A Drosophila Mechanosensory Transduction Channel
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Unless indicated otherwise all wild-type recordings were made from the Canton S strain. Other strains and stocks were obtained from either the Bloomington Drosophila Stock Center J. Szidonya or L. Lorenz or were generated in our laboratory. Mechanosensory mutants were prepared for recording and pipette solutions were as described (7). In this preparation we clamped the transepithelial potential (TEP) at +40 mV the average TEP for wild-type bristles. The recording configuration consisted of two electrodes: a reference electrode placed in the thorax of the fly and a recording/stimulation electrode slipped over the end of a cut bristle thus making a circuit across the sensory epithelium through the hollow bristle. Voltage and current responses were measured through a modified headstage with a voltage-clamp amplifier (AxoPatch 1-D Axon Instruments Foster City CA) in either current- or voltage-clamp mode with series resistance and capacitance compensation. Responses were low-pass filtered at half-power frequencies of 100 to 10 000 Hz before sampling at intervals of 6 to 2000 μs. After subtraction of 4-mV liquid junction potential the resting potential across the mechanosensory epithelium was on average 41 mV. Movements of the recording electrode were driven by a piezoelectrical stage (PZS-100HS Burleigh Instruments Fishers NY). To eliminate mechanical resonance of the pipette input signals driving the piezoelectrical device were low-pass filtered at a half-power frequency of 100 Hz (10 kHz for Fig. 2C; 1 kHz for action potentials) with an eight-pole Bessel filter (Model 3282 Krohn-Hite Avon MA). In Figs. 1 through 7 the stimulus trace represents the driving voltage to the piezoelectric device. Displacements of the stimulus probe were calibrated with an etched micrometer grid. Bristles were displaced over a range of ±35 μm. The bristle position faithfully followed that of the stimulating electrode.
R. G. Walker T. A. Keil C. S. Zuker unpublished observations.
M. Kernan D. Cowan C. Zuker unpublished results.
nompC corresponds to l(2)25Dc. DNA cloning sequencing characterization of mutant alleles and Drosophila melanogaster (Dm) transformations were performed as described by
. nompC cDNAs were identified with a 0.7-kb probe from exon 12 to screen an antennal cDNA library. Ce-nompC gene structure and protein sequence were predicted by the program FGENESH and modified by the deletion of a 70–amino acid sequence (the end of exon 8 and the beginning of exon 9) which introduced a hydrophobic segment in the midst of an ANK repeat that was inconsistent with Dm-NOMPC's structure. Extension of exon 19 by 54 base pairs produced 18 additional amino acid residues with homology to Dm-NOMPC followed by three stop codons. A translational fusion of Ce-NOMPC and GFP was constructed with a GFP expression vector pPD95.81. A 6.2-kb Ce-nompC sequence was amplified by long-range PCR from genomic DNA (wild-type strain N2) with a primer 4.5 kb upstream of the presumptive initiator methionine and a primer corresponding to the end of the third exon. Clones were sequenced at the site of insertion to ensure proper orientation of the insert within the vector. Germ line transformation was performed as described (15). Worms from six independent transgenic lines were viewed by fluorescence microscopy; cell position and morphology were used to identify neurons.
Single-letter abbreviations for the amino acid residues are as follows: A Ala; C Cys; D Asp; E Glu; F Phe; G Gly; H His; I Ile; K Lys; L Leu; M Met; N Asn; P Pro; Q Gln; R Arg; S Ser; T Thr; V Val; W Trp; and Y Tyr.
See Web fig. 1 available at www.sciencemag.org/feature/data/1048845.shl.
R. G. Walker and C. S. Zuker unpublished observations.
E. R. Sawin thesis Massachusetts Institute of Technology (1996).
Using a variety of screening strategies we searched genomic and cDNA libraries for homologs of NOMPC but did not identify additional related molecules. Searches of the >90%-complete Drosophila genomic sequence also failed to identify NOMPC homologs. Additional related channels however may reside in the unsequenced gaps or a different type of channel may mediate the current remaining in nompC mutants.
We acknowledge M. Kernan for introducing us to Drosophila mechanotransduction and for his pioneering and inspiring genetic studies including the isolation of nomp mutants. We thank K. Jalink for assistance in constructing the voltage-clamp apparatus J. Szidonya and L. Lorenz for providing fly stocks T. Keil for electron microscopy R. Terracol for sharing her chromosomal walk and L. Vosshall for providing an antennal cDNA library. We also thank K. Scott for her invaluable assistance with in situ hybridizations and C. Bargmann and D. Tobin for their hospitality expertise and advice with the C. elegans experiments. S. Emr P. Gillespie K. Scott R. Tsien and members of our laboratory kindly proffered constructive comments on the manuscript. R.G.W. is a postdoctoral fellow of the American Cancer Society (PF-4470); A.T.W. was supported by NIH training grant 5T32GM08107. C.S.Z. is an Investigator of the Howard Hughes Medical Institute.