Potassium Channel mRNA Research Articles

Squid express conserved ADAR orthologs that possess novel features.

The coleoid cephalopods display unusually extensive mRNA recoding by adenosine deamination, yet the underlying mechanisms are not well understood. Because the adenosine deaminases that act on RNA (ADAR) enzymes catalyze this form of RNA editing, the structure and function of the cephalopod orthologs may provide clues. Recent genome sequencing projects have provided blueprints for the full complement of coleoid cephalopod ADARs. Previous results from our laboratory have shown that squid express an ADAR2 homolog, with two splice variants named sqADAR2a and sqADAR2b and that these messages are extensively edited. Based on octopus and squid genomes, transcriptomes, and cDNA cloning, we discovered that two additional ADAR homologs are expressed in coleoids. The first is orthologous to vertebrate ADAR1. Unlike other ADAR1s, however, it contains a novel N-terminal domain of 641 aa that is predicted to be disordered, contains 67 phosphorylation motifs, and has an amino acid composition that is unusually high in serines and basic amino acids. mRNAs encoding sqADAR1 are themselves extensively edited. A third ADAR-like enzyme, sqADAR/D-like, which is not orthologous to any of the vertebrate isoforms, is also present. Messages encoding sqADAR/D-like are not edited. Studies using recombinant sqADARs suggest that only sqADAR1 and sqADAR2 are active adenosine deaminases, both on perfect duplex dsRNA and on a squid potassium channel mRNA substrate known to be edited in vivo. sqADAR/D-like shows no activity on these substrates. Overall, these results reveal some unique features in sqADARs that may contribute to the high-level RNA recoding observed in cephalopods.

Identification of MicroRNA-Potassium Channel Messenger RNA Interactions in the Brain of Rats With Post-traumatic Epilepsy.

Background: Dysregulated expression of microRNAs and potassium channels have been reported for their contributions to seizure onset. However, the microRNA–potassium channel gene interactions in traumatic brain injury-induced post-traumatic epilepsy (PTE) remain unknown.Methods: PTE was induced in male rats by intracranial injection with ferrous chloride (0.1 mol/L, 1 μl/min) at the right frontal cortex. Electroencephalography was recorded at 60 min, as well as day 1, 7, and 30, and the behavioral seizures were assessed before injection and at different time points after injection. Rats were killed on day 30 after injection. The right frontal cortex samples were collected and subjected to high throughput messenger RNA (mRNA) and microRNA sequencing. A network of differentially expressed potassium channel mRNAs and microRNAs was constructed using OryCun2.0 and subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. The differential mRNA and microRNA expressions were verified using quantitative real-time-PCR. The microRNA–mRNA was subject to the Pearson correlation analysis.Results: A PTE rat model was successfully established, as evidenced by behavioral seizures and epileptiform discharges on electroencephalography in PTE rats compared with sham rats. Among the 91 mRNAs and 40 microRNAs that were significantly differentially expressed in the PTE rat brain, 4 mRNAs and 10 microRNAs were associated with potassium channels. Except for potassium calcium-activated channel subfamily N member 2, the other three potassium channel mRNAs were negatively correlated with seven microRNAs. These microRNA–mRNA pairs were enriched in annotations and pathways related to neuronal ion channels and neuroinflammation. Quantitative real-time-PCR and correlation analysis verified negative correlations in miR-449a-5p-KCNH2, miR-98-5p-KCNH2, miR-98-5p-KCNK15, miR-19b-3p-KCNK15, and miR-301a-3p-KCNK15 pairs.Conclusion: We identified microRNA–potassium channel mRNA interactions associated with PTE, providing potential diagnostic markers and therapeutic targets for PTE.

