Monitoring ATP release from individual cells with a biosensor

Abstract

Adenosine triphosphate (ATP) is a neurotransmitter/neuromodulator in both central and peripheral nervous systems. Particularly in the taste bud, a peripheral taste organ, ATP serves as an afferent neurotransmitter. To examine the mechanism that mediates ATP secretion in taste cells, we elaborated an approach for monitoring ATP in an extracellular medium by employing a biosensor, that is, cells responsive to ATP. Two lines of ATP-sensitive cells, HEK-293 and COS-1, which endogenously express P2Y receptors, were employed. In addition, HEK-293 cells transfected with P2X₃ receptors were also used. By most relevant parameters (threshold response, inactivation kinetics of ATP responses, and refractory period), COS-1 cells were more suitable as an ATP sensor than HEK-293 cells, both native and transfected. For the HEK-293 cell-based biosensor, one of pitfalls was that they were highly responsive to mechanical disturbances, e.g., solution flux elicited by application of a chemical stimulus, owing to the expression of mechanosensitive Ca²⁺-permeable cation channels. In COS-1 cells, ATP-dependent Ca²⁺ transients were generated mostly due to Ca²⁺ release, the feature allowing one to control the activity of ATP-releasing cells electrophysiologically and to monitor the ATP secretion by Ca²⁺ responses of the ATP-biosensor. By using this technique, it was demonstrated that individual taste cells of a mouse released ATP in response to membrane depolarization.