Shaker Gene Research Articles

Identification and Characterization of Shaker Potassium Channel Gene Family and Response to Salt and Chilling Stress in Rice.

Shaker potassium channel proteins are a class of voltage-gated ion channels responsible for K+ uptake and translocation, playing a crucial role in plant growth and salt tolerance. In this study, bioinformatic analysis was performed to identify the members within the Shaker gene family. Moreover, the expression patterns of rice Shaker(OsShaker) K+ channel genes were analyzed in different tissues and salt treatment by RT-qPCR. The results revealed that there were eight OsShaker K+ channel genes distributed on chromosomes 1, 2, 5, 6 and 7 in rice, and their promoters contained a variety of cis-regulatory elements, including hormone-responsive, light-responsive, and stress-responsive elements, etc. Most of the OsShaker K+ channel genes were expressed in all tissues of rice, but at different levels in different tissues. In addition, the expression of OsShaker K+ channel genes differed in the timing, organization and intensity of response to salt and chilling stress. In conclusion, our findings provide a reference for the understanding of OsShaker K+ channel genes, as well as their potential functions in response to salt and chilling stress in rice.

Genome-wide identification of kiwifruit K+ channel Shaker family members and their response to low-K+ stress

Background‘Hongyang’ kiwifruit (Actinidia chinensis cv ‘Hongyang’) is a high-quality variety of A. chinensis with the advantages of high yield, early ripening, and high stress tolerance. Studies have confirmed that the Shaker K+ genes family is involved in plant uptake and distribution of potassium (K+).ResultsTwenty-eight Shaker genes were identified and analyzed from the ‘Hongyang’ kiwifruit (A. chinensis cv ‘Hongyang’) genome. Subcellular localization results showed that more than one-third of the AcShaker genes were on the cell membrane. Phylogenetic analysis indicated that the AcShaker genes were divided into six subfamilies (I-VI). Conservative model, gene structure, and structural domain analyses showed that AcShaker genes of the same subfamily have similar sequence features and structure. The promoter cis-elements of the AcShaker genes were classified into hormone-associated cis-elements and environmentally stress-associated cis-elements. The results of chromosomal localization and duplicated gene analysis demonstrated that AcShaker genes were distributed on 18 chromosomes, and segmental duplication was the prime mode of gene duplication in the AcShaker family. GO enrichment analysis manifested that the ion-conducting pathway of the AcShaker family plays a crucial role in regulating plant growth and development and adversity stress. Compared with the transcriptome data of the control group, all AcShaker genes were expressed under low-K+stress, and the expression differences of three genes (AcShaker15, AcShaker17, and AcShaker22) were highly significant. The qRT-PCR results showed a high correlation with the transcriptome data, which indicated that these three differentially expressed genes could regulate low-K+ stress and reduce K+ damage in kiwifruit plants, thus improving the resistance to low-K+ stress. Comparison between the salt stress and control transcriptomic data revealed that the expression of AcShaker11 and AcShaker18 genes was significantly different and lower under salt stress, indicating that both genes could be involved in salt stress resistance in kiwifruit.ConclusionThe results showed that 28 AcShaker genes were identified and all expressed under low K+ stress, among which AcShaker22 was differentially and significantly upregulated. The AcShaker22 gene can be used as a candidate gene to cultivate new varieties of kiwifruit resistant to low K+ and provide a reference for exploring more properties and functions of the AcShaker genes.

Targeting Mosquitoes through Generation of an Insecticidal RNAi Yeast Strain Using Cas-CLOVER and Super PiggyBac Engineering in Saccharomyces cerevisiae.