A Single Prior Injection of Methamphetamine Enhances Methamphetamine Self-Administration (SA) and Blocks SA-Induced Changes in DNA Methylation and mRNA Expression of Potassium Channels in the Rat Nucleus Accumbens

The transition from occasional to escalated psychostimulant use is accelerated by prior drug exposure. These behavioral observations may be related to long-lasting transcriptional and/or epigenetic changes induced by the drug pre-exposure. Herein, we investigated if a single methamphetamine (METH) injection would enhance METH self-administration (SA) and impact any METH SA-induced epigenetic or transcriptional alterations. We thus injected a single METH dose (10 mg/kg) or saline to rats before training them to self-administer METH or saline. There were three experimental groups in SA experiments: (1) a single saline injection followed by saline SA (SS); (2) a single saline injection followed by METH SA (SM); and (3) a single METH injection followed by METH SA (MM). METH-pretreated rats escalated METH SA earlier and took more METH than saline-pretreated animals. Both groups showed similar incubation of cue-induced METH craving. Because compulsive METH takers and METH-abstinent rats show differences in potassium (K+) channel mRNA levels in their nucleus accumbens (NAc), we wondered if K+ channel expression might also help to distinguish between SM and MM groups. We found increases in mRNA and protein expression of shaker-related voltage-gated K+ channels (Kv1: Kcna1, Kcna3, and Kcna6) and calcium-activated K+ channels (Kcnn1) in the SM compared to MM rats. SM rats also showed decreased DNA methylation at the CpG-rich sites near the promoter region of Kcna1, Kcna3 and Kcnn1 genes in comparison to MM rats. Together, these results provide additional evidence for potentially using K+ channels as therapeutic targets against METH use disorder.

Herbal Anti-Hyperglycemic Compound Increases Expression of Glucose Transporter Molecules in Diabetic Rats

Background: In previous studies, Sharma et al. has already isolated an anti-hyperglycemic compound from the fruit pulp of Eugenia jambolana using HPLC and other chromatographic techniques. However, the effect of antihyperglycemic compound (FIIc) on the expression of Glucose transporters and Kv 1.3 potassium channel in Streptozotocin-Nicotinamide induced diabetic rats has not been studied so far. Objective: To study the effect of HPLC purified herbal anti-hyperglycemic compound (FIIc) on the expression of GLUT-4, GLUT-8 and Kv 1.3 potassium channel in Streptozotocin-Nicotinamide induced diabetic rats. Methods: 24 Male Wistar rats were taken and diabetes was induced in group B, C and D rats (n=6 each) by injecting Streptozotocin at a dose of 45 mg/kg of body weight 15 minutes after the administration of Nicotinamide at a dose of 230 mg/kg of body weight, intra-peritoneally to overnight fasted rats. Active compound (FIIc) was orally administered to group C and Pioglitazone to group D at a dose of 20 mg/kg of body weight for 6 weeks respectively. Serum was separated for the estimation of Adiponectin and TNF alpha at week 0 and week 6 of the study. Real time mRNA expression of GLUT-4, GLUT-8 and Kv 1.3 potassium channel was measured and compared between healthy and diabetic control rats. Expression of GLUT-4, GLUT-8 and Kv 1.3 potassium channel was also measured at protein level through Immunohistochemistry and compared between healthy and diabetic controls. Results: After treatment with FIIc for 6 weeks there was a 1.28 folds increase in GLUT-4 mRNA expression in skeletal muscles and 2.67 folds increase in GLUT-8 mRNA expression in liver tissues of group C rats as compared to group B rats. However, Kv 1.3 potassium channel mRNA expression was found to be at par among the four study groups. TNF alpha levels were found to be significantly decreased in group C rats as compared to group B. A slight increase in serum Adiponectin level was observed in group C as compared to group B, which was found to be statistically insignificant. Conclusion: FIIc treatment for 6 weeks significantly increases the expression of GLUT-4, GLUT-8 mRNA expression in liver and skeletal muscles leading to increased peripheral insulin sensitivity.

