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 alpha1 subunit EGL-19 and alpha2/delta subunit UNC-36 affect the functional properties of C. elegans mechanosensory neurons. Using the protein-based optical indicator cameleon, we recorded calcium transients from cultured mechanosensory neurons in response to transient depolarization. We observed that in these cultured cells, calcium transients induced by extracellular potassium were significantly reduced by a reduction-of-function mutation in egl-19 and significantly reduced by L-type calcium channel inhibitors; thus, a main source of touch neuron calcium transients appeared to be influx of extracellular calcium through L-type channels. Transients did not depend directly on intracellular calcium stores, although a store-independent 2-APB and gadolinium-sensitive calcium flux was detected. The transients were also significantly reduced by mutations in unc-36, which encodes the main neuronal alpha2/delta 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 responses to smaller depolarizations. These experiments suggest a central role for EGL-19 and UNC-36 in excitability and functional activity of the mechanosensory neurons.

Touch sensitivity in Caenorhabditis elegans

“…Calcium imaging experiments of ALM neurons in vivo show that this influx is dependent on mec-4 and mec-2 as well as egl-19, which encodes an α-subunit of an L-type voltagegated calcium channel [82]. Imaging of cultured touch receptor neurons also suggests that UNC-36, an α 2 /δ subunit of L-type channels, may function as an accessory subunit to enhance calcium influx and voltage sensitivity [28]. The role of this calcium influx in response to the MRC is unclear.…”

“…The role of this calcium influx in response to the MRC is unclear. egl-19 reduction-of-function mutants and unc-36 null mutants are not fully touch sensitive; in addition, they have subtle defects in touch neuron migration and axon guidance that preclude attributing the cause of touch insensitivity to reduced calcium influx [28]. C. elegans lacks voltage-gated sodium channels, so voltage-gated calcium channels may contribute to the propagation of action potentials down the touch neuron process.…”

A Method for Culturing Embryonic <em>C. elegans</em> Cells

“…The functional role of voltage-gated Ca 2+ channels (VGCC) in neurons and glia has been investigated in cultured C. elegans cells by calcium imaging 20,21,41 . The application of calcium imaging in culture has allowed the use of solutions containing high concentrations of KCl to induce cell depolarization and of calcium channel blockers to discern the contribution of different types of channels to calcium influx.…”

Agarose Microchambers for Long-term Calcium Imaging of <em>Caenorhabditis elegans</em>

“…Calcium imaging provides a functional readout for the activity of excitable cells and is established for C. elegans [16][17][18][19][20] . Calcium enters the cell via channels in the plasma membrane that open upon depolarization.…”

“…While ratiometric sensors are less sensitive to movement or expression artifacts, non-ratiometric sensors typically have a higher dynamic range. Both ratiometric and non-ratiometric sensors have been useful to study the activity of excitable cells in C. elegans [16][17][18][19][20]23,24 .…”