Worm Breeder's Gazette 14(5): 38 (February 1, 1997)

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-36 mutations reveal a new role for S6 in voltage-gated potassium channels

Duncan Johnstone1, Aguan Wei2, Lawrence Salkoff2, James H. Thomas1

1 Program in Molecular and Cellular Biology and Department of Genetics, University of Washington, Box 357360, Seattle, WA 98195
2 Department of Anatomy and Neurobiology and Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110

We have been examining two gain-of-function Mac-d mutations (muscle
activation defective) in egl-36, n728 and n2332, which confer defects in
egg-laying and enteric muscle contraction (1).  Both alleles are
phenotypically similar, affecting the same tissues with approximately
the same severity.  We refined the map position of both alleles to the
interval between vab-3 and egl-15 on the X chromosome.  Using sequence
information from the genome consortium, we noted that a predicted
potassium channel, R07A4.1, lies within this interval.  We hypothesized
that the defects in egl-36 mutants could result from constitutive
activation of a potassium channel, leading to inappropriate potassium
efflux and suppression of muscle excitation.  As described below, by
sequencing DNA from egl-36 mutants we have established that egl-36
encodes this potassium channel, a member of the voltage-gated Shaw
subfamily (Kv3.1 in mammals).

The two dominant mutations in egl-36 cause amino acid substitutions. 
The n2332 mutation results in a P435>S substitution in the predicted
sixth transmembrane domain (S6).  This proline is conserved in all known
Shaw family members from C. elegans to humans.  The n728 mutation
causes an E138>K substitution within the cytoplasmic T1 association
domain.  The T1 domain is thought to mediate multimerization between
members each voltage-gated subfamily (2).  This residue is either E or D
in all known Shaw subfamily members.

We have reverted the Egl phenotype in both egl-36(n2332) and
egl-36(n728).  We isolated two revertants of n2332, saS57 and sa629, in
an F1 screen of 30,000 mutagenized haploid genomes.  Both mutations also
revert the Exp phenotype.  In preliminary analysis, the phenotype
of these egl-36(lf) alleles is grossly wild type.  sa577 results in a
stop codon before the first transmembrane domain and thus is probably a
null mutation.  sa629 results in a stop within the extracellular loop
between the S5 and pore segments.  We isolated two wild type revertants
of n728, sa630 and sa631, in an F2 screen of 3400 mutagenized haploid
genomes.  Both are tightly linked to egl-36(n728), and we are currently
determining their sequence changes.

We have examined the macroscopic current of the egl-36 K+ channel by
cRNA injection into Xenopus oocytes.  We find that egl-36 forms
functional homomultimeric K+ channels that require strong depolarization
to open (V50=+70mV, see figure), and have a slow rate of activation
and no inactivation.  Preliminary analysis at the single channel level
shows a large unitary conductance (30-70 pS) with brief open times,
similar to Drosophila Shaw.  Using site-directed mutagenesis, we
generated an n2332-bearing cDNA construct and examined its properties. 
The effects of n2332 on channel activity are surprising and indicate a
new role for S6 in channel regulation.  First, the voltage sensitivity
of channel activation is shifted dramatically to more negative
potentials (V50=+23mV, see figure).  Second, the rate of channel
activation is extremely rapid when compared to the wild type channel. 
Mutations in S6 previously have been reported to affect unitary
conductance, inactivation and ion selectivity, but not voltage
sensitivity or the kinetics of activation (3).  Moreover, a negative
shift in voltage sensitivity is a highly unusual result, and is
particularly surprising for a region not previously thought to be
involved in voltage sensitivity.  We speculate that the P435>S
substitution in n2332 is stabilizing the open state of the Shaw channel,
which normally is characterized at the single channel level by brief
openings.  The observed negative shift in voltage sensitivity and
increased rate of channel activation are consistent with our initial
hypothesis that egl-36(gof) mutations cause inappropriate and excessive
potassium currents in egg-laying and enteric muscle membrane.

(1) Reiner, D.J. et al. (1995) Genetics 141:961. Trent, C. et al. (1983) 
    Genetics 104:619.
(2) Shen, N.V. and Pfaffinger, P.J. (1995) Neuron 14:625.
(3) Sather, W.A. et al. (1994) Curr Op in Neurobio 4:313. Kukulgan, M.   
    et al. (1995) Am J of Physiology 268:C535.