BK potassium channel mRNA level changes in gills of Atlantic salmon after brackish water transfer

BK potassium channels are implicated in a variety of physiological processes and they have been characterized in many species from Drosophila to humans although detailed studies about their properties and functions in the osmoregulation of teleosts are still lacking. In this study, we have cloned 911 bp corresponding to a partial Salmo salar BK channel coding sequence with high level of homology to BK channel sequences from other teleosts. Also, we have followed the BK gene expression pattern in gills of Atlantic salmon during brackish water (BW) acclimation. Interestingly, transfer from freshwater to BW induced an upregulation in mRNA BK channel expression in gills 7 days after transfer to BW, followed by a decrease in the expression. These results are the first describing mRNA changes of BK channel in gills of Atlantic salmon during BW acclimation, and they could suggest a possible role for BK channel during this adaptive response. Additionally, our results could position BK potassium channels expressed in the gills of Salmo salar as new candidates to be a marker of smoltification process, which may complement, and possibly outperform NKA activity measurement, the current gold standard of the Chilean industry.

Renal and colonic potassium transporters in the pregnant rat.

Gestational potassium retention, most of which occurs during late pregnancy, is essential for fetal development. The purpose of this study was to examine mechanisms underlying changes in potassium handling by the kidney and colon in pregnancy. We found that potassium intake and renal excretion increased in late pregnancy while fecal potassium excretion remained unchanged and that pregnant rats exhibited net potassium retention. By quantitative PCR we found markedly increased H+-K+-ATPase type 2 (HKA2) mRNA expression in the cortex and outer medullary of late pregnant vs. virgin. Renal outer medullary potassium channel (ROMK) mRNA was unchanged in the cortex, but apical ROMK abundance (by immunofluorescence) was decreased in pregnant vs. virgin in the distal convoluted tubule (DCT) and connecting tubule (CNT). Big potassium-α (BKα) channel-α protein abundance in intercalated cells in the cortex and outer medullary collecting ducts (by immunohistochemistry) fell in late pregnancy. In the distal colon we found increased HKA2 mRNA and protein abundance (Western blot) and decreased BKα protein with no observed changes in mRNA. Therefore, the potassium retention of pregnancy is likely to be due to increased collecting duct potassium reabsorption (via increased HKA2), decreased potassium secretion (via decreased ROMK and BK), as well as increased colonic reabsorption via HKA2.

Differential Expression of Ion Channels and Transporters During Hepatocellular Carcinoma Development.

Ion channels and transporters are potential markers and therapeutic targets for several cancers. However, their expression during hepatocellular carcinoma (HCC) development remains unclear. To investigate the mRNA expression of Na(+), K(+) and Ca(2+) channels and ABC transporters during rat HCC development, as well as Abcc3 protein in human liver biopsies. Wistar rats were treated with diethylnitrosamine (DEN) and developed both cirrhosis (12weeks of treatment) and either pre-neoplastic lesions (16weeks of treatment) or multinodular HCC (16weeks of treatment plus 2weeks DEN-free). The mRNA expression of 12 ion channels and two ABC transporters was studied using real-time RT-PCR. Tumor-containing or tumor-free liver sections were isolated by laser-capture microdissection. Abcc3 protein expression was studied by immunohistochemistry in healthy, cirrhotic and HCC human biopsies. We observed expression changes in seven genes. Kcna3, Kcnn4, Kcnrg and Kcnj11 potassium channel mRNA expression reached peak values at the end of DEN treatment, while Scn2a1 sodium channel, Trpc6 calcium channel and Abcc3 transporter mRNA expression reached their highest levels in the presence of HCC (18weeks). Whereas Kcnn4 and Scn2a1 channel expression was similar in non-tumor and tumor tissue, the Abcc3 transporter and Kcna3 potassium channels were preferentially overexpressed in the tumor sections. We observed differential Abcc3 protein subcellular localization and expression in human samples. The ion channel/transporter expression profile observed suggests that these genes are potential early markers or therapeutic targets of HCC. The differential localization of Abcc3 may be useful in the diagnosis of cirrhosis and HCC.

A long noncoding RNA contributes to neuropathic pain by silencing Kcna2 in primary afferent neurons.