The global deployment of RNAi yeast insecticides involves transitioning from the use of laboratory yeast strains to more robust strains that are suitable for scaled fermentation. In this investigation, the RNA-guided Cas-CLOVER system was used in combination with Piggybac transposase to produce robust Saccharomyces cerevisiae strains with multiple integrated copies of the Sh.463 short hairpin RNA (shRNA) insecticide expression cassette. This enabled the constitutive high-level expression of an insecticidal shRNA corresponding to a target sequence that is conserved in mosquito Shaker genes, but which is not found in non-target organisms. Top-expressing Cas-CLOVER strains performed well in insecticide trials conducted on Aedes, Culex, and Anopheles larvae and adult mosquitoes, which died following consumption of the yeast. Scaled fermentation facilitated the kilogram-scale production of the yeast, which was subsequently heat-killed and dried. These studies indicate that RNAi yeast insecticide production can be scaled, an advancement that may one day facilitate the global distribution of this new mosquito control intervention.

Potassium ion channel gene family provides new insights into powdery mildew responses in Triticum aestivum

AbstractThe chromosomal localization, gene structure, transmembrane proteins, and transcriptome of the wheat Shaker family genes were analysed. The expression patterns of wheat Shaker genes under different stresses were investigated using reverse transcription quantitative PCR (RT‐qPCR). The effect of potassium ion treatment on wheat disease severity caused by powdery mildew was evaluated. To further investigate whether wheat Shaker genes also have inhibitory effects on other pathogens, Nicotiana benthamiana transiently overexpressing Shaker genes was inoculated with Phytophthora infestans and the effects on P. infestans infection judged by leaf lesion diameter. A total of 25 Shaker genes were identified in wheat (TaShaker genes) and were classified into four groups based on phylogenetic analysis. Chromosome mapping results indicated that the 25 TaShaker genes were distributed on 14 chromosomes in wheat (2B, 4A, 4B, 4D, 5A, 5B, and 5D). Gene structure analysis revealed the untranslated region structures were missing in some TaShaker genes, with the number of introns ranging from 7 to 11. Protein transmembrane prediction analysis indicated that there were multiple TaShaker transmembrane proteins. The results of the RT‐qPCR analysis indicated that TaShaker genes were not only involved in regulating drought stress, but could also resist infection by Fusarium graminearum and powdery mildew. This study also found that spraying potassium ion solutions at the seedling stage could reduce the severity of powdery mildew infection in wheat. Moreover, transient overexpression of TaShaker5.1‐3A in N. benthamiana leaves could inhibit P. infestans infection.

Potassium channel Shaker play a protective role against cardiac aging in Drosophila.

Potassium channels, which are the most diverse group of the ion channel family, play an important role in the repolarization of cardiomyocytes. Recent studies showed that potassium channels, such as KCNQ and HERG/eag, play an important role in regulating adult heart function through shaping the action potential and maintaining the rhythm of cardiac contraction. The potassium channel protein Shaker is the first voltage-gated potassium channel found in Drosophila to maintain the electrical excitability of neurons and muscle cells, but its role in adult cardiac function is still unclear. In this study, Drosophila was used as a model to study the role of Shaker channel in the maintenance of cardiac function under stress and aging. The incidence of heart failure was observed in shaker mutant after external electrical pacing, which simulates cardiac stress. Additionally, The cardiac-specific driver hand4.2 Gal4 was used to specifically knock down the expression of the potassium channel shaker in Drosophila. The cardiac parameter was analyzed at 1, 3, 5 weeks of age on cardiac specific knockdown of shaker using Drosophila adult cardiac physiological assay. The results showed that the mutation of shaker gene seriously affect the cardiac function under stress, demonstrated by significant increase in heart failure rate under electrical stimulation. In addition, cardiac specific knockdown of shaker increased the incidence of arrhythmias in Drosophila at the age of 5 weeks. Cardiac-specific knockdown of shaker reduces life span. Therefore, the results of this study suggest a vital role of the potassium channel shaker in maintaining normal cardiac function during aging.

