Studies have shown that voltage gated calcium channels (VGCCs) are exquisitely regulated by a number of factors including modification or modulation of the pore forming 1-subunit. We are using C. elegans to identify novel genes that modulate the activity of VGCCs. We have focused our analysis on the predicted high voltage activated channel 1-subunit encoded by the
unc-2 gene and the low voltage activated (LVA) channel encoded by
cca-1. Previous genetic and pharmacological studies have shown
unc-2 to be involved in a number of neural processes consistent with a presynaptic role. Transgenic animals that express an
unc-2::GFP construct show that gene expression is restricted to the nervous system, with the exception of specific pharyngeal muscle cells. Sequence analysis of 17 independent
unc-2 mutants identified nine nonsense mutations and eight missense mutations. By using genetic and behavioral analyses we have been able to define an allelic series of the missense mutants. One approach we used was to examine the effects of different
unc-2 mutant backgrounds on the asymmetric expression of an
str-2::GFP array in the AWC sister sensory neurons.
unc-2 nonsense mutants consistently exhibited a 50% loss of asymmetric expression, whereas different missense mutants showed a variety of
str-2::GFP expression patterns ranging from 4 to 80%. Those alleles that showed loss of asymmetric expression greater than 50% are interpreted to represent recessive gain of function mutations. We are now attempting to examine whether the
unc-2 missense mutants affect the electrophysiological behaviour of UNC-2. Mutations in
unc-36, which encodes an 2-VGCC subunit, caused a 95% reduction in asymmetric
str-2::GFP expression suggesting that other 1-subunit genes may participate in this calcium dependent pathway. However, when
str-2::GFP expression was examined in other 1-subunit encoding mutants asymmetric expression was unaffected. This finding suggests that
unc-36 may function in roles other than to modulate the activity of VGCC 1-subunits such as
unc-2. Regulation of LVA or T-type VGCC activity is largely unknown. Analysis of two deletion mutants in the LVA 1-subunit gene
cca-1 have shown that the gene is required for controlling pharyngeal pumping. Consistent with this finding is that GFP fluorescence is observed in the pharyngeal MC neuron in worms that express a
cca-1::GFP construct. Genetic analysis of
unc-2 cca-1 double mutants suggests that these genes may act in some of the same pathways including defecation and egg laying. (Funded by the CIHR).