Worm Breeder's Gazette 14(3): 50 (June 1, 1996)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

egl-19(n2368sd) may cause myotonia by affecting voltage-dependent inactivation of L-type Ca2+ channels

Raymond Lee, Leon Avery

Department of Biochemistry, UT-Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75235-9038

        We have shown previously that egl-19 (aka. eat-12, pat-5) plays
a key role in controlling muscle contraction and depolarization (Lee et
al., WBG 13(1): 45). Putative null mutations of egl-19 lead to a Pat
phenotype, suggesting that embryonic muscles are unable to contract.
Partial loss-of-function alleles show feeble contraction of pharyngeal,
body, and egg-laying muscles while gain-of-function alleles lead to
muscle hypercontraction. Based on the electrophysiological analysis of
the pharyngeal phenotype, we argue that these contraction defects can be
explained at least in part by defects in muscle excitation
(loss-of-function) and repolarization (gain-of-function).
        We have cloned egl-19 (Lobel et al., WBG 13(2): 71). It encodes
a polypeptide of 1,787 amino acids and shares more than 60% sequence
identity with the alpha1 subunit of L-type voltage-activated Ca2+
channels. To address the question of how egl-19 affects muscle
excitation, we localized its expression by constructing an egl-19::GFP
translational fusion. EGL-19::GFP was first detected in body muscles in
1-1/2-fold embryos, before the onset of embryonic rolling. This result
is consistent with the Pat phenotype seen in null mutants, suggesting a
cell-autonomous muscle defect. By hatching, GFP fluorescence is found in
pharyngeal muscles pm3, pm4, pm5, and pm7; in body muscles; and in the
anal depressor muscle. The muscle expression pattern is again consistent
with a muscle cell-autonomous defect caused by mutations. Interestingly,
however, we also found expression in the nervous system including the
pharyngeal neuron M4 and many neurons in the nerve ring, the ventral
nerve cord, and the pre-anal ganglion. We do not know the significance
of the apparent nervous system expression since most of the phenotypes
of egl-19 mutants are consistent with defects of muscle function. (This
is not surprising, as failure of muscle would hide most nervous system
phenotypes.) One exception is that two gain-of-function alleles have a
Daf-d phenotype. Although some dauers do form, their modification
appears incomplete as they are less resistant to SDS treatment. We could
not detect expression in egg-laying muscles. It may be that the level is
too low, or that the promoter fragment we used lacks elements for vulval
muscle expression.
        In order to learn at a molecular level how gain-of-function
mutations of egl-19 affect muscle repolarization, we sequenced the
coding regions of ad695sd and n2368sd. We did not find any mutation in
the ORF of ad695sd. One possiblity is that ad695sd may affect gene
expression. In egl-19(n2368sd), however, we found a G->A mutation in the
putative 6th transmembrane domain of repeat I (IS6). This mutation would
change a conserved glycine residue to an arginine. The IS6 region has
previously been shown to determine the rate of voltage-dependent
inactivation of L-type channels expressed in Xenopus oocytes (Zhang et
al., Nature 372: 97). The myotonic phenotype (delayed
relaxation/repolarization) of n2368sd can be reasonably explained by
slower inactivation of muscle Ca2+ channels.