Effects of Voltage-Gated Calcium Channel Subunit Genes on Calcium Influx in Cultured C. elegans Mechanosensory Neurons

ABSTRACT: Voltage-gated calcium channels (VGCCs) serve as a critical link between electrical signaling and diverse cellular processes in neurons. We have exploited recent advances in genetically encoded calcium sensors and in culture techniques to investigate how the VGCC a1 subunit EGL-19 and a2/d subunit UNC-36 affect the functional properties of C. elegans mechanosensory neu-rons. Using the protein-based optical indicator camel-eon, we recorded calcium transients from cultured mechanosensory neurons in response to transient depo-larization. We observed that in these cultured cells, cal-cium transients induced by extracellular potassium were significantly reduced by a reduction-of-function muta-tion in egl-19 and significantly reduced by L-type cal-cium channel inhibitors; thus, a main source of touch neuron calcium transients appeared to be influx of extracellular calcium through L-type channels. Transi-ents did not depend directly on intracellular calcium stores, although a store-independent 2-APB and gado-linium-sensitive calcium flux was detected. The transi-ents were also significantly reduced by mutations in unc-36, which encodes the main neuronal a2/d subunit in C. elegans. Interestingly, while egl-19 mutations resulted in similar reductions in calcium influx at all stimulus strengths, unc-36 mutations preferentially affected re-sponses to smaller depolarizations. These experiments suggest a central role for EGL-19 and UNC-36 in excit-ability and functional activity of the mechanosensor