Characterization of a dual-action adulticidal and larvicidal interfering RNA pesticide targeting the Shaker gene of multiple disease vector mosquitoes

The existing mosquito pesticide repertoire faces great challenges to sustainability, and new classes of pesticides are vitally needed to address established and emerging mosquito-borne infectious diseases. RNA interference- (RNAi-) based pesticides are emerging as a promising new biorational mosquito control strategy. In this investigation, we describe characterization of an interfering RNA pesticide (IRP) corresponding to the mosquito Shaker (Sh) gene, which encodes an evolutionarily conserved voltage-gated potassium channel subunit. Delivery of the IRP to Aedes aegypti adult mosquitoes in the form of siRNA that was injected or provided as an attractive toxic sugar bait (ATSB) led to Sh gene silencing that resulted in severe neural and behavioral defects and high levels of adult mortality. Likewise, when provided to A. aegypti larvae in the form of short hairpin RNA (shRNA) expressed in Saccharomyces cerevisiae (baker’s yeast) that had been formulated into a dried inactivated yeast tablet, the yeast IRP induced neural defects and larval death. Although the Sh IRP lacks a known target site in humans or other non-target organisms, conservation of the target site in the Sh genes of multiple mosquito species suggested that it may function as a biorational broad-range mosquito insecticide. In support of this, the Sh IRP induced both adult and larval mortality in treated Aedes albopictus, Anopheles gambiae, and Culex quinquefasciatus mosquitoes, but was not toxic to non-target arthropods. These studies indicated that IRPs targeting Sh could one day be used in integrated biorational mosquito control programs for the prevention of multiple mosquito-borne illnesses. The results of this investigation also suggest that the species-specificity of ATSB technology, a new paradigm for vector control, could be enhanced through the use of RNAi-based pesticides.

Hypnotic effects of a novel anti-insomnia formula on Drosophila insomnia model.

To assess the biological effects of the six-herb mixture Anti-Insomia Formula (AIF) extract using caffeine-induced insomnia Drosophila model and short-sleep mutants. Caffeineinduced insomnia wild-type Drosophila and short-sleep mutant flies minisleep (mns) and Hyperkinetic(Y) (Hk(Y)) were used to assess the hypnotic effects of the AIF in vivo. The night time activity, the amount of night time sleep and the number of sleep bouts were determined using Drosophila activity monitoring system. Sleep was defined as any period of uninterrupted behavioral immobility (0 count per minute) lasting > 5 min. Night time sleep was calculated by summing up the sleep time in the dark period. Number of sleep bouts was calculated by counting the number of sleep episodes in the dark period. AIF at the dosage of 50 mg/mL, effectively attenuated caffeine-induced wakefulness (P<0.01) in wild-type Canton-S flies as indicated by the reduction of the sleep bouts, night time activities and increase of the amount of night time sleep. AIF also significantly reduced sleeping time of short-sleep Hk(Y) mutant flies (P<0.01). However, AIF did not produce similar effect in mns mutants. AIF might be able to rescue the abnormal condition caused by mutated modulatory subunit of the tetrameric potassium channel, but not rescuing the abnormal nerve firing caused by Shaker gene mutation. This study provides the scientific evidence to support the use of AIF in Chinese medicine for promoting sleep quality in insomnia.

Expanded Functional Diversity of Shaker K+ Channels in Cnidarians Is Driven by Gene Expansion

The genome of the cnidarian Nematostella vectensis (starlet sea anemone) provides a molecular genetic view into the first nervous systems, which appeared in a late common ancestor of cnidarians and bilaterians. Nematostella has a surprisingly large and diverse set of neuronal signaling genes including paralogs of most neuronal signaling molecules found in higher metazoans. Several ion channel gene families are highly expanded in the sea anemone, including three subfamilies of the Shaker K+ channel gene family: Shaker (Kv1), Shaw (Kv3) and Shal (Kv4). In order to better understand the physiological significance of these voltage-gated K+ channel expansions, we analyzed the function of 18 members of the 20 gene Shaker subfamily in Nematostella. Six of the Nematostella Shaker genes express functional homotetrameric K+ channels in vitro. These include functional orthologs of bilaterian Shakers and channels with an unusually high threshold for voltage activation. We identified 11 Nematostella Shaker genes with a distinct “silent” or “regulatory” phenotype; these encode subunits that function only in heteromeric channels and serve to further diversify Nematostella Shaker channel gating properties. Subunits with the regulatory phenotype have not previously been found in the Shaker subfamily, but have evolved independently in the Shab (Kv2) family in vertebrates and the Shal family in a cnidarian. Phylogenetic analysis indicates that regulatory subunits were present in ancestral cnidarians, but have continued to diversity at a high rate after the split between anthozoans and hydrozoans. Comparison of Shaker family gene complements from diverse metazoan species reveals frequent, large scale duplication has produced highly unique sets of Shaker channels in the major metazoan lineages.