Neuropathic pain is a refractory disease characterized by maladaptive changes in gene transcription and translation in the sensory pathway. Long noncoding RNAs (lncRNAs) are emerging as new players in gene regulation, but how lncRNAs operate in the development of neuropathic pain is unclear. Here we identify a conserved lncRNA, named Kcna2 antisense RNA, for a voltage-dependent potassium channel mRNA, Kcna2, in first-order sensory neurons of rat dorsal root ganglion (DRG). Peripheral nerve injury increased Kcna2 antisense RNA expression in injured DRG through activation of myeloid zinc finger protein 1, a transcription factor that binds to the Kcna2 antisense RNA gene promoter. Mimicking this increase downregulated Kcna2, reduced total voltage-gated potassium current, increased excitability in DRG neurons and produced neuropathic pain symptoms. Blocking this increase reversed nerve injury-induced downregulation of DRG Kcna2 and attenuated development and maintenance of neuropathic pain. These findings suggest endogenous Kcna2 antisense RNA as a therapeutic target for the treatment of neuropathic pain.

Huntington disease skeletal muscle is hyperexcitable owing to chloride and potassium channel dysfunction

Huntington disease is a progressive and fatal genetic disorder with debilitating motor and cognitive defects. Chorea, rigidity, dystonia, and muscle weakness are characteristic motor defects of the disease that are commonly attributed to central neurodegeneration. However, no previous study has examined the membrane properties that control contraction in Huntington disease muscle. We show primary defects in ex vivo adult skeletal muscle from the R6/2 transgenic mouse model of Huntington disease. Action potentials in diseased fibers are more easily triggered and prolonged than in fibers from WT littermates. Furthermore, some action potentials in the diseased fibers self-trigger. These defects occur because of decreases in the resting chloride and potassium conductances. Consistent with this, the expression of the muscle chloride channel, ClC-1, in Huntington disease muscle was compromised by improper splicing and a corresponding reduction in total Clcn1 (gene for ClC-1) mRNA. Additionally, the total Kcnj2 (gene for the Kir2.1 potassium channel) mRNA was reduced in disease muscle. The resulting muscle hyperexcitability causes involuntary and prolonged contractions that may contribute to the chorea, rigidity, and dystonia that characterize Huntington disease.

277. Time-Dependent Effects of Synthetic Double-Stranded RNA on Eag1 Potassium Channel mRNA Content from Human Glioma Cells

277. Time-Dependent Effects of Synthetic Double-Stranded RNA on Eag1 Potassium Channel mRNA Content from Human Glioma Cells

226: A novel approach to understanding cardiac physiology using a computer model

Physiological myocardial hypertrophy is an important adaptive response to the increased workload of pregnancy. The action potentials(AP) in pregnancy, through unknown molecular mechanisms, are found to have a longer QT-interval dispersion. Using a computer model of myocardial function, we sought to quantify changes in sodium and potassium channel mRNA expression during pregnancy to determine if this accounts for observed changes in the AP.

Profiling Voltage-gated Potassium Channel mRNA Expression in Nigral Neurons using Single-cell RT-PCR Techniques

In mammalian central nervous system, different types of neurons with diverse molecular and functional characteristics are intermingled with each other, difficult to separate and also not easily identified by their morphology. Thus, it is often difficult to analyze gene expression in a specific neuron type. Here we document a procedure that combines whole-cell patch clamp recording techniques with single-cell reverse transcription polymerase chain reaction (scRT-PCR) to profile mRNA expression in different types of neurons in the substantial nigra. Electrophysiological techniques are first used to record the neurophysiological and functional properties of individual neurons. Then, the cytoplasm of single electrophysiologically characterized nigral neurons is aspirated and subjected to scRT-PCR analysis to obtain mRNA expression profiles for neurotransmitter synthesis enzymes, receptors, and ion channels. The high selectivity and sensitivity make this method particularly useful when immunohistochemistry can not be used due to a lack of suitable antibody or low expression level of the protein. This method is also applicable to neurons in other brain areas.

