A Novel Optical Intracellular Imaging Approach for Potassium Dynamics in Astrocytes

Astrocytes fulfill a central role in regulating K(+) and glutamate, both released by neurons into the extracellular space during activity. Glial glutamate uptake is a secondary active process that involves the influx of three Na(+) ions and one proton and the efflux of one K(+) ion. Thus, intracellular K(+) concentration ([K(+)](i)) is potentially influenced both by extracellular K(+) concentration ([K(+)](o)) fluctuations and glutamate transport in astrocytes. We evaluated the impact of these K(+) ion movements on [K(+)](i) in primary mouse astrocytes by microspectrofluorimetry. We established a new noninvasive and reliable approach to monitor and quantify [K(+)](i) using the recently developed K(+) sensitive fluorescent indicator Asante Potassium Green-1 (APG-1). An in situ calibration procedure enabled us to estimate the resting [K(+)](i) at 133±1 mM. We first investigated the dependency of [K(+)](i) levels on [K(+)](o). We found that [K(+)](i) followed [K(+)](o) changes nearly proportionally in the range 3–10 mM, which is consistent with previously reported microelectrode measurements of intracellular K(+) concentration changes in astrocytes. We then found that glutamate superfusion caused a reversible drop of [K(+)](i) that depended on the glutamate concentration with an apparent EC(50) of 11.1±1.4 µM, corresponding to the affinity of astrocyte glutamate transporters. The amplitude of the [K(+)](i) drop was found to be 2.3±0.1 mM for 200 µM glutamate applications. Overall, this study shows that the fluorescent K(+) indicator APG-1 is a powerful new tool for addressing important questions regarding fine [K(+)](i) regulation with excellent spatial resolution.