The LIM-Homeodomain Protein Islet Dictates Motor Neuron Electrical Properties by Regulating K+ Channel Expression

SummaryNeuron electrical properties are critical to function and generally subtype specific, as are patterns of axonal and dendritic projections. Specification of motoneuron morphology and axon pathfinding has been studied extensively, implicating the combinatorial action of Lim-homeodomain transcription factors. However, the specification of electrical properties is not understood. Here, we address the key issues of whether the same transcription factors that specify morphology also determine subtype specific electrical properties. We show that Drosophila motoneuron subtypes express different K+ currents and that these are regulated by the conserved Lim-homeodomain transcription factor Islet. Specifically, Islet is sufficient to repress a Shaker-mediated A-type K+ current, most likely due to a direct transcriptional effect. A reduction in Shaker increases the frequency of action potential firing. Our results demonstrate the deterministic role of Islet on the excitability patterns characteristic of motoneuron subtypes.

Seymour Benzer 1921–2007

Seymour Benzer 1921–2007

Reduced sleep in Drosophila Shaker mutants

Most of us sleep 7-8 h per night, and if we are deprived of sleep our performance suffers greatly; however, a few do well with just 3-4 h of sleep-a trait that seems to run in families. Determining which genes underlie this phenotype could shed light on the mechanisms and functions of sleep. To do so, we performed mutagenesis in Drosophila melanogaster, because flies also sleep for many hours and, when sleep deprived, show sleep rebound and performance impairments. By screening 9,000 mutant lines, we found minisleep (mns), a line that sleeps for one-third of the wild-type amount. We show that mns flies perform normally in a number of tasks, have preserved sleep homeostasis, but are not impaired by sleep deprivation. We then show that mns flies carry a point mutation in a conserved domain of the Shaker gene. Moreover, after crossing out genetic modifiers accumulated over many generations, other Shaker alleles also become short sleepers and fail to complement the mns phenotype. Finally, we show that short-sleeping Shaker flies have a reduced lifespan. Shaker, which encodes a voltage-dependent potassium channel controlling membrane repolarization and transmitter release, may thus regulate sleep need or efficiency.

Structural Requirements for Rapid Inactivation and Voltage Dependence in Splice Variants of Lobster Shaker Potassium Channels

We studied the properties of currents generated in Xenopus oocytes by nine splice variants of the spiny lobster Shaker gene. These isoforms differ in their amino termini and in the P-loop region of the pore. Both the voltage dependence and kinetic properties of the currents varied significantly, depending on which amino terminus was present. A cluster of net positive charges at the N-terminus was not necessary for rapid inactivation: negatively charged N-termini also inactivated rapidly. There was no obvious correlation between N-terminus length and inactivation rate. These N-terminal effects were additive with a separate set of voltage and kinetic properties controlled by the two alternative P-loop exons.

Regulation of K+ channel activities in plants: from physiological to molecular aspects.

Plant voltage-gated channels belonging to the Shaker family participate in sustained K+ transport processes at the cell and whole plant levels, such as K+ uptake from the soil solution, long-distance K+ transport in the xylem and phloem, and K+ fluxes in guard cells during stomatal movements. The attention here is focused on the regulation of these transport systems by protein-protein interactions. Clues to the identity of the regulatory mechanisms have been provided by electrophysiological approaches in planta or in heterologous systems, and through analogies with their animal counterparts. It has been shown that, like their animal homologues, plant voltage-gated channels can assemble as homo- or heterotetramers associating polypeptides encoded by different Shaker genes, and that they can bind auxiliary subunits homologous to those identified in mammals. Furthermore, several regulatory processes (involving, for example, protein kinases and phosphatases, G proteins, 14-3-3s, or syntaxins) might be common to plant and animal Shakers. However, the molecular identification of plant channel partners is still at its beginning. This paper reviews current knowledge on plant K+ channel regulation at the physiological and molecular levels, in the light of the corresponding knowledge in animal cells, and discusses perspectives for the deciphering of regulatory networks in the future.