Profiling Voltage-gated Potassium Channel mRNA Expression in Nigral Neurons using Single-cell RT-PCR Techniques

In mammalian central nervous system, different types of neurons with diverse molecular and functional characteristics are intermingled with each other, difficult to separate and also not easily identified by their morphology. Thus, it is often difficult to analyze gene expression in a specific neuron type. Here we document a procedure that combines whole-cell patch clamp recording techniques with single-cell reverse transcription polymerase chain reaction (scRT-PCR) to profile mRNA expression in different types of neurons in the substantial nigra. Electrophysiological techniques are first used to record the neurophysiological and functional properties of individual neurons. Then, the cytoplasm of single electrophysiologically characterized nigral neurons is aspirated and subjected to scRT-PCR analysis to obtain mRNA expression profiles for neurotransmitter synthesis enzymes, receptors, and ion channels. The high selectivity and sensitivity make this method particularly useful when immunohistochemistry can not be used due to a lack of suitable antibody or low expression level of the protein. This method is also applicable to neurons in other brain areas.

Gating Differs Between an Antarctic and a Tropical Kv1 Channel Because of RNA Editing

In squid, shaker-like potassium channel mRNA's are extensively edited by adenosine deamination. Because A-to-I RNA editing tends to recode for smaller amino acids, it could create more flexible proteins and be involved in cold adaptation. In this study we compare editing patterns and their functional consequences for a Kv1 K+ channel mRNA from two species of octopus: Pareledone sp., from the extreme cold of Antarctica and O. vulgaris from tropical inshore waters of Puerto Rico. From each species, the same Kv1 gene was cloned and sequenced. An editing map was created by sequencing 50 cDNA clones and identifying sites with A/G variation. At the genomic level, the channels were nearly identical, differing by four amino acids. By contrast, their editing patterns differed substantially. In total there were 13 non-silent editing sites, with five sites unique to one species or the other. At four sites editing percentages differed by more than 50% between species: N40S and S54G are edited more in O. vulgaris while N105G and I321V are edited more in Pareledone. We tested the eletrophysiological effects of differences at both the genomic and RNA editing levels. Channels were expressed in Xenopus oocytes and their kinetics of activation, deactivation, and inactivation, along with their voltage-dependence, were compared. The two genomically encoded channels were nearly identical, although the activation kinetics were slightly faster for the Antarctic channel. Individual editing sites, however, changed multiple parameters. The “tropical” edits N40S and S54G, both in the tetramerization domain, slowed activation and accelerated inactivation. The “Antarctic” edit I321V shifted the voltage dependence of activation by +12 mV and more than doubled the rate of deactivation. I321V, which is in the S5 helix, appears to be a candidate for cold-adaptation.

Bringing down the ROS: a new therapeutic approach for PPHN

Although infants with persistent pulmonary hypertension of the newborn (PPHN) experience some relief and therapeutic benefit from current therapies, over 50% have a limited or transient response and significant morbidity. There is no consistency in the best first line treatment throughout hospitals in the United States. Ventilation with high levels of oxygen or inhaled nitric oxide (NO) are typical strategies for improving the extracorporeal membrane oxygen, although they remain unproven to increase survival rates. While oxygen may stimulate endothelial nitric oxide synthase (eNOS) and NO production dilating the pulmonary vasculature, it also fuels the production of reactive oxygen species (ROS). ROS is likely to have counterproductive effects; in addition to stimulating vascular smooth muscle cell proliferation and increasing vascular tone, ROS may directly regulate eNOS and NO. The recent article by Farrow and colleagues (3) in AJP-Lung investigates the role of ROS on eNOS. By using recombinant human superoxide dismutase (rhSOD), they observed 1) increased eNOS activity and expression, 2) increased tetrahydrobiopterin (BH4), a cofactor critical to the function of eNOS, and 3 )a decrease in oxidative stress, in addition to the stimulation of NO production and ultimately pulmonary vasodilatation. The observations they made may be paramount to increasing the survival of infants with PPHN and may lead to an adapted treatment regimen that addresses the pitfalls of current therapeutic approaches. PPHN When the pulmonary circulation fails to respond to natural stimuli, including increased oxygen tension, ventilation, and shear stress, it does not undergo the shift from the high resistance state in utero to a postnatal low resistance system, enabling efficient pulmonary gas exchange and oxygenation. Impaired NO-cGMP signaling has been shown to be critical to the regulation of pulmonary circulation in the newborn, and clinical strategies have involved administration of inhaled NO since the early 1990s (6, 10). While effective in immediate relief due to vasodilatation, the infants can enter an inhaled NO dependency state, and thus inhaled NO proffers poor long-term relief. The necessity for extensive research into the regulation of perinatal circulation and the changes that occur upon ventilation have led to improved and more specific therapeutic approaches for infants with PPHN over the past 30 years. Despite this, PPHN is still associated with significant shortterm and long-term morbidity. Farrow et al. (3) strive to dissect the signaling pathways, determining the impact of ROS and elucidating the potential of decreasing oxidative stress as a therapeutic approach in PPHN. This study is published in a milieu of recent publications exploring the functional abnor

