affinity, second-generation sensor and used it to discover that ATP release from astrocytes after HOS can be affected by the presence of neurons.