Blockage of one class of potassium channel alters the effectiveness of halothane in a brain circuit of Drosophila.

At concentrations comparable to those used in the clinic, halothane has profound effects on a neuronal pathway devoted to the escape reflex of the fruit fly, Drosophila melanogaster. We studied the influence of the potassium channel that is encoded by the Shaker gene on the halothane sensitivity of this circuit. Shaker channels were specifically inactivated either by genetic means, using strains with two different severe Shaker mutations, or by pharmacologic means, using ingestion of millimolar concentrations of 4-aminopyridine. In all cases, halothane potency decreased substantially. To ensure that the genetic alteration was specific, both mutations were studied as stocks that had been repeatedly backcrossed to a control strain. The specificity of the pharmacologic inhibition was demonstrated by the fact that 4-aminopyridine had no effect on halothane potency in a Shaker mutant. Quantitative differences in the effects of channel inhibition between males and females suggested a sexual dimorphism in the functional brain anatomy of the reflex circuit.

Blockage of One Class of Potassium Channel Alters the Effectiveness of Halothane in a Brain Circuit of Drosophila

At concentrations comparable to those used in the clinic, halothane has profound effects on a neuronal pathway devoted to the escape reflex of the fruit fly, Drosophila melanogaster. We studied the influence of the potassium channel that is encoded by the Shaker gene on the halothane sensitivity of this circuit. Shaker channels were specifically inactivated either by genetic means, using strains with two different severe Shaker mutations, or by pharmacologic means, using ingestion of millimolar concentrations of 4-aminopyridine. In all cases, halothane potency decreased substantially. To ensure that the genetic alteration was specific, both mutations were studied as stocks that had been repeatedly backcrossed to a control strain. The specificity of the pharmacologic inhibition was demonstrated by the fact that 4-aminopyridine had no effect on halothane potency in a Shaker mutant. Quantitative differences in the effects of channel inhibition between males and females suggested a sexual dimorphism in the functional brain anatomy of the reflex circuit.

Overexpression of a Shaker-type potassium channel in mammalian central nervous system dysregulates native potassium channel gene expression.

The nervous system maintains a delicate balance between excitation and inhibition, partly through the complex interplay between voltage-gated sodium and potassium ion channels. Because K+ channel blockade or gene deletion causes hyperexcitability, it is generally assumed that increases in K+ channel gene expression should reduce neuronal network excitability. We have tested this hypothesis by creating a transgenic mouse that expresses a Shaker-type K+ channel gene. Paradoxically, we find that addition of the extra K+ channel gene results in a hyperexcitable rather than a hypoexcitable phenotype. The presence of the transgene leads to a complex deregulation of endogenous Shaker genes in the adult central nervous system as well as an increase in network excitability that includes spontaneous cortical spike and wave discharges and a lower threshold for epileptiform bursting in isolated hippocampal slices. These data suggest that an increase in K+ channel gene dosage leads to dysregulation of normal K+ channel gene expression, and it may underlie a mechanism contributing to the pathogenesis of human aneuploidies such as Down syndrome.