Alternative polyadenylation signals in the 3′ non-coding region of a voltage-gated potassium channel gene are major determinants of mRNA isoform expression

We investigated the role of the 3′ non-coding region of a mouse voltage-gated potassium channel mRNA (mKv1.4 mRNA) in post-transcriptional regulation of gene expression. In contrast to an earlier report from studies carried out in Xenopus oocytes, we found that 3′ non-coding region sequences of mKv1.4 mRNAs did not significantly affect expression of a heterologous reporter RNA in vitro or in mammalian cells/cell lines. Instead, our data revealed a possible role for alternative polyadenylation mediated by distinct determinants ∼ 0.2 kb and ∼ 1.2 kb downstream of the Kv1.4 coding region. The use of the downstream polyadenylation signal correlated with the synthesis of a larger Kv1.4 mRNA isoform that was more abundantly expressed than the smaller mRNA species, whose expression was regulated by the upstream polyadenylation signal. Our results suggest that the relative strengths of the polyadenylation signals are major determinants of overall Kv1.4 mRNA abundance in cells.

Regulation of ROMK (Kir 1.1) Channel Expression in Kidney Thick Ascending Limb by Hypertonicity: Role of TonEBP and MAPK Pathways

The present study assessed the mechanisms by which hypertonicity caused by NaCl enhances the renal outer medullary potassium channel (ROMK) mRNA abundance in rat kidney medullary thick ascending limb (MTAL) and in cultured mouse TAL cells. Using the run-off technique, we observed that the ROMK gene transcription rate in nuclei isolated from MTAL fragments was enhanced ∼40% by a high NaCl medium. In MTAL fragments, hypertonicity (450 mosm) caused by NaCl, not by mannitol or urea, enhanced both ROMK mRNA abundance and tonicity-responsive enhancer binding protein (TonEBP) total abundance and nuclear localization. In an immortalized mouse TAL cell culture in which ROMK is apically expressed, hypertonicity caused by both NaCl and mannitol, not urea, enhanced both ROMK mRNA abundance and TonEBP total abundance and nuclear localization. Confocal microscopy confirmed an increased nuclear translocation of TonEBP in response to NaCl-induced hypertonicity. Finally, inhibition of the p38 MAPK pathway by SB203580 and of the ERK pathway by PD98059 abolished the NaCl-induced stimulation of TonEBP and ROMK. These results establish that mRNA expression of ROMK is augmented in the MTAL by NaCl-induced hypertonicity through stimulation of ROMK gene transcription, and that TonEBP and the p38 MAPK and ERK pathways are involved in this effect.

Molecular analyses of KCNQ1–5 potassium channel mRNAs in rat and guinea pig inner ears: expression, cloning, and alternative splicing

