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. 2017 Dec;31(4):325-336.
doi: 10.1080/01677063.2017.1393076. Epub 2017 Nov 9.

Generation and Characterization of New Alleles of Quiver (Qvr) That Encodes an Extracellular Modulator of the Shaker Potassium Channel

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Free PMC article

Generation and Characterization of New Alleles of Quiver (Qvr) That Encodes an Extracellular Modulator of the Shaker Potassium Channel

Hongyu Ruan et al. J Neurogenet. .
Free PMC article

Abstract

Our earlier genetic screen uncovered a paraquat-sensitive leg-shaking mutant quiver1 (qvr1), whose gene product interacts with the Shaker (Sh) K+ channel. We also mapped the qvr locus to EY04063 and noticed altered day-night activity patterns in these mutants. Such circadian behavioral defects were independently reported by another group, who employed the qvr1 allele we supplied them, and attributed the extreme restless phenotype of EY04063 to the qvr gene. However, their report adopted a new noncanonical gene name sleepless (sss) for qvr. In addition to qvr1 and qvrEY, our continuous effort since the early 2000s generated a number of novel recessive qvr alleles, including ethyl methanesulfonate (EMS)-induced mutations qvr2 and qvr3, and P-element excision lines qvrip6 (imprecise jumpout), qvrrv7, and qvrrv9 (revertants) derived from qvrEY. Distinct from the original intron-located qvr1 allele that generates abnormal-sized mRNAs, qvr2, and qvr3 had their lesion sites in exons 6 and 7, respectively, producing nearly normal-sized mRNA products. A set of RNA-editing sites are nearby the lesion sites of qvr3 and qvrEY on exon 7. Except for the revertants, all qvr alleles display a clear ether-induced leg-shaking phenotype just like Sh, and weakened climbing abilities to varying degrees. Unlike Sh, all shaking qvr alleles (except for qvrf01257) displayed a unique activity-dependent enhancement in excitatory junction potentials (EJPs) at larval neuromuscular junctions (NMJs) at very low stimulus frequencies, with qvrEY displaying the largest EJP and more significant NMJ overgrowth than other alleles. Our detailed characterization of a collection of qvr alleles helps to establish links between novel molecular lesions and different behavioral and physiological consequences, revealing how modifications of the qvr gene lead to a wide spectrum of phenotypes, including neuromuscular hyperexcitability, defective motor ability and activity-rest cycles.

Keywords: Ly-6/neurotoxin superfamily; RNA editing; Shaker; ether-induced leg shaking; potassium current inactivation; qvr/sss; sleepless; synaptic plasticity.

Figures

Figure 1
Figure 1. Abnormal motor behaviors in qvr and Sh flies
(A) Ether-induced leg shaking in qvr and Sh flies. qvrip6 displayed vigorous leg-shaking similar to Sh133, but qvrf0 allele showed only mild shaking. WT (CS) flies do not shake. Dark field photo micrograph, 50-ms exposure. (B, C) Weakened climbing ability in qvr flies. Number of replicates indicated in (C). 10 flies per trial, see methods. ***, **, and * indicate p < 0.001, 0.01, and 0.05 in comparison with CS in t-test with sequential Bonferroni adjustment for multiple comparisons. Error bars indicate SEMs.
Figure 2
Figure 2. Exon and sequence information of qvr alleles
(A) Exons and lesion sites of qvr locus. Locations of PCR primers, P1(EY04063) and P2(f01257) insertions are indicated. Open and filled arrows indicate the positions of the primer sets for RT-PCR and qRT-PCR shown in Figure 3B and 3C, respectively. Enlarged box: zoom-in view of exon 5, 6, 7 and part of 3′ untranslated region. Arrow heads indicate locations of the 5′, 3′, and flanking (F) primer sets used in Figure 3A. (B) The DNA sequence information for exons 6 and 7, with lesion sites of qvr1, qvr2, qvr3, and qvrEY indicated. Glycosylation, GPI anchor, and RNA-editing sites are also indicated.
Figure 3
Figure 3. Molecular characterization of qvr alleles
(A) Genomic PCR analysis for qvrEY and jumpout lines. The locations of 5′ end, 3′ end and flanking (F) primers are indicated in Figure 2A. (B) Reverse-transcriptase PCR results of different qvr alleles. Note 3 bands in qvr1 but a single band in qvr2 and qvr3. (C) qRT-PCR studies show the overexpression of qvr PCR product in qvr1, unaltered expression level in qvr2, increased expression in qvr3, and decreased expression in qvrEY as well as qvrip6. Three independent sets of experiments were done, with controls using CS, ry+5, and qvrrv7, respectively. Mutant data were normalized to controls. Number of replicates indicated. For the primers used in (B) and (C), see Figure 2A and Method.
Figure 4
Figure 4. Low-frequency use-dependent enhancement of transmitter release in qvr alleles
(A1 and A2) Representative EJP recordings over prolonged low-frequency (0.2–0.5 Hz) repetitive stimulation (A1) and high frequency (5–20 Hz, A2) for WT (CS), Sh133, Sh120, qvr1 and qvrEY (0.1 mM Ca2+). Recording durations and stimulus numbers are indicated in A1. Note the gradual augmentation rising to a plateau-like, steady-state level in qvr EJPs with both low (0.2 Hz) and higher frequency (5 Hz) of repetitive stimulation, whereas augmentation in WT occurred only after prolonged high-frequency (20 Hz) stimulation, toward the last 5 s of the 50-s stimulus train as shown in A2. The A2 display is a scanned reproduction of chart recorder traces with a limited temporal resolution of about 0.3 s. (B1 and B2) Expanded individual EJPs evoked at 1st, 5th, and 20th stimuli at 0.2–0.5 Hz (B1) and at the initial (0th) and 10th seconds of 5–20 Hz (B2) repetitive stimulation. Same data sets correspond to that shown in A1 and A2, respectively. Note that WT EJPs only displayed quantal fluctuations in response to low-frequency stimulation (0.5 Hz), and that qvrEY showed the greatest 1st EJP among qvr alleles. (C) Summary statistics of 1st EJP (i.e. without prior stimulation, filled bars) and steady-state EJP (st-st EJP, open bars), measured from responses to low-frequency (0.2–0.5 Hz) repetitive stimulation of the indicated genotypes (see A1 traces for example). Significant differences between the 1st and st-st EJPs were found in qvr alleles (*, p < 0.05; **, p < 0.01; ***, p < 0.001, paired Students’ t-tests with Bonferroni correction), but not in WT, Sh alleles, qvr revertants (qvrrv7 and qvrrv9), and qvrf01257 (qvrF0). Among all qvr alleles, qvrEY displayed the greatest 1st EJP (+, p < 0.05, one-way ANOVA). Error bars indicate SEMs.
Figure 5
Figure 5. Mild synaptic overgrowth in qvr alleles
(A) Sample images of anti-HRP immunostaining of NMJs in muscle 12 and 13 of 3rd instar larvae. Scale bar: 50 μm. (B) Type Ib bouton counts for muscle 12 and 13 NMJs. Filled bars indicate control lines. One way ANOVA and Tukey HSD tests were performed. * p < 0.05. Error bars indicate SEMs.
Figure 6
Figure 6. Electrophysiological recordings from amputated legs demonstrating spiking activity correlated with ether-induced shaking
(A) Recording configuration. See Methods for description. (B-D) Representative traces from WT, Sh133, and qvr2. Note the rhythmic firing (~20 Hz) in Sh133 and qvr2. Dots indicate action potential spikes.

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