Voltage-gated calcium channels are protein complexes that mediate calcium transients essential for muscle contraction and synaptic transmission. Although the properties of the pore-forming alpha-1 subunit have been studied extensively in vertebrate systems, less is known about the role of the associated subunits. We are using recently developed calcium imaging techniques to investigate the effects of loss of function in one of these subunits, the putative a 2 subunit encoded by
unc-36 .
unc-36 has several loss of function alleles and encodes an 85kD protein with 28% similarity to the human a 2/ d 1 subunit CACNA2D1. Loss of function alleles of
unc-36 produce a moderately severe uncoordinated phenotype and reduced rates of pharyngeal pumping, indicating that the a 2 subunit is functionally important in C. elegans . Although other studies have implicated a role for
unc-36 in neurons, we are initially characterizing the role of
unc-36 in the pharyngeal muscles. Preliminary GFP expression data indicates that
unc-36 is expressed in the pharyngeal muscles. We have chosen the pharynx as our model system because it is well-characterized electrically and pharmacologically. We are using the genetically encoded cameleon calcium sensor driven by the pharyngeal-specific
myo-2 promoter to record calcium transients in intact worms. We quantify the pumping behavior by measuring the pumping frequency, the duration of each transient, and the rate of increase in the calcium. In the two alleles we have tested,
unc-36(
e251) and
unc-36(
ad698) , we found that the duration of transients is similar to that of wild-type but that the rate of calcium increase is 30-40% greater. This suggests that the role of
unc-36 on the pharynx is to reduce the calcium flux. The simplest explanation is that the pharyngeal channels are decreased in number or conductivity in the presence of UNC-36 and suggests a possible negative regulatory role for the a 2 subunit in muscles. Alternatively, UNC-36 could be acting directly in neurons and the observed effect in muscle could be an acute or developmental response to the altered neuronal input. The reduced rate of pumping in
unc-36 mutants is consistent with this hypothesis: the pharyngeal nervous system is required for rapid pharyngeal pumping. This suggests that UNC-36 may facilitate the activity of calcium channels in neurons. To distinguish between these two hypotheses, we are eliminating synaptic input by conducting experiments in a
snt-1 background.
snt-1 encodes the only known synaptotagmin in C. elegans and is thus thought to be essential for evoked neurotransmitter release. Experiments on
unc-36 mutants in this background should allow us to distinguish between the effect of UNC-36 in muscle and in the nervous system. Results from these experiments can be verified by laser ablation of the MC neuron, the only neuron in the pharyngeal nervous system responsible for inducing rapid pharyngeal pumping. We also intend to rescue the
unc-36 mutants with the pharynx specific
myo-2::
unc-36 construct to further characterize the effect UNC-36 in the nervous system.