Program of the 2001 International Worm Meeting - Sternberg Lab ...

821
821. Effect of the alpha 2 Ca 2+
channel subunit UNC-36 on calcium
transients in pharyngeal muscles.
Christian Frøkjær-Jensen 1,2 , Rex
Kerr 1 , Roger Y. Tsien 3,4 , William R.
Schafer 1
1 Division of Biology, University of California,
San Diego, La Jolla, California 92093
2 Niels Bohr Institute, University of
Copenhagen, DK-2100, Copenhagen
3 Division of Pharmacology, University of
California, San Diego, La Jolla, California
92093
4 Howard Hughes Medical Institute, University
of California, San Diego, La Jolla, California
92093
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
α2 subunit encoded by unc-36. unc-36 has
several loss of function alleles and encodes an
85kD protein with 28% similarity to the human
α2/δ1 subunit CACNA2D1. Loss of function
alleles of unc-36 produce a moderately severe
uncoordinated phenotype and reduced rates of
pharyngeal pumping, indicating that the α2
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,
821
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 α2 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.