Cloning and expression of Shaker α- and β-subunits during inner ear development

Sensory cells of the chicken cochlea exhibit different ion channels relative to their position along the epithelium. One of these channels conducts an A-type potassium current which is found primarily in `short' hair cells. Here, we report the first full length cloning and developmental expression of Shaker genes from this endorgan. Clones were obtained by screening a chicken ( Gallus gallus) cochlea cDNA library, using probes made from RHK1 (i.e., Kvα1.4) cDNA, a Shaker homologue isolated from rat heart, and hKvβ1.2 cDNA, a β homologue isolated from human heart. Sequence analysis revealed a chick homologue of Kvα1.4, with a deduced amino acid similarity of 76–79% to mammalian Kvα1.4, and a chick homologue of Kvβ1.1, with a similarity of 95% to mammalian Kvβ1.1. In addition, we isolated a variant of cKvα1.4 (cKvα1.4 (m)) that differs in its untranslated regions and shows complete similarity in its coding region, except for the deletion of a single nucleotide. During development of the inner ear, reverse transcription–polymerase chain reaction (RT-PCR) studies show that the β-subunit is expressed as early as embryonic day 3, whereas α- and β-subunits are coexpressed on embryonic days 7 to 10, 14, and in adult.

Tissue-specific alternative splicing of Shaker potassium channel transcripts results from distinct modes of regulating 3? splice choice

Alternative splicing of precursor RNA enables a single gene to encode multiple protein isoforms with different functional characteristics and tissue distributions. Differential splicing of Drosophila Shaker (Sh) gene transcripts regulates the tissue-specific expression of kinetically distinct potassium ion channels throughout development. Regulation of Sh alternative splicing is being examined in germline transformants using lacZ as a reporter gene. P-element constructs were generated in which one or both of the two mutually exclusive Sh 3' acceptor sites were positioned in the same translational reading frame as the lacZ coding sequences. The constructs were introduced into the germline and the transgenic animals examined for tissue-specific beta-galactosidase expression patterns. Some tissues exhibit "promiscuous" splicing; these tissues are competent to splice to either 3' acceptor even when both are present on the same pre-mRNA. In other tissues splice choice results from competition between the two 3' sites; these tissues can splice to either site when it is the only available 3' acceptor, but when given a choice will splice to only one of the two 3' acceptors. In some tissues, splicing occurs exclusively at only one of the 3' acceptor sites; these tissues are not competent to splice to one of the sites even if it is the only 3' acceptor present on the pre-mRNA. These results suggests that multiple, distinct regulatory modes are operating to control tissue-specific alternative splicing of Sh 3' domains and are discussed in terms of potential underlying mechanisms for regulating the tissue-specific expression of alternatively spliced genes.

Different photoreceptors within the same retina express unique combinations of potassium channels

Single electrode current and voltage clamp recordings in Calliphora, and whole-cell voltage clamp recordings in Drosophila were used to characterise the voltage-gated K channels in both major classes of photoreceptors, R7/8 (long visual fibres, LVFs) and R1-6 (short visual fibres, SVFs). R7/8 were identified by their unique spectral properties, ca. 3–4 fold higher input resistances and 3–4 fold lower cell capacitance. In Calliphora SVFs possess both fast and slow activating delayed rectifier potassium conductances. Drosophila SVFs possess a slowly inactivating delayed rectifier (IKs), a very rapidly inactivating A channel encoded by the Shaker gene (IA), and, in a minority of cells, a third K conductance with intermediate kinetics (IKf). In both specs the LVFs lack the slowest component, but exhibit the faster K conductance(s) with properties indistinguishable from those in the SVFs. These findings add to established evidence demonstrating the significant role played by potassium channels in tuning the photoreceptor membrane. The results also suggest that R1-6 photoreceptors and R7/8 form inputs to visual subsystems tuned to different temporal frequencies.

Unidirectional K+ fluxes through recombinant Shaker potassium channels expressed in single Xenopus oocytes.

We describe a method to evaluate the ratio of ionic fluxes through recombinant channels expressed in a single Xenopus oocyte. A potassium channel encoded by the Drosophila Shaker gene tested by this method exhibited flux ratios far from those expected for independent ion movement. At a fixed extracellular concentration of 25 mM K+, this channel showed single-file diffusion with an Ussing flux-ratio exponent, n', of 3.4 at a membrane potential of -30 mV. There was an apparent, small voltage dependence of this parameter with n' values of 2.4 at -15 and -5 mV. These results indicate that the pore in these channels can simultaneously accommodate at least four K+ ions. If each of these K+ ions is in contact with two water molecules, the minimum length of the pore is 24 A.