Confocal ratiometric voltage imaging of cultured human keratinocytes reveals layer-specific responses to ATP

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Abstract
Recent evidence suggests that changes in membrane potential influence the proliferation and differentiation of keratinocytes. To further elucidate the role of changes in membrane potential for their biological fate, the electrical behavior of keratinocytes needs to be studied under complex conditions such as multilayered cultures. However, electrophysiological recordings from cells in the various layers of a complex culture would be extremely difficult. Given the high spatial resolution of confocal imaging and the availability of novel voltage-sensitive dyes, we combined these methods in an attempt to develop a viable alternative for recording membrane potentials in more complex tissue systems. As a first step, we used confocal ratiometric imaging of fluorescence resonance energy transfer (FRET)-based voltage-sensitive dyes. We then validated this approach by comparing the optically recorded voltage signals in HaCaT keratinocytes with the electrophysiological signals obtained by whole cell recordings of the same preparation. We demonstrate 1) that optical recordings allow precise multisite measurements of voltage changes evoked by the extracellular signaling molecules ATP and bradykinin and 2) that responsiveness to ATP differs in various layers of cultured keratinocytes.