Cytosolic calcium transients: Spatial localization and role in drosophila photoreceptor cell function

Drosophila phototransduction is a phosphoinositide-mediated and Ca²⁺-regulated signaling cascade ideal for the dissection of feedback regulatory mechanisms. To study the roles of intracellular Ca²⁺ ([Ca²⁺]_i) in this process, we developed novel techniques for the measurement of [Ca²⁺]_i in intact photoreceptors. We genetically engineered flies that express a UV-specific rhodopsin in place of the normal rhodopsin, so that long wavelength light can be used to image [Ca²⁺]_i changes while minimally exciting the photoreceptor cells. We show that activation with UV generates [Ca²⁺]_i increases that are spatially localized to the rhabdomeres and that are entirely dependent on the influx of extracellular Ca²⁺. Application of intracellular Ca²⁺ chelators of varying affinities demonstrates that the Ca²⁺ influx initially generates a large-amplitude transient that is crucial for negative regulation. Internal Ca²⁺ stores were revealed by discharging them with thapsigargin. But, in contrast to proposals that IP₃-sensitive stores mediate phototransduction, thapsigargin does not mimic or acutely interfere with photoexcitation. Finally, we identify a photoreceptor-specific PKC as essential for normal kinetics of [Ca²⁺]_i recovery.