Conclusion: Expression of neuronal Kcnq gene family transcripts in the inner ear provides further evidence for cochlear M-type currents and for complex molecular heterogeneities of voltage-gated potassium channels composed of various KCNQ subunits and/or alternative splice variants. Furthermore, important roles in regulation of cellular excitability in the auditory system, and hearing disorders related to (hyper)excitability, e.g. tinnitus, are implied. Background: Voltage-gated potassium channels play key roles in hearing, as evidenced by deafness resulting from disruption of genes encoding, for example, KCNQ1 or KCNQ4 subunits. Other members of the Kcnq gene family (Kcnq2, 3, and 5) are the molecular correlates of M currents, which regulate neuronal excitability. The expression of the latter has not previously been thoroughly investigated in the inner ear. Objective: The aim of this study was to identify genetic correlates of M currents, previously identified in cochlear hair cells by electrophysiological methods. Materials and methods: Expression of Kcnq genes was investigated by reverse transcription-polymerase chain reaction (RT-PCR) using subtype-specific primers with total RNA isolated from whole guinea pig or rat cochlea as template. PCR products were confirmed by direct DNA sequencing. Results: All members of the Kcnq family were expressed in guinea pig and rat cochlea. Cochlear expression of Kcnq2 exhibited two alternatively spliced forms, lacking exons 8, 15a, and 8, 12a, 15a, respectively. Novel molecular sequence data, e.g. guinea pig Kcnq cDNA sequences, were deposited in GenBank (AY684985–AY684990).

Structurally Distinct Elements Mediate Internal Ribosome Entry within the 5′-Noncoding Region of a Voltage-gated Potassium Channel mRNA

The approximately 1.2-kb 5'-noncoding region (5'-NCR) of mRNA species encoding mouse Kv1.4, a member of the Shaker-related subfamily of voltage-gated potassium channels, was shown to mediate internal ribosome entry in cells derived from brain, heart, and skeletal muscle, tissues known to express Kv1.4 mRNA species. We also show that the upstream approximately 1.0 kb and the downstream approximately 0.2 kb of the Kv1.4 5'-NCR independently mediated internal ribosome entry; however, separately, these sequences were less efficient in mediating internal ribosome entry than when together in the complete (and contiguous) 5'-NCR. Using enzymatic structure probing, the 3'-most approximately 0.2 kb was predicted to form three distinct stem-loop structures (stem-loops X, Y, and Z) and two defined single-stranded regions (loops Psi and Omega) in the presence and absence of the upstream approximately 1.0 kb. Although the systematic deletion of sequences within the 3'-most approximately 0.2 kb resulted in distinct changes in expression, enzymatic structure probing indicated that local RNA folding was not completely altered. Structure probing analysis strongly suggested an interaction between stem-loop X and a downstream polypyrimidine tract; however, opposing changes in activity were observed when sequences within these two regions were independently deleted. Moreover, deletions correlating with positive as well as negative changes in expression altered RNase cleavage within stem-loop X, indicating that this structure may be an integral element. Therefore, these findings indicate that Kv1.4 expression is mediated through a complex interplay between many distinct RNA regions.

Tonotopic gradients of membrane and synaptic properties for neurons of the chicken nucleus magnocellularis.

Nucleus magnocellularis (NM) is a division of the avian cochlear nucleus that extracts the timing of auditory signals. We compared the membrane excitability and synaptic transmission along the tonotopic axis of NM. Neurons expressed a Kv1.1 potassium channel mRNA and protein predominantly in the high characteristic frequency (CF) region of NM. In contrast, the expression of Kv1.2 mRNA did not change tonotopically. Neurons also showed tonotopic gradients in resting potential, spike threshold, amplitude, and membrane rectification. All neurons were sensitive to 100 nm dendrotoxin, but the effects were most significant in the high CF neurons. The EPSC recorded by minimal stimulation of auditory nerve fibers (ANFs) was 13 times larger in high CF neurons than in low CF neurons. Moreover, EPSCs were generated in an all-or-none manner in the high CF neurons when stimulus intensity was increased, whereas EPSCs were graded in the low CF neurons, indicating multiple axonal inputs. ANF synaptic terminals were visualized by DiI. ANF formed enfolding end-bulbs of Held around the cell body in the high and middle CF region but not in the low CF region. These observations indicate coordinated gradients of neuronal properties both presynaptically and postsynaptically along the tonotopic axis. Such specializations may be suitable for extracting and preserving the timing information of auditory signals over a wide range of acoustic frequencies.