Identification and Modulation of Voltage-Gated Ca2+Currents in Zebrafish Rohon-Beard Neurons

Abstract

Electrically excitable cells have voltage-dependent ion channels on the plasma membrane that regulate membrane permeability to specific ions. Voltage-gated Ca(2+) channels (VGCCs) are especially important as Ca(2+) serves as both a charge carrier and second messenger. Zebrafish (Danio rerio) are an important model vertebrate for studies of neuronal excitability, circuits, and behavior. However, electrophysiological properties of zebrafish VGCCs remain largely unexplored because a suitable preparation for whole cell voltage-clamp studies is lacking. Rohon-Beard (R-B) sensory neurons represent an attractive candidate for this purpose because of their relatively large somata and functional homology to mammalian dorsal root ganglia (DRG) neurons. Transgenic zebrafish expressing green fluorescent protein in R-B neurons, (Isl2b:EGFP)(ZC7), were used to identify dissociated neurons suitable for whole cell patch-clamp experiments. Based on biophysical and pharmacological properties, zebrafish R-B neurons express both high- and low-voltage-gated Ca(2+) current (HVA- and LVA-I(Ca), respectively). Ni(+)-sensitive LVA-I(Ca) occur in the minority of R-B neurons (30%) and ω-conotoxin GVIA-sensitive Ca(V)2.2 (N-type) Ca(2+) channels underlie the vast majority (90%) of HVA-I(Ca). To identify G protein coupled receptors (GPCRs) that modulate HVA-I(Ca), a panel of neurotransmitters was screened. Application of GABA/baclofen or serotonin produced a voltage-dependent inhibition while application of the mu-opioid agonist DAMGO resulted in a voltage-independent inhibition. Unlike in mammalian neurons, GPCR-mediated voltage-dependent modulation of I(Ca) appears to be transduced primarily via a cholera toxin-sensitive Gα subunit. These results provide the basis for using the zebrafish model system to understanding Ca(2+) channel function, and in turn, how Ca(2+) channels contribute to mechanosensory function.