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.
|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. [THE ORIGINAL DOCUMENT CONTAINS A FIGURE.]